CA2370426A1 - Identification of modulators of the marr family proteins - Google Patents

Abstract

Assays for identifying compounds which modulate the ability of MarR family polypeptides to bind to DNA and to negatively or positively regulate the transcription of genetic loci are disclosed herein. The instant assays detec t the ability of compounds to bind to and/or regulate the activity of MarR family polypeptides. The invention provides, inter alia, MarR family polypeptides and methods of their use.

Description

2 ~ 02370426 2001-10-22 IDENTIFICATION OF MODULATORS OF THE MARR FAMILY PROTEINS
Government Funding This work was funded, in part, by I~'IH grant GM 51661. The government may, therefore, have certain rights to this invention.
Background of the Invention Multidrug resistance in microbes is generally attributed to the acquisition of multiple transposons and plasmids bearing genetic determinants for different mechanisms of resistance (Gold et al. 1996. :'~~. Engl. J. Med. 335:1445).
However, descriptions of intrinsic mechanisms that confer multidrug resistance have begun to emerge. The first of these was a chromosomally encoded multiple antibiotic resistance (mar) locus in Escherichia coli (George and Levy. 1983. J. Bacteriol. 155:531;
George and Levy 1983. J. Bacteriol. 155:541).
The multiple antibiotic resistance (mar) operon of Escherichia coli is a chromosomally encoded locus that controls an adaptational response to antibiotics and other environmental hazards (Alekshun, M.N. and Levy, S.B. 1997. Antimicrob.
Agents Chemother. 10: 2067-2075). This control is accomplished on the genomic level by MarA, a transcriptional activator encoded within the marRAB operon, that regulates the expression of multiple genes on the E coli chromosome (Alekshun, M.N. and Levy, S.B. 1997. Antimicrob. Agents Chemother. 10: 2067-2075).
In the absence of an appropriate stimulus, MarR negatively regulates expression of the marRAB operon (Cohen, S.P., et al. 1993. J. Bacteriol. 175: 1484-1492.;
Martin, R.G. and Rosner, J.L. 1995. Proc. Natl. Acad. Sci. 92: 5456-5460; Seoane, A.S.
and Levy, S.B. 1995. J. Bacteriol. 177: 3414-3419., 1995). DNA footprinting experiments suggest that MarR dimerizes at two locations, sites I and II, within the mar operator (mar0) (Martin and Rosner, 1995, supra). Site I is positioned among the -35 and -10 hexamers and site II spans the putative MarR ribosome binding site (reviewed in Alekshun, M.N. and Levy, S.B. 1997. Antimicrob. Agents Chemother. 10: 2067-2075).

MarR is a member of a newly recognized family of regulatory proteins (Alekshun, M.N. and Levy, S.B. 1997. Antimicrob. Agents Chemother. 10: 2067-2075.
Sulavik, M.C., et al. 1995. Mol. Med. 1: 436-446) and many functional homologues have been identified in a variety of important human pathogens and have been found to regulate a variety of different processes. For example, some MarR homologues have been found to control expression of multiple antibiotic resistance operons, some regulate tissue-specific adhesive properties, some control expression of a cryptic hemolysin, some regulate protease production, and some regulate sporulation. Insight into the mechanism by which the MarR family of proteins is regulated would be of great value in controlling functions in microbes that are regulated by this family of proteins, for example, antibiotic resistance and virulence. and ultimately controlling them.
Summary The present invention represents an important advance in controlling microbial processes by demonstrating that multiple compounds bind to and directly affect the function of MarR as well as by elucidating the domains of MarR which are critical in mediating its function. Accordingly, the invention provides, inter alia, MarR
family polypeptides and methods of their use. This new understanding of how MarR
works to regulate gene transcription will be invaluable to understanding and ultimately controlling microbial processes that are regulated by this protein. Moreover, since MarR
is a member of a family of proteins, deciphering the function and structure of MarR
allows control of related proteins performing other essential functions in bacteria.
Accordingly, in one aspect, the invention pertains to a method for identifying a compound that modulates MarR family polypeptide activity or expression, by contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate the activity or expression of the MarR family polypeptide to thereby identify a compound that modulates MarR family polypeptide activity or expression.
In one embodiment, the ability of the compound to modulate MarR family polypeptide activity is detected. In another embodiment, the ability of the compound to modulate MarR family polypeptide expression is detected.

-, In another aspect, the invention pertains to a method for identifying a compound that modulates the ability of a compound to modulate the ability of a MarR
family polypeptide to interact with a MarR binding partner, by contacting a MarR
family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate the ability of the MarR family polypeptide to interact with a MarR binding partner to thereby identify a compound that modulates the ability of a MarR family polypeptide to interact with a MarR binding partner.
In one embodiment, the MarR binding partner is a DNA molecule. In another embodiment, the MarR binding partner is a polypeptide.
In still another aspect, the invention pertains to a method for identifying a compound that modulates MDR, by contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate MDR to thereby identify a compound that modulates MDR.
In one embodiment, the MarR family polypeptide is expressed in a cell. In another embodiment, the MarR family polvpeptide is an isolated polypeptide.
In another embodiment, the DNA binding activity of the MarR family polypeptide is measured by detecting transcription from a gene locus regulated by a MarR family polypeptide.
In one embodiment, the MarR family polypeptide is derived from a protein selected from the group consisting of: MarR, Ec 17kd, MprA(EmrR), and MexR.
In one embodiment, the MarR family polypeptide is an E. coli MarR
polypeptide.
In yet another embodiment, the MarR family polypeptide comprises a MarR
family polypeptide helix-turn-helix domain corresponding to about amino acids 61-80 or about 97-116 of MarR.
In one embodiment, the polypeptide comprises an amino acid sequence corresponding to about amino acid 1 to about amino acid 41 of MarR.
In yet another embodiment, the polypeptide comprises an amino acid sequence corresponding to about amino acid 41 to about amino acid 144 of MarR.

In still another embodiment, the step of detecting the MarR family polypeptide activity comprises detecting transcription from a marRAB responsive promoter.
In one embodiment, the step of detecting comprises detecting the ability of the compound to modulate the binding of MarR to mar0.
In one embodiment, the marRAB responsive promoter is Pmarll. In another embodiment, the marRAB responsive promoter is linked to a reporter gene. In yet another embodiment, the reporter gene is selected from the group consisting of lacZ, phoA, or green fluorescence protein.
In one embodiment, the step of detecting comprises detecting the amount of reporter gene product produced by the cell. In another embodiment, the step of assaying comprises detecting the amount of RNA produced by the cell.
In one embodiment, the step of detecting comprises detecting the activity of the reporter gene product.
In another embodiment, the step of detecting comprises detecting the ability of an antibody to bind to the reporter gene product.
In one aspect, the invention pertains to a method for identifying a compound that modulates MDR, comprising: screening a library of bacteriophage displaying on their surface a MarR polypeptide, the polypeptide sequence being specified by a nucleic acid molecule contained within the bacteriophage, for the ability to bind a compound to obtain those compounds having affinity for the MarR polypeptide; said method by contacting the phage which displays the MarR polypeptide with a compound so that the polypeptide can form a complex with a compound having an affinity for the polypeptide;
contacting the complex of the polypeptide and bound compound with an agent that dissociates the bacteriophage from the compound; and identifying the compounds that bound to the polypeptide to thereby identify a compound that modulates MDR.
In one embodiment, the compound is an antibiotic compound. In another embodiment, the compound is non-antibiotic compound. In another embodiment, the compound is a candidate disinfectant or antiseptic compound. In yet another embodiment, the compound is derived from a library of compounds.

Brief Description of the Drawings Figure 1 shows the genetic organization of the Escherichia coli mar locus.
Expression of the two transcriptional units. containing marC and marRAB, is under the control of independent promoters (Pmarl and Pmarll) located within a centrally positioned promoter/operator region (marPlO). The repressor MarR negatively controls marRAB expression by binding to mar0. MarA activates transcription of marRAB
by binding to Pmarll. MarB has unknown function as does MarC which encodes a putative inner membrane protein with multiple transmembrane spanning helices.
Figure 2 illustrates the structures of some chemical inducers that directly affect MarR function in vitro.
Figure 3 shows a map of the plasmid pSup-Test. Expression of ccdB is positively controlled by the marRAB promoter (Pmarll), 196 by upstream of ccdB, and negatively regulated by MarR binding to the mar operator (mar0) and LacR to the lac operator (lac0). The sequence of mar0 is given and within it sites I and II, the known MarR binding regions, are underlined. The locations of the two SspI
recognition sequences are indicated and the SspI restriction site in mar0 is boldfaced.
Figure 4 shows locations of several mutations in MarR. The thick horizontal line represents full length MarR and the scale above it depicts residues in the protein. The full-length sequence of MarR is available (Cohen, S. P., et al. 1993. J.
Bacteriol. 175:
1484-1492.). Solid dots and asterisks on the line designate nonsense and Pro to Ser missense mutations, respectively. Vertical bars above the line indicate mutations that result in moderately active MarR; vertical bars below the line mark positions of negative complementing traps-dominant mutations. Broken bars below the line depict mutations that result in near wild type repressor activities. The first 41 amino acids are presumed to participate in the oligomerization of the repressor. The positions of the putative helix-turn-helix (HTH) motifs for DNA binding are indicated.

Figure 5 compares sequences of the two putative helix-turn-helix (HTH) motifs in MarR comprising residues 61-80 (MarR-M) in the middle and 97-116 (MarR-C) in the C-terminus of the full length protein with other known HTH structures. The MarA, TrpR, Fis, TetR, and y8 resolvase crystal structures have been previously characterized (Patio, C. O. and Sauer, R. T. 1984. Ann. Rev. Biochem. 53: 293-321.; Patio, C. O. and Sauer, R. T. 1992. Annu. Rev. Biochem. 61: 1053-1095; Rhee, S., et al. 1998.
Proc. Natl.
Acad Sci. USA 18: 10413-10418.). Amino acids are given their standard single letter abbreviations. Residues where MarR traps-dominant mutations were isolated are underlined and positions that are normally subject to stereochemical constraints are italicized. The helix-turn-helix consensus sequence designation (Con) at the top of the alignment was adapted from a previous report (Kelley, R. L. and Yanofsky, C.
1985.
Proc. Natl. Acad Sci. USA 82: 482-487.) and indicates if an amino acid is exposed to solvent (J), partially exposed (X), completely buried (B), non-branched chain (b), glycine or alanine (O), or makes contacts with DNA (~) (Branden, C. and Tooze, J.
1991. Introduction to protein structure. New York, NY, USA; London, England:
Garland Publishing, Inc.; Patio and Sauer, 1984, supra; Patio and Sauer, 1992, supra).
Residues that do not conform to the consensus sequence in either the putative MarR
HTHs or HTH motifs of known crystal structure are indicated in a boldfaced font. The putative helix-turn-helix in MarR as proposed previously (MarR/CinR
(Dalrymple, B. P.
and Swadling, Y. 1997. 143: 1203-1210.)) is also shown.
Figure 6 shows diagrams of the two helix-turn-helix motifs in MarR
representing amino acids 61-80 (MarR-M) and 97-116 (MarR-C) of the full length protein.
Residues that are well conserved among known HTH motifs are circled. In the traps-dominant mutants, wild type residues were changed to the amino acids in the rectangles.
Figure 7 shows the mar operator (mar0) sequence (not drawn to scale). MarR
protects two regions (half sites) in mar0 , sites I and II (Martin, R. G. and Rosner, J. L.
1995. Proc. Natl. Acad Sci. 92: 5456-5460.) or direct repeats (DR)-l and 1' (Cohen, S.
et al. 1993. J Bacteriol. 175: 1484-1492.), from nuclease digestion (Martin and Rosner, 1995, supra) that extend (indicated by the brackets) beyond the sequences shown here.

_ '7 _ Sites I and II have a dyad axis of symmetw (indicated by the broken lined boxes) and are composed of two sub-elements (represented by arrows above or below those sequences).
In this figure, the top strand of the double helix (5'-3') is contained within solid boxes and its complement (3'-5') is not.
Figure 8 shows the sequence of Pmarlllmar0. The locations of the -35/-10 hexamers sequences and MarR ribosome binding site are indicated and the SspI
restriction enzyme recognition sequence in site I of mar0 is in boldfaced font.
Figure 9 shows the locations of the MarR superrepressor mutations identified in this study. The designations in parenthesis refer to the single letter code for the wild type residue and is followed by the location of that amino acid in the full length MarR
and the single letter code for the mutation isolated at this point. The numbers in parenthesis represent the number of independent isolates at that site. The checkered box indicates the region of amino acid homology among the MarR family members.
Figure 10 shows MarR repressor activity assayed in the Pmarll IacZ reporter strain E coli SPC105 (marR+) without or with salicylate (hatched bar). The lower the LacZ activity, the stronger the activity of MarR mutants and decreased response to salicylate.
Detailed Description The present invention represents an advance in controlling processes in microbes regulated by the MarR family of proteins, e.g., their ability to grow, cause infection, and to resist drugs. The examples presented herein represent the first demonstration that multiple, structurally unrelated compounds can interact directly with MarR and affect its function. Prior to the instant invention, only sodium salicylate had been found to directly affect the activity of MarR. Radioactive salicylic acid was demonstrated to bind MarR using equilibrium dialysis assays, while other compounds, e.g., chloramphenicol and tetracycline, were not (Martin, R.G. and Rosner. 1995. Supra). These results suggested that compounds other than salicylates, rather than directly acting on MarR, _g_ induced multidrug resistance (MDR) indirectly. Surprisingly, the instant examples show that structurally diverse compounds can interact with MarR. In addition, critical domains of MarR are identified herein. Accordingly, the present invention provides, inter alia, assays for identifying compounds that interact with MarR and, thus, lead to the development of MDR in bacteria.
Before further description of the invention, certain terms employed in the specification, examples and appended claims are, for convenience, collected here.
I. Defrnitions The language "MarR family polypeptide" as used herein includes molecules related to MarR, e.g., having certain shared structural and functional features. MarR
family polypeptides share amino acid sequence similarity with MarR. MarR
family members, in addition to having similarity to MarR, all bind to DNA and regulate transcription. While some MarR family members negatively control transcription (e.g., MarR), others have positive/activator functions (e.g., SIyA, BadR, NhhD, and MexR).
MarR family polypeptides comprise DNA and protein binding domains. In addition, MarR family polypeptides can interact with a variety of structurally unrelated compounds that regulate their activity.
Exemplary MarR family members are taught in the art and can be found, e.g., in Sulavik et al. (1995. Molecular Medicine. 1:436) or Miller and Sulavik. (1996.
Molecular Microbiology. 21:441 ) in which alignments of MarR and related proteins are shown. Exemplary MarR family polypeptides are also illustrated in the following chart:

MarR Family Polypeptides Gram-negative Gram-positive cid-fast Escherichia coli acillus subtilus ycobacterium MarR YdcH tuberculosis SIyA YhbI 14.7kD

EmrR (MprA) YkmA Rv1404 PapX YkoM Rv0737 PrsX Orf7 Rv0042c HpcR YfiV Yz08 (15.6kD) Ec 17kD YetL

YdgJ ycobacterium leprae Slamonella typhimuriumYwoH Yz08 (15.6kD) MarR YwaE

SIyA YwhA rchaea EmrR Hpr ethanobacterium YybA thermoautotrophicum Pseudomonas YxaD MTH313 aeruginosa YsmB

MexR YusO ulfolobus solfataricus YpoP Lrs 14 Erwinia chrysanthemiYkvE

PecS rchaeoglobus fulgidus acillus frrmus CinR

Rhodopseudomonas Orf7 palustris urple non-sulfur BadR taphylococcus hodobacter capsulatus sciuri Orfl 45 Pete Burkholderia Orfl 41 pseudomallei inorhizobium meliloti OrfE utyrivibrio fibrisolvensSIyA (E293909) CinR

Orf158 hodococcus iodochrous NhhD
Orfl peucetius SUBSTITUTE SHEET (RULE 26) Thus, MarR family polypeptides are "structurally related" to one or more of the MarR molecules set forth in the Table above. This structural relatedness can be demonstrated by sequence similarity between two MarR family nucleotide sequences or between the amino acid sequences of two MarR family polypeptides. As used herein, the term "MarR family polypeptide" includes polypeptides specified by MarR
family genes. In isolating or identifying other MarR family molecules, sequence similarity can be shown, e.g., by generating alignments as described in more detail below.
Preferably, a MarR family polypeptide is MarR. Other preferred MarR family polypeptides include: EmrR, Ec 17kD, and MexR.
As used herein, the term "nucleic acid molecule(s)" includes polyribonucleotides or polydeoxribonucleotides, which may be unmodified RNA or DNA or modified RNA
or DNA. As such, "nucleic acid molecule(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions. single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
In addition, "nucleic acid molecule" as used herein refers to triple-stranded regions comprising RNA
or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
As used herein, the term "nucleic acid molecule" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "nucleic acid molecule(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are nucleic acid molecules as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "nucleic acid molecule(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of nucleic acid molecules, as well as the chemical forms of DNA and RNA
characteristic of WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 viruses and cells, including, for example. simple and complex cells. "Nucleic acid molecule(s)" also embraces short nucleic acid molecules often referred to as oligonucleotide(s).
Preferred MarR family nucleic acid molecules are isolated. An "isolated"
nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regard to genomic DNA, (e.g. whether chromosomal or episomal) the term "isolated" includes nucleic acid molecules which are separated from flanking DNA sequences with which the DNA
is naturally associated. Preferably, an "isolated" nucleic acid molecule is free of sequences which naturally flank the nucleic acid molecule (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) in the DNA (e.g., chromosomal or episomal) of the organism from which the nucleic acid molecule is derived. As such, isolated DNA is not in its naturally occurring state (although. as described in more detail below, its sequence may be naturally occurring in the sense that has not been altered (e.g., mutated) from its naturally occurring form). For example, in various embodiments, an isolated MarR
family nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb, 0.1 kb, or O.OSkb of nucleotide sequences which naturally flank the nucleic acid molecule in DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium ~i~hen produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" MarR family nucleic acid molecule may, however, be linked to other nucleotide sequences that do not normally flank the MarR family sequences in genomic DNA (e.g., the MarR family nucleotide sequences may be linked to vector sequences). In certain preferred embodiments, an "isolated" nucleic acid molecule, such as a cDNA molecule, also may be free of other cellular material. However, it is not necessary for the MarR family nucleic acid molecule to be free of other cellular material to be considered "isolated" (e.g., an MarR family DNA molecule separated from other chromosomal DNA and inserted into another bacterial cell would still be considered to be "isolated").

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 As used herein, "polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
"Polypeptide(s)" refers to both short chains. commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins.
Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
"Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications. but also by chemical modification techniques.
Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone. the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-1 S ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative. covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues. hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, Proteins--Structure And Molecular Properties, 2"d Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification Of Proteins, B. C.
Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol.
182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 cyclic, with or without branching. Cyclic. branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
As used herein, an "isolated polypeptide" or "isolated protein" refers to a polypeptide or protein that is substantially free of other polypeptides, proteins, cellular material and culture medium when isolated from cells or produced by recombinant DNA
techniques, or chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" polypeptide or biologically active portion thereof is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the MarR family polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of MarR family polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of MarR family polypeptide having less than about 30% (by dry weight) of non- MarR family polypeptide (also referred to herein as a "contaminating polypeptide"), more preferably less than about 20% of non- MarR family polypeptide, still more preferably less than about 10% of non- MarR family polypeptide, and most preferably less than about 5%
non- MarR family polypeptide. When the MarR family polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of MarR family polypeptide in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of MarR family polypeptide having less than about 30% (by dry weight) of chemical precursors or non- MarR family chemicals, more preferably less than about 20% chemical precursors or non-MarR

family chemicals, still more preferably less than about 10% chemical precursors or non-MarR family chemicals, and most preferably less than about 5% chemical precursors or non- MarR family chemicals.
Preferred MarR family nucleic acid molecules and polypeptides are "naturally occurring." As used herein, a "naturally-occurring" molecule refers to an MarR
family molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural MarR family polypeptide). In addition, naturally or non-naturally occurring variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to modulate adaptation to stress and/or virulence in a microbe. Such variants can be made, e.g., by mutation using techniques that are known in the art.
Alternatively, variants can be chemically synthesized.
As used herein the term "variant(s)" includes nucleic acid molecules or polypeptides that differ in sequence from a reference nucleic acid molecule or polypeptide, but retains its essential properties. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference nucleic acid molecule. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions in any combination. A variant of a nucleic acid molecule or polypeptide may be naturally occurnng, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acid molecules and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
For example, it will be understood that the MarR family polypeptides described herein are also meant to include equivalents thereof. Such variants can be made, e.g., by mutation using techniques that are known in the art. Alternatively, variants can be chemically synthesized. For instance, mutant forms of MarR family polypeptides which are functionally equivalent, (e.g., have the ability to bind to DNA and to regulate transcription from an operon) can be made using techniques which are well known in the art. Mutations can include, e.g., at least one of a discrete point mutation which can give rise to a substitution, or by at least one deletion or insertion. For example, random mutagenesis can be used. Mutations can also be made by random mutagenesis or using cassette mutagenesis. For the former, the entire coding region of a molecule is mutagenized by one of several methods (chemical, PCR, doped oligonucleotide synthesis) and that collection of randomly mutated molecules is subjected to selection or screening procedures. In the latter, discrete regions of a polypeptide, corresponding either to defined structural or functional determinants are subjected to saturating or semi-random mutagenesis and these mutagenized cassettes are re-introduced into the context of the otherwise wild type allele. In one embodiment, PCR mutagenesis can be used.
For example, Megaprimer PCR can be used (O.H. Landt, 1990. Gene 96:125-128).
In certain embodiments, such variants have at least about 25, 30, 35, 40, 45, 50, 1 S or 60% or more amino acid identity with a naturally occurring MarR family polypeptide.
In preferred embodiments, such variants have at least about 70% amino acid identity with a naturally occurring MarR family polypeptide. In more preferred embodiments, such variants have at least about 80% amino acid identity with a naturally occurring MarR family polypeptide. In particularly preferred embodiments, such variants have at least about 90% amino acid identity and preferably at least about 95% amino acid identity with a naturally occurring MarR family polypeptide.
In yet other embodiments, a nucleic acid molecule encoding a variant of an MarR family polypeptide is capable of hybridizing under stringent conditions to a nucleic molecule encoding a naturally occurring MarR family polypeptide.
Preferred MarR family nucleic acid molecules and MarR family polypeptides are "naturally occurring." As used herein, a "naturally-occurring" molecule refers to an MaxR family polypeptide encoded by a nucleotide sequence that occurs in nature (e.g., encodes a natural MarR family polypeptide). Such molecules can be obtained from other microbes, e.g., based on their sequence similarity to the MarR family molecules described herein.

WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 In addition, naturally or non-naturally occurring variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to modulate microbial responses to environmental stress and, thereby, modulate microbial adaptation to stress and/or microbial virulence are also within the scope of the invention.
Such variants can be made, e.g., by mutation using techniques which are known in the art. Alternatively, variants can be chemically synthesized.
The language "MarR family polypeptide activity" as used herein includes the ability to bind to, and control gene expression. Other MarR family polypeptide activities are described in more detail below.
As used herein, the language "marR family promoter" includes a promoter which is positively or negatively regulated by a MarR family polypeptide.
Preferably, the promoter is a marRAB family promoter. A marRAB family promoter initiates transcription of an operon in a microbe and is structurally or functionally related to the marRAB promoter, e.g., is bound by MarA or a protein related to MarA.
Preferably, the marRAB family promoter is a marRAB promoter. For example, in the mar operon, several promoters are marRAB family promoters as defined herein, e.g., the 405-by Thal fragment from the mar0 region is a marRAB family promoter (Cohen et al. 1993.
J.
Bact. 175:7856). In addition, MarA has been shown to bind to a 16 by MarA
binding site (referred to as the "marbox" within mar0 (Martin et al. 1996. J.
Bacteriol.
178:2216). MarA also initiates transcription from the acrAB; micF; mlr 1, 2, 3; slp; nfo;
inaA; fpr; sodA; soi-17,19; zwf fumC; or rpsF promoters (Alekshun and Levy.
1997.
Antimicrobial Agents and Chemother. 41:2067). Other marRAB family promoters are known in the art and include: araBAD, araE, araFGH and araC, which are activated by AraC; Pm, which is activated by XyIS; melAB which is activated by MeIR; and oriC
which is bound by Rob.
The term "interact" or "bind" includes close contact between molecules that results in a measurable effect, e.g., on the conformation and/or activity of at least one of the molecules involved in the interaction. For example, a first molecule can be said to interact with a second when it inhibits the binding of the second molecule to a target binding partner (a molecule such as a nucleic acid or polypeptide molecule to which that second molecule normally binds, e.g., in a cell), or when it alters the activity of the WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 second molecule, e.g., by steric interaction with a domain of the second molecule that mediates its activity. For example, a DNA binding domain of a MarR family polypeptide can interact with DNA and alter the level of transcription of DNA or with a polypeptide molecule. Likewise, compounds can interact with (e.g., by binding) to an MarR
family polypeptide and alter the activity of the MarR family polypeptide or can interact with (e.g., by binding) to an MarR family nucleic acid molecule and alter transcription of an MarR family polypeptide from that nucleic acid molecule.
As used herein, the term "multiple drug resistance (MDR)" includes resistance to both antibiotic and non-antibiotic compounds. MDR results from the increased transcription of a chromosomal or plasmid encoded genetic locus in an organism, e.g., a marRAB locus, that results in the ability of the organism to minimize the toxic effects of a compound to which it has been exposed. as well as to other non-related compounds, e.g., by stimulating an efflux pumps) or microbiological catabolic or metabolic processes.
As used herein the term "reporter gene" includes any gene which encodes an easily detectable product which is operably linked to a promoter, e.g., a marRAB family promoter. By operably linked it is meant that under appropriate conditions an RNA
polymerase may bind to the promoter of the regulatory region and proceed to transcribe the nucleotide sequence of the reporter gene. In preferred embodiments, a reporter gene construct consists of marRAB family promoter linked to a reporter gene. In certain embodiments, however, it may be desirable to include other sequences, e.g, transcriptional regulatory sequences, in the reporter gene construct. For example, modulation of the activity of the promoter may be affected by altering the RNA
polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA. Thus, sequences which are herein collectively referred to as transcriptional regulatory elements or sequences may also be included in the reporter gene construct. In addition, the construct may include sequences of nucleotides that alter translation of the resulting mRNA, thereby altering the amount of reporter gene product.

WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 As used herein the term "compound" includes any reagent which is tested using the assays of the invention to determine whether it modulates a MarR family polypeptide activity. More than one compound, e.g., a plurality of compounds, can be tested at the same time for their ability to modulate a MarR family polypeptide activity in a screening assay.
Compounds that can be tested in the subject assays include antibiotic and non-antibiotic compounds. In one embodiment. compounds include candidate detergent or disinfectant compounds. Exemplary compounds which can be screened for activity include, but are not limited to, peptides, non-peptidic compounds, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides), and natural product extract libraries. The term "non-peptidic compound" is intended to encompass compounds that are comprised, at least in part, of molecular structures different from naturally-occurring L-amino acid residues linked by natural peptide bonds. However, "non-peptidic compounds" are intended to include compounds composed, in whole or in part, of peptidomimetic structures, such as D-amino acids, non-naturally-occurring L-amino acids, modified peptide backbones and the like, as well as compounds that are composed, in whole or in part, of molecular structures unrelated to naturally-occurring L-amino acid residues linked by natural peptide bonds. "Non-peptidic compounds" also are intended to include natural products.
Il. MarR family polypeptides and their functional domains MarR family member polypeptide sequences are "structurally related" to one or more known MarR family members, preferably to MarR. This structural relatedness is shown by sequence similarity between two MarR family polypeptide sequences or between two MarR family nucleotide sequences. Sequence similarity can be shown, e.g., by optimally aligning MarR family member sequences using an alignment program for purposes of comparison and comparing corresponding positions. To determine the degree of similarity between sequences, they will be aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for nucleic acid molecule for optimal alignment with the other protein or nucleic acid molecules). The amino acid residues or bases and corresponding amino acid positions or bases are then WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 compared. When a position in one sequence is occupied by the same amino acid residue or by the same base as the corresponding position in the other sequence, then the molecules are identical at that position. If amino acid residues are not identical, they may be similar. As used herein, an amino acid residue is "similar" to another amino acid residue if the two amino acid residues are members of the same family of residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art (see, for example, Altschul et al. 1990. J. Mol.
Biol.
215:403) including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenvlalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine. isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). The degree (percentage) of similarity between sequences, therefore, is a function of the number of identical or similar positions shared by two sequences (i.e., % homology = # of identical or similar positions/total # of positions x 100). Alignment strategies are well known in the art; see, for example, Altschul et al. supra for optimal sequence alignment.
MarR family polypeptides share some amino acid sequence similarity with MarR. The nucleic acid and amino acid sequences of MarR as well as other MarR
family polypeptides are available in the art. For example, the nucleic acid and amino acid sequence of MarR can be found, e.g., on GeneBank (accession number M96235 or in Cohen et al. 1993. J. Bacteriol. 175:1484. or in SEQ ID NO:1).
The nucleic acid and protein sequences of MarR can be used as "query sequences" to perform a search against databases (e.g., either public or private) to, for example, identify other MaxR family members having related sequences. Such searches can be performed, e.g., using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to MarR family nucleic acid molecules. BLAST
protein searches can be performed with the XBLAST program, score = 50, wordlength =
3 to obtain amino acid sequences homologous to MarR protein molecules of the WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 invention. To obtain gapped alignments for comparison purposes, Gapped BLAST
can be utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402.
When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
MarR family members can also be identified as being structurally similiar based on their ability to specifically hybridize to nucleic acid sequences specifying MarR. Such stringent conditions are known to those skilled in the art and can be found e.g., in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. Conditions for hybridizations are largely dependent on the melting temperature Tm that is observed for half of the molecules of a substantially pure population of a double-stranded nucleic acid. Tm is the temperature in °C at which half the molecules of a given sequence are melted or single-stranded. For nucleic acids of sequence 11 to 23 bases, the Tm can be estimated in degrees C
as 2(number of A+T residues) + 4(number of C+G residues). Hybridization or annealing of nucleic acid molecules should be conducted at a temperature lower than the Tm, e.g., 15 °C, 20°C, 25°C or 30°C lower than the Tm. The effect of salt concentration (in M of NaCI) can also be calculated, see for example, Brown, A., "Hybridization" pp.
503-506, in The Encyclopedia ofMolec. Biol., J. Kendrew, Ed., Blackwell, Oxford (1994).
Preferably, the nucleic acid sequence of a MarR family member identified in this way is at least about 10%, 20%, more preferably at least about 30%, more preferably at least about 40% identical and most preferably at least about 50%, or 60%, 70%, 80%, 90% or more identical or more with a MarR nucleotide sequence, e.g., with the entire length of the nucleotide sequence or a portion thereof. In another embodiment, a MarR
family member nucleic acid molecule has at least about 10%, 20%, 30%, 40%
identity and most preferably at least about 50%, 60%, 70%, 80%, 90% or more identity with a nucleic acid molecule comprising at least about 100, 200, 300, 400, 500, or more contiguous nucleotides of a MarR family member, e.g., as listed in the Table above.

WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 Preferably, MarR family members have an amino acid sequence at least about 20%, more preferably at least about 30%. more preferably at least about 40%
identical and most preferably at least about 50%, or 60%, 70%, 80%, 90% or more identical with a MarR amino acid sequence. However, it will be understood that the level of sequence similarity among microbial regulators of gene transcription, even though members of the same family, is not necessarily high. This is particularly true in the case of divergent genomes where the level of sequence identity may be low, e.g., less than 20%
(e.g., B.
burgdorferi as compared e.g., to B. subtilis). For example, the level of amino acid sequence homology between MarR and Pecs is about 31 % and the level of amino acid sequence homology between MarR and PapX is about 28% when determined as described above. Accordingly, structural similarity among MarR family members can also be determined based on "three-dimensional correspondence" of amino acid residues. As used herein, the language "three-dimensional correspondence" is meant to includes residues which spatially correspond, e.g., are in the same functional position of a MarR family protein member as determined, e.g., by x-ray crystallography, but which may not correspond when aligned using a linear alignment program. The language "three-dimensional correspondence" also includes residues which perform the same function, e.g., bind to DNA or bind the same cofactor, as determined, e.g., by mutational analysis.
Preferred MarR family polypeptides include: MarR, EmrR, Ecl7kD, MexR, PapX , SIyA, Hpr, PecS, Hpr, MprA, or (EmrR). In a more preferred embodiment, a MarR family polypeptide is selected from the group consisting of: MarR, EmrR, Ecl7kD, and MexR . In a particularly preferred embodiment, a MarR family polypeptide is MarR.
In addition to sharing structural similarity, MarR family members have a MarR
family polypeptide activity, i.e., they bind to DNA and regulate transcription. Some MarR family members positively regulate transcription (e.g., SIyA, BadR, NhhD, or MexR), while others negatively regulate transcription (e.g., MarR). While all MarR
family members bind to DNA and regulate transcription, the different loci controlled by 3D each family member regulate different processes in microbes. For example, MarR
family polypeptides can control the expression of microbial loci involved in:
regulation WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 of antibiotic resistance [e.g., MarR (Cohen et al. 1993. J. Bacteriol.
175:1484), EmrR
(Lomovskaya and Lewis. 1992. Proc. Natl. Acad. Sci. 89:8938), and Ecl7kD
(Sulavik et al. 1995. Mol. Med. 1:436), and MexR (Poole et al. 1996. Antimicrob. Agents.
Chemother. 40:2021 )], regulation of tissue-specific adhesive properties [e.g., PapX
(Marklund et al., 1992. Mol. Microbiol. 6:2225)], regulation of expression of a cryptic hemolysin [e.g., SIyA (Ludwig et al. 1995 249:4740)], regulation of protease production [e.g., Hpr from B. subtilis (Perago and Hoch. 1988. J. Bacteriol. 170:2560) and PecS
from Erwinia chrysanthemi (Reverchon et al., 1994. Mol. Microbiol. 11:1127)]
and regulation of sporulation [e.g., Hpr (Perego and Hoch. 1988. J. Bacteriol.
170:2560)], regulation of the breakdown of plant materials [e.g., CinR (Dalymple and Swadling 1997 Microbiology)] sensing of phenolic compounds [(e.g., Sulvik et al. 1995.
Mol.
Med. 1:436], and repress marRAB expression when introduced into E. coli [e.g., Ecl7kd (Marklund et al. 1992. Mol. Microbiol. 6:2225) and MprA (EmrR) (del Castillo et al., 1991. J. Bacteriol. 173:3924)]. The activiy of MarR family polypeptides is antagonized by salicylate (Lomovskaya et al., 1995. J. Bacteriol. 177:2328; Sulavik et al.
I 995. Mol.
Med. 1:436).
Preferred MarR family polypeptide activities include regulation of multiple drug resistance and/or regulation of virulence.
In addition to full length MarR family polypeptide fragments MarR family polypeptide and their use are also within the scope of the invention. As used herein, a fragment of a MarR family polypeptide refers to a portion of a full-length MarR family polypeptide which is useful in a screening assay to identify compounds which modulate a biological activity (e.g., DNA binding) of a MarR family polypeptide.
Accordingly, MarR family polypeptides for use in the instant screening assays can be full length MarR
family member proteins or fragments thereof. Thus, a MarR family polypeptide can comprise, consist essentially of, or consist of an amino acid sequence derived from the full length amino acid sequence of a MarR family member.
Full length MarR family polypeptides comprise both DNA interacting domains and protein interacting domains. Accordingly, in one embodiment, a polypeptide comprising a MarR family polypeptide DNA interacting domain can be used in a screening assay . For example, compounds can be tested for their ability to modulate, WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 e.g., interfere with and reduce the ability of a MarR family polypeptide to directly bind to DNA. Alternatively, compounds can be tested for their ability to alter downstream effects of this DNA binding, e.g., to alter the ability of a MarR family polypeptide to regulate transcription of an operon.
In another embodiment, a polypeptide comprising a MarR family member protein interacting domain can be used in a screening assay . For example, compounds can be tested for their ability to bind to a protein interactive domain of a MarR family polypeptide. Such binding can lead to allosteric changes in the polypeptide that interfere with or enhance the ability of the a MarR family polypeptide to interact with DNA or can block or alter the ability of other proteins (which are necessary for the MarR family polypeptide to regulate transcription) to interact with MarR. Alternatively, compounds can be tested for their ability to alter events downstream of such an interaction, e.g., the ability of a compound to bind to a polypeptide and alter the ability of the polypeptide to regulate transcription of an operon can be tested.
Full length MarR family polypeptides also comprise MarR family polypeptide helix-turn-helix domains. As used herein. the language "helix-turn-helix domain"
includes the art recognized definition of the term. Helix-turn helix (HTH) domains have been implicated in DNA binding (Ann Rev. of Biochem. 1984. 53:293). An example of a consensus sequence of a helix-turn-helix domain can be found in Brunelle and Schleif (1989. J. Mol. Biol. 209:607). The domain has been illustrated by the sequence JJJBbJJXOXJJJJBJJXX (adapted from Kelley and Yanofsky, 1985). This sequence indicates if an amino acid is exposed to solvent (J), partially exposed (X), completely buried (B), non-branched chain (b), glycine or alanine (O), or makes contacts within DNA (bold) (Branden, C. and Tooze, J. ( 1991 ) In Introduction to Protein Structure.
New York, NY USA; London, England: Garland Publishing, Inc., pp. 87-111.;
Pabo, C.O. and Sauer, R.T. (1984) Ann. Rev. Biochem. 53: 293-321.; Pabo, C.O. and Sauer, R.T. (1992) Anrcu. Rev. Biochem. 61: 1053-1095).
In the case of MarR, the two helix-turn-helix domains appear to be in the middle and C-terminal DNA binding domain of the polypeptide and comprise from about amino acids 61-80 and from about amino acids 97-116 of MarR. Given the HTH domains of MarR and the particular MarR mutants identified and characterized herein, the locations of HTH domains of other MarR family members which correspond to those identified in MarR and which can be used in the subject screening assays can be identified by one of skill in the art. For example, using the MarR polypeptide sequence and an alignment program (e.g., ALIGN, BLAST, or MultAlin) or based on three dimensional alignments, one can predict helix-turn-helix domains in the MarR family members and use them in screening assays. The first and second helm-turn-helix domains of MarR are shown in Figure 5.
Accordingly, in one embodiment. a polypeptide for use in a screening assay comprises a DNA interacting domain of a MarR family polypeptide. In one embodiment, such as polypeptide for use in a screening assay comprises a sequence that corresponds to the amino acid sequence shown from about amino acid 41 to about amino acid 144 of MarR. In a preferred embodiment, a MarR family polypeptide comprises from between about amino acid 41 to about amino acid 144 of MarR.
In one embodiment, a polypeptide for use in a screening assay comprises a protein interacting domain of a MarR family polypeptide. In one embodiment, such a polypeptide comprises a protein interacting domain comprising an amino acid sequence that corresponds to the amino acid sequence shown from about amino acid 1 to about amino acid 41 of MarR. In a preferred embodiment, a MarR family polypeptide comprises a protein interacting sequence shown from about amino acid 1 to about amino acid 41 of MarR.
In one embodiment, a polypeptide for use in a screening assay comprises a helix-turn-helix domain of a MarR family polypeptide. In one embodiment, a polypeptide comprises a helix-turn-helix domain comprising a sequence that corresponds to the amino acid sequence shown from about amino acid 61 to about amino acid 80 of MarR
and/or from about amino acid 97 to about amino acid 116 of MarR. In a preferred embodiment, a polypeptide comprises an amino acid sequence from about amino acid 61 to about amino acid 80 of MarR andlor from about amino acid 97 to about amino acid 116 of MarR.
In certain embodiments, other polypeptide sequences may also be present, e.g., portions of a MarR family polypeptide that are not absolutely required for MarR
function. In another embodiment non-MarR family polypeptide sequences (i.e., derived WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 - 25 _ from a different protein) can be present, e.g., sequences that might facilitate immobilizing the MarR family member polypeptide or portion thereof on a support, or, alternatively, might facilitate the purification of the domain. Accordingly, in one embodiment, a polypeptide for use in screening compounds consists essentially of a protein binding domain of a MarR family polypeptide, e.g., a polypeptide comprising an amino acid sequence that corresponds to the amino acid sequence shown from between about amino acid 1 to about amino acid 41 of MarR. In another embodiment, a polypeptide for use in screening compounds consists essentially of a DNA
interacting domain, e.g., a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence shown from about amino acid 41 to about amino acid 144 of MarR.
In another embodiment, a polypeptide for use in screening consists essentially of an HTH domain from a MarR family polypeptide, e.g., having an amino acid sequence that corresponds to that shown from about amino acids 61-80 or from about amino acids 97-116 of MarR. In preferred embodiments. a polypeptide for use in the subject screening assays consists essentially of a portion of a MarR polypeptide.
Preferred MarR family polypeptides are "naturally occurring." As used herein, a "naturally-occurring" molecule refers to an MarR family molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural MarR family protein).
In addition, naturally or non-naturally occurring variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to bind to DNA and regulate transcription. Such variants can be made, e.g., by mutation using techniques which are known in the art. Alternatively, variants can be chemically synthesized.
For example, it will be understood that the MaxR family polypeptides described herein, are also meant to include equivalents thereof. For instance, mutant forms of MarR family polypeptides which are functionally equivalent, (e.g., have the ability to bind to DNA and to regulate transcription from an operon) can be made using techniques which are well known in the art. Mutations can include, e.g., at least one of a discrete point mutation which can give rise to a substitution, or by at least one deletion or insertion. For example, random mutagenesis can be used. Mutations can be made, e.g., by random mutagenesis or using cassette mutagenesis. For the former, the entire coding WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 region of a molecule is mutagenized by one of several methods (chemical, PCR, doped oligonucleotide synthesis) and that collection of randomly mutated molecules is subjected to selection or screening procedures. In the latter, discrete regions of a protein, corresponding either to defined structural or functional determinants (e.g., the first or second helix of a helix-turn-helix domain) are subjected to saturating or semi-random mutagenesis and these mutagenized cassettes are re-introduced into the context of the otherwise wild type allele. In one embodiment, PCR mutagenesis can be used.
For example, Megaprimer PCR can be used (O.H. Landt, Gene 96:125-128).
In addition, other portions of the above described polypeptides suitable for use in the claimed assays, such as those which retain their function (e.g., the ability to bind to DNA, to regulate transcription from an operon) or those which are critical for binding to regulatory molecules (such as test compounds) can be easily determined by one of ordinary skill in the art, e.g, using standard truncation or mutagenesis techniques and used in the instant assays. Exemplary techniques are described by Gallegos et al. ( 1996.
J. Bacteriol. 178:6427).
In addition to MarR family polypeptides comprising only naturally-occurring amino acids, MarR family peptidomimetics are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics" (Fauchere, J. (1986) Adv. Drug Res.
15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J.
Med. Chem 30: 1229, which are incorporated herein by reference) and are usually developed with the aid of computerized molecular modeling.
Peptide mimetics that axe structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as MarR family, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting o~ -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2S0-, by methods known in the art and further described in the following references: Spatola, A.F. in "Chemistry and Biochemistry of Amino Acids, WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);
Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley, J. S., Trends Pharm Sci (1980) pp.

(general review); Hudson, D. et al., Int J Pept Prot Res (1979) 14:177-185 (-CH2NH-, CH2CH2-); Spatola, A. F. et al., Life Sci (1986) 38:1243-1249 (-CH2-S); Hann, M. M., J Chem Soc Perkin Trans I (1982) 307-314 (-CH-CH-, cis and traps); Almquist, R. G. et al., J Med Chem (1980) 23:1392-1398 (-COCH2-); Jennings-White, C. et al., Tetrahedron Lett (1982) 23:2533 (-COCH2-); Szelke, M. et al., European Appln.
EP
45665 (1982) CA: 97:39405 (1982)(-CH(OH)CH2-); Holladay, M. W. et al., Tetrahedron Lett (1983) 24:4401-4404 (-C(OH)CH2-); and Hruby, V. J., Life Sci (1982) 31:189-199 (-CH2-S-); each of which is incorporated herein by reference. A
particularly preferred non-peptide linkage is -CH2NH-.
Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering positions) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) to which the peptidomimetic binds to produce the therapeutic effect.
Derivitization (e.g., labelling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
Systematic substitution of one or more amino acids of an MarR family amino acid sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising an MarR family amino acid sequence or a substantially identical sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387, incorporated herein by reference); for example, by WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
The amino acid sequences of MarR family polypeptides identified herein will enable those of skill in the art to produce polypeptides corresponding to MarR
family peptide sequences and sequence variants thereof. Such polypeptides may be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding an MarR family peptide sequence, frequently as part of a larger polypeptide.
Alternatively, such peptides may be synthesized by chemical methods. Methods for expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. ( 1969) J. Am. Chem. Soc. 91: 501; Chaiken I. M.
( 1981 ) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989) Science 243: 187;
Merrifield, B. (1986) Science 232: 342; Kent, S. B. H. (1988) Ann. Rev. Biochem. 57: 957;
and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference).
Peptides can be produced, typically by direct chemical synthesis, and used e.g., as agonists or antagonists of an MarR familymolecule, e.g., to modulate binding of an MarR family polypeptide and a molecule with which it normally interacts.
Peptides can be produced as modified peptides, with nonpeptide moieties attached bycovalent linkage to the N-terminus and/or C-terminus. In certain preferred embodiments, either the carboxy-terminus or the amino-terminus, or both, are chemically modified.
The most common modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively. Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, may be incorporated into various embodiments of the invention. Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence can provide advantageous physical. chemical, biochemical, and pharmacological WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 _29_ properties, such as: enhanced stability. increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others. Peptides may be used to control bacterial growth, e.g, to treat infection or to clean surfaces.
It shall be understood that the instant invention also pertains to isolated MarR
family member polypeptides, portions thereof; and the nucleic acid molecules encoding them, including naturally occurring and mutant forms.
III. Preparation of MarR Family Polypeptides Preferred MarR family polypeptides for use in screening assays are "isolated or recombinant" polypeptides. In one embodiment, MarR family polypeptides can be made from nucleic acid molecules. Nucleic acid molecules encoding MarR
family polypeptides can be used to produce MarR family polypeptides for use in the instant assays. For example, nucleic acid molecules encoding a MarR family polypeptide can be isolated (e.g., isolated from the sequences which naturally flank it in the genome and from cellular components) and can be used to produce a MarR
family polypeptide. In one embodiment, a nucleic acid molecule which has been (1) amplified in vitro by, for example, polymerase chain reaction (PCR); (2) recombinantly produced by cloning, or (3) purified, as by cleavage and gel separation; or (4) synthesized by, for example, chemical synthesis can be used to produce MarR family polypeptides.
As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
Nucleic acid molecules specifying MarR family polypeptides can be placed in a vector. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. The term "expression vector"
includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a promoter). In the present specification, "plasmid" and "vector" are used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.

Exemplary expression vectors for expression of a gene encoding a MarR family polypeptide and capable of replication in a bacterium, such as a bacterium from a genus selected from the group consisting of: Escherichia, Bacillus, Streptomyces, Streptococcus, or in a cell of a simple eukaryotic fungus such as a Saccharomyces or, Pichia, or in a cell of a eukaryotic organism such as an insect, a bird, a mammal, or a plant, are known in the art. Such vectors may carry functional replication-specifying sequences (replicons) both for a host for expression, for example a Streptomyces, and for a host, for example, E. coli, for genetic manipulations and vector construction. See e.g.
U.S.P.N 4,745,056. Suitable vectors for a variety of organisms are described in Ausubel, F. et al., Short Protocols in Molecular Biology, Wiley, New York (1995), and for example, for Pichia, can be obtained from Invitrogen (Carlsbad, CA).
Useful expression control sequences. include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is ~ directed by T7 RNA polymerise, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. A useful translational enhancer sequence is described in U.S.P.N. 4,820,639.
It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
"Transcriptional regulatory sequence" is a generic term to refer to DNA
sequences, such as initiation signals, enhancers, operators, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
It will also be understood that a recombinant gene encoding a MarR family polypeptide can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring MarR family gene. Exemplary regulatory sequences are described in Goeddel;
Gene WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 Expression Technology: Methods in En~~mology 185, Academic Press, San Diego, CA
(1990). For instance, any of a wide variety of expression control sequences, that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding the MarR family proteins of this invention.
Appropriate vectors are widely available commercially and it is within the knowledge and discretion of one of ordinary skill in the art to choose a vector which is appropriate for use with a given microbial cell. The sequences encoding MarR
family polypeptides can be introduced into a cell on a self replicating vector or may be introduced into the chromosome of a microbe using homologous recombination or by an insertion element such as a transposon.
Such vectors can be introduced into cells using standard techniques, e.g., transformation or transfection. The terms "transformation" and "transfection"
mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient or "host" cell.
The term "transduction" means transfer of a nucleic acid sequence, preferably DNA, from a donor to a recipient cell, by means of infection with a virus previously grown in the donor, preferably a bacteriophage. Nucleic acids can also be introduced into microbial cells by transformation using calcium chloride or electroporation.
"Cells," "host cells," "recipient cells. are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. In preferred embodiments, cells used to express MarR family polypeptides for purification, e.g., host cells, comprise a mutation which renders any endogenous MarR family polypeptide nonfunctional or causes the endogenous polypeptide to not be expressed. In other embodiments, mutations may also be made in other related genes of the host cell, such that there will be no interference from the endogenous host loci.
Purification of a MarR family polypeptides, e.g., recombinantly expressed polypeptides, can be accomplished using techniques known in the art. For example, if the MarR family polypeptide is expressed in a form that is secreted from cells, the medium can be collected. Alternatively, if the MarR family polypeptide is expressed in a form that is retained by cells, the host cells can be lysed to release the MarR family polypeptide. Such spent medium or cell lysate can be used to concentrate and purify the WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 MarR family polypeptide. For example, the medium or lysate can be passed over a column, e.g., a column to which antibodies specific for the MarR family member polypeptide have been bound. Alternativel~~. such antibodies can be specific for a non-MarR family member polypeptide which has been fused to the MarR family polypeptide (e.g., as a tag) to facilitate purification of the MarR family member polypeptide. Other means of purifying MarR family member polypeptides are known in the art.
IV. Methods of Identifying Compounds which Interact With MarR Polypeptides The invention provides a method (also referred to herein as a "screening assay") to identify those compounds which modulate (enhance (agonists) or block (antagonists)) the action of MarR family polypeptides or nucleic acid molecules, particularly those compounds that are bacteriostatic and/or bactericidal or prevent the infectious process.
The subject screening assays can be used to identify modulators, i.e., candidate or test compounds or agents (e.g., polypeptides, peptides, peptidomimetics, small molecules or other drugs) which modulate MarR family polypeptides, i.e., have a stimulatory or inhibitory effect on, for example, MarR family polypeptide expression or MarR
family polypeptide activity. Test compounds may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 ( 1991 ).
MarR family polypeptide agonists and antagonists can be assayed in a variety of ways. For example, in one embodiment. the invention provides for methods for identifying a compound which modulates an MarR family molecule, e.g., by detecting or measuring the ability of the compound to interact with an MarR family nucleic acid molecule or an MarR family polypeptide or the ability of a compound to modulate the activity or expression of an MarR family polypeptide. Furthermore, the ability of a compound to modulate the binding of an MarR family polypeptide or MarR family nucleic acid molecule to a molecule to which they normally bind, e.g., an MarR
family binding polypeptide can be tested.
Compounds for testing in the instant methods can be derived from a variety of different sources and can be known or can be novel. Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 _»_ compounds. The encoded proteins, upon expression, can be used as a target for the screening of antibacterial drugs. In another embodiment, antisense nucleic acid molecules or nucleic acid molecules that encode for dominant negative MarR
family mutants can also be tested in the subject assays.
In one embodiment, libraries of compounds are tested in the instant methods.
In another embodiment, known compounds are tested in the instant methods. In another embodiment, compounds among the list of compounds generally regarded as safe (GRAS) by the Environmental Protection Agency are tested in the instant methods.
In one embodiment, a library of compounds can be screened in the subject assays. A recent trend in medicinal chemistry includes the production of mixtures of compounds, referred to as libraries. While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds. such as benzodiazepines (Bunin et al. 1992.
J. Am. Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA
90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al.
1993. Science. 261:1303), and hydantoins (DeWitt et al. supra). Rebek et al.
have described an approach for the synthesis of molecular libraries of small organic molecules with a diversity of 104-105 (Carell et al. 1994. Angew. Chem. Int. Ed. Engl.
33:2059;
Carell et al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).
The compounds for screening in the assays of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. Anticancer Drug Des. 1997.
12:145).
Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63), and natural product extract libraries. In one embodiment, the test compound is a peptide or peptidomimetic. In another, preferred embodiment, the compounds are small, organic non-peptidic compounds.
Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in: Erb et al. 1994. Proc. Natl. Acad. Sci. USA 91:11422;
Horwell et al. 1996 Immunopharmacology 33:68: and in Gallop et al. 1994. J. Med. Chem.
37:1233. Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421 ), or on beads (Lam ( 1991 ) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);
(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J.
Mol. Biol.
222:301-310); (Ladner supra). Other types of peptide libraries may also be expressed, see, for example, U.S. Patents 5,270,181 and x,292,646). In still another embodiment, combinatorial polypeptides can be produced from a cDNA library.
The efficacy of the agonist or antagonist can be assessed by generating dose response curves from data obtained using various concentrations of the test modulating agent. Moreover, a control assay can also be performed to provide a baseline for comparison.
A variety of different techniques can be used to perform such an assay, e.g., by determining whether a compound modulates binding of a MarR family protein to a molecule with which it normally interacts, (a binding partner such as DNA, e.g., a marRAB promoter, or a polypeptide). For example, the ability of a compound to decrease binding of a MarR family polypeptide to DNA, e.g., to decrease the binding of MarR to mar0, or the ability of the compound to reduce MarR family polypeptide-mediated regulation of transcription from such a promoter can be measured. As described in more detail below, either whole cell or cell free assay systems can be employed.
These assays are useful in the identification of compounds which will lead to an alteration in the expression of genetic loci in microbes that are controlled by MarR
family members, e.g., loci which mediate MDR or virulence. If compounds are identified as interfering with transcription of a desirable gene or gene locus, the use of such compounds can be minimized, whereas if compounds are identified as enhancing transcription of an undesirable gene or gene locus, the use of such compounds can be promoted. For example, compounds that interfere with the activity of MarR and promote MDR can be identified and their use as antimicrobials or disinfectants curtailed.
As described in more detail below, either whole cell or cell free assay systems can be employed.
1. Whole Cell Assays In one embodiment of the invention. the subject screening assays can be performed using whole cells. In one embodiment of the invention, the step of determining whether a compound modulates. e.g., reduces the activity or expression of an MarR family polypeptide comprises contacting a cell expressing an MarR
family polypeptide with a compound and measuring the ability of the compound to modulate the activity or expression of an MarR family- polypeptide.
In another embodiment, modulators of MarR family polypeptide expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of MarR family polypeptide mRNA or protein in the cell is determined. The level of expression of MarR family polypeptide mRNA or protein in the presence of the candidate compound is compared to the level of expression of MarR family polypeptide mRNA or polypeptide in the absence of the candidate compound. The candidate compound can then be identified as a modulator of MarR family polypeptide expression based on this comparison. For example, when expression of MarR family polypeptide mRNA or protein is greater (e.g., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of MarR family polypeptide mRNA or protein expression.
Alternatively, when expression of MarR family polypeptide mRNA or protein is less (e.g., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of MarR family mRNA or protein expression. The level of MarR family mRNA or protein expression in the cells can be determined by methods described herein for detecting MarR family mRNA or protein.

WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 To measure expression of an MarR family polypeptide, transcription of an MarR
family gene can be measured in control cells which have not been treated with the compound and compared with that of test cells which have been treated with the compound. For example, cells which express endogenous MarR family polypeptides or which are engineered to express or overexpress recombinant MarR family polypeptides can be caused to express or overexpress a recombinant MarR family polypeptide in the presence and absence of a test modulating agent of interest, with the assay scoring for modulation in MarR family polypeptide responses by the target cell mediated by the test agent. For example, as with the cell-free assays, modulating agents which produce a change, e.g., a statistically significant change in MarR family polypeptide -dependent responses (either an increase or decrease) can be identified.
Recombinant expression vectors that can be used for expression of MarR family polypeptide are known in the art (see discussions above). In one embodiment, within the expression vector the MarR family polypeptide -coding sequences are operatively linked to regulatory sequences that allow for constitutive or inducible expression of MarR
family polypeptide in the indicator cell(s). Use of a recombinant expression vector that allows for constitutive or inducible expression of MarR family polypeptide in a cell can be used for identification of compounds that enhance or inhibit the activity or expression of MarR family polypeptide. In an alternative embodiment, within the expression vector the MarR family polypeptide coding sequences are operatively linked to regulatory sequences of the endogenous MarR family polypeptide gene (i.e., the promoter regulatory region derived from the endogenous gene). Use of a recombinant expression vector in which MarR family polypeptide expression is controlled by the endogenous regulatory sequences is preferred for identification of compounds that enhance or inhibit the transcriptional expression of MarR family polypeptide.
In one embodiment of the invention, the step of determining whether a compound reduces the activity of a MarR family polypeptide comprises contacting the cell with a compound and detecting the ability of the compound to increase transcription from a marRAB promoter. In such an assay, since the MarR family polypeptide regulates transcription (either positively, e.g., SIyA, BadR, NhhD, or MexR, or negatively, e.g., MarR), a compound would be identified based on its ability to WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 _37_ modulate, e.g. increase or decrease, the control level of transcription as compared to the level of transcription in a cell which has not been treated with the compound.
In one embodiment, the level of transcription can be determined by measuring the amount of RNA produced by the cell. For example, RNA can be isolated from cells which express an MarR family polypeptide and that have been incubated in the presence or absence of compound. Northern blots using probes specific for the sequences to be detected can then be performed using techniques known in the art. Numerous other, art-recognized techniques can be used. For example, western blot analysis can be used to test for MarR family. For example, in another embodiment, transcription of specific RNA molecules can be detected using the polymerase chain reaction, for example by making cDNA copies of the RNA transcript to be measured and amplifying and measuring them. In another embodiment. RNAse protection assays, such as S 1 nuclease mapping or RNase mapping can be used to detect the level of transcription of a gene. In another embodiment, primer extension can be used.
Sequences which can be detected include sequences which are regulated by a MarR family member. In one embodiment. sequences not normally regulated by a MarR
family member can be regulated by a MarR family polypeptide by linking them to a promoter that is regulated by a MarR family member polypeptide. For example, sequences can be linked to a marRAB family promoter, including, for example, endogenous sequences or reporter gene sequences. Exemplary endogenous sequences which can be detected include: acrAB; micF; mlr l, 2, 3; slp; inaA; fpr; sodA;
soi-17,19;
zwf, fumC; or rpsF; another example, would be araBAD, araE, araFGH and araC, which are activated by AraC; Pm, which is activated by XyIS; melAB which is activated by MeIR; and oriC which is bound by Rob. as well as sequences from genetic loci that are identified using the assays described infra.
In yet other embodiments, the ability of a compound to induce a change in transcription from a marRAB promoter can be accomplished by detecting the amount of a polypeptide produced by the cell. Polypeptides which can be detected include any polypeptides which are produced upon the activation of a MarR family responsive promoter, including, for example, both endogenous sequences and reporter gene sequences. Exemplary endogenous polypeptides which can be detected include:
AcrAB;

WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 _38_ Mlr 1,2,3; Slp; InaA; Fpr; SodA; Soi-17.19: Zwf; FumC; or RpsF (Alekshun and Levy.
1997. Antimicrobial Agents and Chemother. 41:2067). Others are known in the art and include: AraBAD, AraE, AraFGH and AraC, which are activated by AraC, as well as polypeptides translated from genetic loci that are identified using the assays described infra. In one embodiment, the amount of polypeptide made by a cell can be detected using an antibody against that polypeptide. In other embodiments, the activity of such a polypeptide can be measured.
In yet another embodiment, the ability of a compound to modulate a MarR
family polypeptide activity, e,g., virulence. multidrug resistance, or microbiological metabolism can be tested by detecting the ability of the compound to affect the a virulence or drug resistance phenotype in a microbe, e.g. by testing the ability of the microbe to resist antibiotics or to cause infection.
In yet other embodiments, the abiliy of a compound to induce a change in transcription or translation of an MarR family polypeptide can be accomplished by measuring the amount of MarR family pol~~peptide produced by the cell.
Polypeptides which can be detected include any polypeptides which are produced upon the activation of an MarR family responsive promoter, including, for example, both endogenous sequences and reporter gene sequences. In one embodiment, the amount of polypeptide made by a cell can be detected using an antibody against that polypeptide. In other embodiments, the activity of such a polypeptide can be measured.
In one embodiment, other sequences which are regulated by an MarR family promoter (e.g., a promoter having sequence identity with a promoter that regulates expression of an MarR family gene set forth herein) can be detected. In one embodiment, sequences not normally regulated by an MarR family promoter can be assayed by linking them to a promoter that regulates transcription of an MarR
family polypeptide.
In preferred embodiments, to provide a convenient readout of the transcription from an MarR family promoter, such a promoter is linked to a reporter gene, the transcription of which is readily detectable. For example, a bacterial cell, e.g., an E. coli cell, can be transformed as taught in Cohen et al. 1993. J. Bacteriol.
175:7856.

WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 Examples of reporter genes include. but are not limited to, CAT
(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase;
firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); PhoA, alkaline phosphatase (Toh et al. (1989) Eur. J.
Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol.
216:362-368) and green fluorescent polypeptide (LT.S. patent 5,491,084; W096/23898).
In yet another embodiment, the ability of a compound to modulate the binding of an MarR family polypeptide to an MarR family binding partner can be determined.
Exemplary MarR family binding partners include nucleic acid molecules, such as DNA, as well as polypeptides that are downstream of MppA, a periplasmic binding protein in E. coli which functions upstream of MarA in a signal transduction pathway (Li and Park.
1999. J. of Bacteriology. 181:4842) and related molecules. MarR family binding polypeptides can be identified using techniques which are known in the art.
For example, in the case of binding polypeptides that interact with MarR family polypeptides, interaction trap assays or two hybrid screening assays can be used.
MarR family binding partners can be identified e.g., e.g., by using an MarR
family polypeptides or portions thereof of the invention as a "bait proteins"
in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317;
Zervos et al.
(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;
and Brent W094/10300), to identify other proteins, which bind to or interact with MarR
family polypeptides ("MarR family -binding polypeptides") and are involved in MarR
family activity. Such MarR family family-binding polypeptides are also likely to be involved in the propagation of signals by the MarR family polypeptides or to associate with MarR family polypeptides and enhance or inhibit their activity.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an MarR family polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an MarR family polypeptide-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the polypeptide which interacts with the MarR family polypeptide.
MarR family binding partners may also be identified in other ways. For example, a library of molecules can be tested for the presence of MarR family binding polypeptides. In one embodiment, the library of molecules can be tested by expressing them in an expression vector, e.g., a bacteriophage. Bacteriophage can be made to display on their surface a plurality of polypeptide sequences, each polypeptide sequence being encoded by a nucleic acid contained within the bacteriophage. The phage expressing these candidate MarR family binding polypeptides can be tested for the ability to bind an immobilized MarR family polypeptide, to obtain those polypeptides having affinity for the MarR family polypeptide. For example, the method can comprise: contacting the immobilized MarR family polypeptide with a sample of the library of bacteriophage so that the MarR family polypeptide can interact with the different polypeptide sequences and bind those having affinity for the MarR
family polypeptide to form a set of complexes consisting of immobilized MarR family polypeptide and bound bacteriophage. The complexes which have not formed a complex can be separated. The complexes of MarR family polypeptide and bound bacteriophage can be contacted with an agent that dissociates the bound bacteriophage from the complexes; arid the dissociated bacteriophage can be isolated and the sequence of the nucleic acid moleculeencoding the displayed polypeptide obtained, so that amino WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 acid sequences of displayed polypeptides with affinity for MarR family polypeptides are obtained.
In the case of MarR family nucleic acid molecules, MarR family binding polypeptides can be identified, e.g., by contacting an MarR family nucleotide sequence with candidate MarR family binding polypeptides (e.g., in the form of microbial extract) under conditions which allow interaction of components of the extract with the MarR
family nucleotide sequence. The ability of the MarR family nucleotide sequence to interact with the components can then be measured to thereby identify a polypeptide that binds to an MarR family nucleotide sequence.
2. Cell-Free Assays The subject screening methods can involve cell-free assays, e.g., using high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix comprising an MarR family molecule and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be an agonist or antagonist. In one embodiment, the reaction mix can further comprise a cellular compartment, such as a membrane, cell envelope or cell wall, or a combination thereof. The ability of the test compound to agonize or antagonize the MarR family polypeptide is reflected in decreased binding of the MarR family polypeptide to an MarR family binding partner or in a decrease in MarR family polypeptide activity.
In many drug screening programs which test libraries of modulating agents and natural extracts, high throughput assays are desirable in order to maximize the number of modulating agents surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test modulating agent. Moreover, the effects of cellular toxicity and/or bioavailability of the test modulating agent can be generally ignored in the in vitro system.

WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 In one embodiment, the ability of a compound to modulate the activity or expression of an MarR family polypeptide is accomplished using isolated MarR
family polypeptides or MarR family nucleic acid molecule in a cell-free system. In such an assay, the step of measuring the ability of a compound to modulate the activity or expression of the MarR family polypeptide is accomplished, for example, by measuring direct binding of the compound to an MarR family polypeptide or MarR family nucleic acid molecule or the ability of the compound to alter the ability of the MarR
family polypeptide to bind to a binding partner to which the MarR family polypeptide normally binds (e.g., protein or DNA).
In yet another embodiment, an assay of the present invention is a cell-free assay in which an MarR family polypeptide or portion thereof is contacted with a test compound and the ability of the test compound to bind to the MarR family polypeptide or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of an MarR family polypeptide can be accomplished, 1 ~ for example, by determining the ability of the MarR family polypeptide to bind to an MarR family target molecule by one of the methods described above for determining direct binding. Determining the ability of the MarR family polypeptide to bind to an MarR family target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C.
(1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705.
As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
In yet another embodiment, the cell-free assay involves contacting an MarR
family polypeptide or biologically active portion thereof with a known compound which binds the MarR family polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the MarR family polypeptide, wherein determining the ability of the test compound to interact with the MarR family polypeptide comprises determining the WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 _ 4; _ ability of the MarR family polypeptide to preferentially bind to or modulate the activity of an MarR family binding partner.
Exemplary MarR family binding partners include nucleic acid molecules, such as DNA, as well as polypeptides that are downstream of MppA, a periplasmic binding protein in E. coli which functions upstream of MarA in a signal transduction pathway (Li and Park. 1999. J. of Bacteriology. 181:4842) and related molecules.
The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of proteins (e.g., MarR family polypeptides or MarR
family binding polypeptides). In the case of cell-free assays in which a membrane-bound form of a polypeptide is used it ma~~ be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-100, Triton~ X-114, Thesit~, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.
For example, compounds can be tested for their ability to directly bind to an MarR family nucleic acid molecule or an MarR family polypeptide or portion thereof, e.g., by using labeled compounds, e.g., radioactively labeled compounds. For example, an MarR family polypeptide sequence can be expressed by a bacteriophage. In this embodiment, phage which display the MarR family polypeptide would then be contacted with a compound so that the polypeptide can interact with and potentially form a complex with the compound. Phage which have formed complexes with compounds can then be separated from those which have not. The complex of the polypeptide and compound can then be contacted with an agent that dissociates the bacteriophage from the compound. Any compounds that bound to the polypeptide can then be isolated and identified.

WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 In another embodiment, the ability of a compound to bind to an MarR family nucleic acid molecule can be measured (e.g.. MarR binding to mar0). For example, gel shift assays or restriction enzyme protection assays can be used. Gel shift assays separate polypeptide-DNA complexes from free DNA by non-denaturing polyacrylamide gel electrophoresis. In such an experiment, the level of binding of a compound to DNA can be determined and compared to that in the absence of compound.
Compounds which change the level of this binding are selected in the screen as modulating the activity of an MarR family polypeptide.
In preferred embodiments, assays will be performed in which direct binding is measured, e.g., protection of mar0 restriction enzyme digestion as described in the appended examples, in order to rule out indirect effects of compounds, e.g., on mRNA
stability.
Other methods of assaying the ability of proteins to bind to DNA, e.g., DNA
footprinting, and nuclease protection are also well known in the art and can be used to test the ability of a compound to bind to an MarR family nucleotide sequence.
In another embodiment, the invention provides a method for identifying compounds that modulate antibiotic resistance by assaying for test compounds that bind to MarR family nucleic acid molecules and interfere, e.g., with gene transcription.
In another embodiment, an MarR family nucleic acid molecule and an MarR
family binding polypeptide that normally binds to that nucleotide sequence are contacted with a test compound to identify compounds that block the interaction of an MarR
family nucleic acid molecule and an MarR family binding polypeptide. For example, in one embodiment, the MarR family nucleotide sequence and/or the MarR family binding polypeptide are contacted under conditions which allow interaction of the compound with at least one of the MarR family nucleic acid molecule and the MarR family binding polypeptide. The ability of the compound to modulate the interaction of the MarR
family nucleotide sequence with the MarR family binding polypeptide is indicative of its ability to modulate an MarR family polypeptide activity.
Determining the ability of the MarR family polypeptide to bind to or interact with an MarR family binding polypeptide can be accomplished, e.g., by direct binding.
In a direct binding assay, the MarR family polypeptide could be coupled with a WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 radioisotope or enzymatic label such that binding of the MarR family polypeptide to an MarR family polypeptide target molecule can be determined by detecting the labeled MarR family polypeptide in a complex. For example MarR family polypeptides can be labeled with 1251, 35S 14C or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
Alternatively, MarR family polypeptide molecules can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
Typically, it will be desirable to immobilize either MarR family polypeptide, an MarR family binding partner or a compound to facilitate separation of complexes from uncomplexed forms, as well as to accommodate automation of the assay. Binding of MarR family polypeptide to an upstream or downstream binding partner, in the presence and absence of a candidate agent, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix. For example, glutathione-S-transferase/ MarR family polypeptide (GST/ MarR family polypeptide) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates, e.g. an 35S-labeled, and the test modulating agent, and the mixture incubated under conditions conducive to complex formation, e.g., at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintilant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of MarR
family polypeptide -binding polypeptide found in the bead fraction quantitated from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, either an MarR family polypeptide or polypeptide to which it binds can be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated MarR family polypeptide molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with MarR family polypeptide but which do not interfere with binding of upstream or downstream elements can be derivatized to the wells of the plate, and MarR
family polypeptide trapped in the wells by antibody conjugation. As above, preparations of an MarR family polypeptide -binding polypeptide and a test modulating agent are incubated in the MarR family polypeptide -presenting wells of the plate, and the amount of complex trapped in the well can be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, 1 S include immunodetection of complexes using antibodies reactive with the MarR family binding polypeptide, or which are reactive with MarR family polypeptide and compete with the binding polypeptide; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with the MarR family binding polypeptide. To illustrate, the MarR family polypeptide can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of protein trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-1-napthol. Likewise, a fusion protein comprising the protein and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the polypeptide, such as anti- MarR
family polypeptide antibodies, can be used. Alternatively, the polypeptide to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 addition to the MarR family polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST
fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharamacia, NJ).
It is also within the scope of this invention to determine the ability of a compound to modulate the interaction between MarR family polypeptide and its target molecule, without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of MarR family polypeptide with its target molecule without the labeling of either MarR family polypeptide or the target molecule. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate 1 S at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between compound and receptor.
This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in methods of reducing drug resistance in microbes, e.g., in vivo or ex vivo. Such agents can be used in methods of treatment (in vivo or ex vivo) or in methods of reducing resistance to drugs in the environment.
V. Microbes Suitable For Testing Numerous different microbes are suitable for use in testing for compounds that affect MarR or as sources of materials for use in the instant assays. The term "microbe"
includes any microorganism having a MarR family member polypeptide. Preferably unicellular microbes including bacteria, fungi, or protozoa. In another embodiment, microbes suitable for use in the invention are multicellular, e.g., parasites or fungi. In preferred embodiments, microbes are pathogenic for humans, animals, or plants.
In one embodiment, microbes causing environmental problems, e.g., fouling or spoilage or that WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 perform useful functions such as breakdown of plant matter are preferred. As such, any of these disclosed microbes may be used as intact cells or as sources of materials for cell-free assays as described herein.
In preferred embodiments, microbes for use in the claimed methods are bacteria, either Gram-negative or Gram-positive bacteria. In a preferred embodiment, any bacteria that are shown to become resistant to antibiotics, e.g., to display MDR are appropriate for use in the claimed methods.
In preferred embodiments, microbes suitable for testing are bacteria from the family Enterobacteriaceae. In more preferred embodiments bacteria of a genus selected from the group consisting of: Escherichia, Proteus, Salmonella, Klebsiella, Providencia, Enterobacter, Burkholderia, Pseudomonas. . Acinetobacter, Aeromonas, Haemophilus, Yersinia, Neisseria, and Erwinia, Rhodopseardomonas, or Burkholderia for use in the claimed assays.
In yet other embodiments, the microbes to be tested are Gram-positive bacteria and are from a genus selected from the group consisting of: Lactobacillus, Azorhizobium, Streptomyces, Pediococcus, Photvbacterium, Bacillus, Enterococcus, Staphylococcus, Clostridium, Streptococcus. Butyrivibrio, Sphingomonas, Rhodococcus, or Streptomyces In yet other embodiments, the microbes to be tested are acid fast bacilli, e.g., from the genus Mycobacterium.
In still other embodiments, the microbes to be tested are, e.g., selected from a genus selected from the group consisting of: Methanobacterium, Sulfolobus, Archaeoglobu, Rhodobacter, or Sinorhizobium.
In other embodiments, the microbes to be tested are fungi. In a preferred embodiment the fungus is from the genus Mucor or Candida, e.g., Mucor racemosus or Candida albicans.
In yet other embodiments, the microbes to be tested are protozoa. In a preferred embodiment the microbe is a malaria or cryptosporidium parasite.

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 VI. Test Compounds Compounds to be tested can include candidate antiinfective compounds. As used herein, the language "antiinfective compound" includes a compound which reduces the ability of a microbe to produce infection in a host or which reduces the ability of a microbe to multiply or remain infective in the environment. Exemplary antiinfective compounds include e.g., disinfectants or antibiotics. Antiinfective compounds include those compounds which are static or cidal for microbes, e.g., an antimicrobial compound which inhibits the proliferation and/or viability of a microbe. Preferred antiinfective compounds increase the susceptibility of microbes to antibiotics or decrease the infectivity or virulence of a microbe.
The term "antibiotics" is art recognized and includes antimicrobial agents synthesized by an organism in nature and isolated from this natural source, and chemically synthesized antibiotics. The term includes but is not limited to:
polyether ionophore such as monensin and nigericin; macrolide antibiotics such as erythromycin and tylosin; aminoglycoside antibiotics such as streptomycin and kanamycin; (3-lactam antibiotics such as penicillin and cephalosporin; and polypeptide antibiotics such as subtilisin and neosporin. Semi-synthetic derivatives of antibiotics, and antibiotics produced by chemical methods are also encompassed by this term. Chemically-derived antimicrobial agents such as isoniazid, trimethoprim, quinolones, fluoroquinolones and sulfa drugs are considered antibacterial drugs, although the term antibiotic has been applied to these. It is within the scope of the screens of the present invention to include compounds derived from natural products and compounds that are chemically synthesized. The term "antibiotic" includes the antimicrobial agents to which the Mar phenotype has been shown to mediate resistance and, as such, includes disinfectants, antiseptics, and surface delivered compounds. For example, antibiotics, biocides, or other type of antibacterial compounds, including agents which induce oxidative stress agents, and organic solvents are included in this term.
The term "biocide" is art recognized and includes an agent that those ordinarily skilled in the art prior to the present invention believed would kill a cell "non-specifically," or a broad spectrum agent whose mechanism of action is unknown, e.g., prior to the present invention, one of ordinary skill in the art would not have expected WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 the agent to be target-specific. Examples of biocides include paraben, chlorbutanol, phenol, alkylating agents such as ethylene oxide and formaldehyde. halides, mercurials and other heavy metals, detergents, acids. alkalis, and chlorhexidine. The term "bactericidal" refers to an agent that can kill a bacterium; "bacteriostatic"
refers to an agent that inhibits the growth of a bacterium.
In contrast to the term "biocide," an antibiotic or an "anti-microbial drug approved for human use" is considered to have a specific molecular target in a microbial cell. Preferably a microbial target of a therapeutic agent is sufficiently different from its physiological counterpart in a subject in need of treatment that the antibiotic or drug has minimal adverse effects on the subj ect.
Compounds for testing in the instant methods can be derived from a variety of different sources and can be in solution or immobilized on a surface. In preferred embodiments, libraries of compounds are tested in the instant methods to identify compounds that modulate the ability of a MarR family polypeptide to negatively regulate transcription of an operon, the transcription of which leads to MDR.
A recent trend in medicinal chemistry includes the production of mixtures of compounds, referred to as libraries. While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds. such as benzodiazepines (Bunin et al. 1992.
J. Am. Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA
90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al.
1993. Science. 261:1303), and hydantoins (DeWitt et al. supra). Rebek et al.
have described an approach for the synthesis of molecular libraries of small organic molecules with a diversity of 104-105 (Carell et al. 1994. Angew. Chem. Int.
Ed. Engl.
33:2059; Carell et al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).
The compounds for screening in the assays of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the 'one-bead one-compound' library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. Anticancer Drug Des. 1997.
12:145).
Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63), and natural product extract libraries. In one embodiment, the test compound is a peptide or peptidomimetic. In another, preferred embodiment, the compounds are small, organic non-peptidic compounds.
Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in: Erb et al. 1994. Proc. Natl. Acad. Sci. USA 91:11422;
Horwell et al. 1996 Immunopharmacology 33:68; and in Gallop et al. 1994. J. Med. Chem.
37:1233. In addition, libraries such as those described in the commonly owned applications U.S.S.N. 08/864,241, U.S.S.I~,T. 08/864,240 and U.S.S.N.
08/835,623 can be used to provide compounds for testing in the present invention. The contents of each of these applications is expressly incorporated herein by this reference.
Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421 ), or on beads (Lam ( 1991 ) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP
'409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);
(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J.
Mol. Biol.
222:301-310); (Ladner supra). Other types of peptide libraries may also be expressed, see, for example, U.S. Patents 5,270,181 and 5,292,646). In still another embodiment, combinatorial polypeptides can be produced from a cDNA library.
In a preferred embodiment, compounds to be tested do not include salicylate or related compounds. Examples of such related compounds include sodium benzoate and acetyl salicylate (Rosner, J.L. 1985. Proc. Natl. Acad. Sci. USA 82:8771).
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, genetics, microbiology, recombinant DNA, and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature. See, for example, Genetics;

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 _j7_ Molecular Cloning A Laboratory Manual. 2nd Ed., ed. by Sambrook, J. et al.
(Cold Spring Harbor Laboratory Press (1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel, F. et al. (Whey, NY (1990); DNA Cloning, Volumes I and II (D.
N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al. U.S.
Patent No: 4,683,195; Nucleic Acid Hybridi=anon (B. D. Hames & S. J. Higgins eds.
(1984)); the treatise, Methods In Enzymoloy (Academic Press, Inc., N.Y);
Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London (1987)); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds. ( 1986)); and Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(1972)).
The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.
The invention is further illustrated by the following examples, which should not be construed as further limiting.
EXAMPLES
Example 1. Structurally diverse compounds bind to MarR
The multiple antibiotic resistance (mar) operon of Escherichia coli is a chromosomally encoded locus that controls an adaptational response to antibiotics and other environmental hazards (Alekshun, M. N. and Levy 1997, Antimicrob. Agents Chemother). This control is accomplished on the genomic level whereby MarA, a transcriptional activator encoded within the marRAB operon, regulates the expression of multiple genes on the E. coli chromosome (Alekshun, M. N. and Levy 1997, Antimicrob. Agents Chemother 10:2067).
In the absence of an appropriate stimulus, MarR negatively regulates expression of the marRAB operon (Cohen et al., 1993. J. Bacteriol. 175: 7856; Martin and Rosner, 1995, Proc. Natl. Acad. Sci. 92: 5456 and Seoane and Levy, 1995, J. Bacteriol.
177:
3414). DNA footprinting experiments suggest that MarR oligomerizes at two locations, sites I and II, within the mar operator (mar0) (Martin and Rosner, 1995, Proc.
Natl.
Acad Sci. 92: 5456). Site I is positioned within the -35 and -10 hexamers and site II

WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 _ j3 _ spans the putative MarR ribosome binding site (reviewed in (Alekshun and Levy, 1997, Antimicrob. Agents Chemother. 10: 2067)).
Several structurally diverse inducing compounds activate marRAB expression in vivo (Ariza et al., 1994, J. Bacteriol. 176: 143; Cohen et al., 1993, J.
Bacteriol. 175:
7856; Martin and Rosner, 1995, Proc. Natl. Acad. Sci. 92: 5456 and Seoane and Levy, 1995, J. Bacteriol. 177: 3414) and the repressor activity of MarR is antagonized by sodium salicylate (Martin and Rosner, 199. Proc. Natl. Acad Sci. 92: 5456).
The present study shows that MarR binds to multiple compounds and is, thus, a multifunctional protein possessing effector molecule recognition as well as DNA
binding properties.
In this study, the relative ability of different chemicals to alter MarR
function was tested in a strain where expression of the lethal ccdB gene product was negatively controlled by MarR. The same chemicals were tested for their effect on the DNA
binding activity of MarR in vitro. Sodium salicylate, benzoic acid, plumbagin, 2,4-dinitrophenol, menadione, and ampicillin all interfered with the interaction of MarR with the mar operator. In addition, antibiotics were found to bind to MarR. For example, the ~i lactam antibiotic cefotaxime was found to bind to and interfere with the activity of MarR. These compounds are presumed to interact directly with MarR to derepress expression of the mar operon. No effect was detected with other known inducers, namely chloramphenicol, tetracycline, or paraquat. These compounds probably induce mar expression in intact cells through an indirect action. These experiments are the first to demonstrate that MarR can interact with many structurally dissimilar chemicals in vitro and suggest that this interaction directly affects the repressor activity of MarR.
In one approach, the inducer concentration needed to achieve derepression of a Pmarll ccdB fusion in the bacterial cell was determined using a "killer"
system.
Alternatively, a restriction enzyme site protection assay was employed to qualitatively measure the MarR-mar0 interaction in the presence of many chemicals in vitro.
These experiments offer insights into the mechanism of MarR derepression in E. coli following exposure to a variety of chemicals.

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 Methods The following methods were used in Example 1:
Bacterial Strains, Plasmids and Genetic Techniques-Escherichia coli DHSa and BL21(DE3), are both wild type (mar+) strains. A low copy number wild type MarR
expression vector was constructed using a modified version of pACT7 (Maneewannakul, K., et al 1992. Mol. Microbiol. 6, 2961-2973). MarR amplified by PCR from chromosomal DNA, isolated from E. coli strain AG100 (George, A.M., et al 1983.
J.
Bacteriol. 155, 531-540), using Taq DNA polymerase according to the manufacturer's protocols (Life Technologies, Gaithersburg. MD). EcoRI and PstI restriction sites were incorporated into the forward and reverse primers, respectively, to facilitate directional cloning into pACT7 in place of the T7-RI~'A polymerase gene following digestion with EcoRI and PstI. In the resulting plasmid. p AC-MarR (WT), transcription of marR is regulated by the lacPl promoter and polypeptide synthesis is governed by the wild type MarR ribosome binding site (AGGG) and translational initiation (GTG) signals (Cohen, S.P., et al 1993. J. Bacteriol. 175, 1484-1492). DNA sequence analysis was performed using an ABI automated DNA sequencer in order to confirm the integrity of the marR
coding sequence.
A high-copy number wild type MarR expression vector was constructed in pETl3a (Studier, F.W., et al 1990. Meth. Enrymol. 185, 60-89), a kanamycin resistance version of pET311 a (Novagen, Madison, WI). PCR amplification of marR was performed as described above using forward and reverse primers containing Vspl and BamHI restriction sites, respectively, to facilitate directional cloning into NdellBamHI
digested pETl3a. In the resulting plasmid, pMarR-WT, expression of MarR is under the control of the T7-RNA polymerase promoter and a strong ribosome binding site.
DNA
sequence analysis was performed as described above.
In order to assay for the function of MarR in whole cells, a Pmarll ccdB
fusion was created in a modified version of pETl ld (Novagen, Madison, WI). pETI ld was digested with EcoRV to remove the majority of lack the vector was purified using the Qiagen gel purification kit (Qiagen, Santa Clarita, CA), and religated.
Pmarll, containing the mar operator (mar0) and promoter sequences, was amplified by PCR
from AG100 chromosomal DNA as described above and blunt-end cloned into the EagI

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 _JJ_ site of pETI ld (lacking lacI). Subsequently. the lac0-ccdB portion of pKIL 18 (Bernard, P., et al 1994. Gene 148, 71-74) was amplified by PCR so as to exclude the tac promotor sequences. XhoI and BsmI restriction sites were incorporated into the forward and reverse primers, respectively, to facilitate directional cloning downstream of Pmarll, into the AvaI- BsmI digested vector. The resulting plasmid was designated pSup-Test (FIG. 3).
Gradient Plate Analysis of DH~ a Containing pSup-Test-The susceptibility of DHSa containing pSup-Test to compounds that induce expression of the marRAB
operon was assayed using gradient plate analysis as previously described (McDermott, P.F., 1998. J. Bacteriol. 180, 2995-2998).
Protein Purification-Wild type MarR was overproduced in E. coli BL 21 (DE3) using pMarR-WT. Cells were grown in LB at 37°C to OD600-~-0.8-1Ø
Isopropyl (3-D-thiogalactoside (IPTG) was then added to a final concentration of 1 mM and after incubation for 3 hr, cells were collected by centrifugation, washed once with phosphate buffered saline (pH 7.0), and frozen at -70°C. The frozen cell pellet was resuspended in 10 mL buffer A [50 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 2.5 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (serine protease inhibitor) (Sigma, St. Louis, MO)] and lysed using sonication. Insoluble matter was removed by centrifugation at 30,000 X g for 1 hour and the supernatant was then loaded onto a SP-sepharose HiTrap column (Pharmacia Biotech, Piscataway, NJ) equilibrated with 50 mM
Tris-HC 1 (pH 7.4). The column was washed with 50 mM Tris-HC 1 (pH 7.4) and MarR
was eluted using a linear gradient of 0-1 m NaC 1 in 50 mM Tris-HC 1 (pH 7.4).
The purified protein eluted at 0.2-0.3 mM NaC l and was dialyzed against 100 volumes of 50 mM Tris-HC 1 (pH 7.4), 100 mM NaC 1, 10% glycerol, and 1 mM
phenylmethlysulfonyl fluoride (serine protease inhibitor) overnight at 4°C. The purified MarR, was judged to be >90% pure on a SDS-PAGE Coomassie stained gel, was stored in aliquots at -70°C
until further use.
Restriction Enzyme Site Protection Assays-The presence of a SspI restriction endonculease recognition sequence within site I, one of two MarR binding sites within mar0, was used to assess qualitatively MarR binding. Reaction mixtures were prepared _56_ according to established protocols (Joachimiak, A.J., et al 1983. Proc. Natl.
Acad Sci.
USA 80, 668-672; Melville, S.B., et al 1996. Proc. Natl. Acad. Sci. USA 93, 1226-1231;
Smith, H.Q. and Somerville, R.L. 1997. J. Bacteriol. 179:5914). To a volume of 18 ql containing 0.2 pg pSup-Test (target DNA. 3.4 nM), 10 mM Tris-HC 1 (pH 7.5), 5 mM
NaCI, 1 mM MgCl2, 0.0025% Triton X-100. and an inducing or non-inducing chemical, was added 2 ~l purified MarR (2.92-3.6 fig. 9.1-11.2 qM (assuming the monomeric form of MarR) (final volume 201). The reaction components were incubated at room temperature for 10 min. Subsequently, 5 units of SspI (New England Biolabs, Beverly, MA) was then added and plasmid DNA was digested for 30 min. at 37°C.
The reaction mixtures were terminated by the addition of 1.5 ~1 stop buffer (0.25M EDTA (pH
8.0), 1% SDS) and 5 ~16X agarose gel loading buffer (0.25% bromophenol blue, 0.25%
xylene cyanol, 30% glycerol) and analyzed on 0.7% agarose (Life Technologies, Gaithersburg, MD) gels. As a control, the mobilities of supercoiled (undigested) and linearized (BamHI digested at the single BamHI site in pSup-Test) pSup-Test were compared in the same gel system. The mobility of uncut pSup-Test was detectably slower (5 kb) than that of the linearized (4.576 kb) vector.
Results To determine the relative concentrations of inducers necessary to inactivate MarR in whole cells, the lethal properties of the ccdB gene product, which is contained within the pKIL plasmids (Bernard, P., et al 1994. Gene 148, 71-74), was exploited.
Expression of the cytotoxic ccdB gene product is lethal only under inducing conditions and its persistence in a sensitive host cell confers a lethal phenotype by inhibiting bacterial topoisomerase II (Bernard, P., et al 1994. Gene 148, 71-74).
In pSup-Test, the lethal ccdB gene was placed under the control of the marRAB
promoter (Pmarll (FIG. 1 )), which contains both the marRAB promoter sites and the wild type MarR binding regions (operator sequences, sites I and II (Martin and Rosner.
1995. Proc. Natl. Acad. Sci. 92:5456)), and lac0 (FIG. 3). Cells bearing this plasmid were used to evaluate inducers of the marRAB operon. Plumbagin and 2,4-dinitrophenol (DNP) were very effective inducers since viable colonies did not form at concentrations greater than 0.01 and 0.05 mM, respectively (Table 1 ). Sodium salicylate, sodium WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 _j7_ benzoate, and menadione were less potent. but were still very effective in inactivating MarR (Table 1 ). In control platings, the growth of cells containing pET 11 d or pmar0 (pSup-Test lacking ccdB) was unaffected by the highest concentrations of inducers tested. These results demonstrate that derepression of mar0-regulated ccdB
synthesis is attributable to MarR inactivation and not the result of specific/non-specific inducer mediated cell death.
An in vitro assay based on protection of restriction enzyme action on mar0 was used to assay MarR repressor activity. In the presence of MarR, the repressor prevents access of SspI to the recognition sequence in site I of mar0 and pSup-Test is digested into a single linear fragment of 4576 by in size. In the absence of MarR, the SspI
recognition sequence in mar0 is not protected and the plasmid is cut into two segments of 3472 (fragment B) and 1104 by (fragment C) in length. A single cut in the non-mar0 SspI restriction sites results in fragment A (476 bp) and indicates protection of the SspI
site in mar0 by MarR. Using serial decreases in the concentration of MarR, deprotection is seen by the conversion of the 4756 by fragment into two smaller bands, fragments B and C. As the concentration of MarR is decreased, site I is no longer protected; the 4576 by fragment is converted simultaneously into two smaller fragments of 3472 and 1104 by in length. Previous studies demonstrated that the MarR-mar0 interaction is highly specific (K~y:.l-5 nM) (Seoane, A.S., et al 1995. J.
Bacteriol. 177, 3414-3419, Martin, R.G., et al (1995) Proc. Natl. Acad. Sci. 92, 5456-5460).
The affinity of MarR for mar0 was estimated by determining the point of 50%
protection, as judged by visual inspection of ethidium bromide stained gels according to established methods (Joachimiak, A.J., et al 1983. Proc. Natl. Acad. Sci. USA
80, 668-672; Melville, S.B., et al 1996. Proc. Natl. Acad Sci. USA 93, 1226-1231 and Smith, H.Q., et al 1997. J. Bacteriol. 179, 5914-5921). Consistently, an average value, ~KD, of 1.9 and 0.95 pM (assuming the monomeric and dimeric forms of MarR, respectively) was obtained. However, this value does not represent a true KD for many reasons. Both competition between MarR and the restriction endonuclease and continual depletion of the amount of target DNA (mar0) due to the restriction enzyme contribute to an underestimate of the true KD. Although non-specific protein-nucleic interactions have been observed for other bacterial transcription factors (Joachimiak, A.J., et al 1983.

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 Proc. Natl. Acad. Sci. USA 80, 668-672 and Winter, R.B., et al 1981. Biochem.
20, 6961-6977), non-specific binding for MarR was not exceedingly significant, since the non-operator SspI recognition sequence was accessible at the highest protein concentrations tested. In these experiments. the off rate for the MarR-mar0 interaction must be sufficiently low in order to protect mar0 from cleavage. The advantage of the restriction enzyme site protection assay over a standard gel-shift assay is that it is performed at equilibrium.
The restriction site protection assay s were repeated in the presence of various inducers to confirm the gradient plate data and to test putative inducers that are potent antibiotics, i.e., tetracycline, chloramphenicol, ampicillin, and norfloxacin.
The DNA
binding activity of MarR in vitro was antagonized by weak acids and oxidative stress agents. As controls, vector (pSup-Test, 3.4 nM) alone with and without digestion with SspI, and pSup-Test in the presence of MarR (2.92-3.6 fig, 9.1-11.2 ~M, assuming the monomeric form of MarR) digested with SspI were electrophoresed along with the samples. Protection assays in the presence of chloramphenicol, tetracycline, ampicillin, and paraquat were performed with these chemicals at a concentration of 5 mM.
Plumbagin and 2,4-dinitrophenol were tested at concentrations of 0.25, 0.5, and 1 mM;
menadione was tested at concentrations of 0.8, 2, and 5 mM. Sodium salicylate and sodium benzoate were tested at concentrations of 0.8, 2, and 5 mM. For sodium salicylate, 2mM of this chemical affected the DNA binding activity of MarR and this property was more pronounced at a higher concentration. Previous experiments demonstrated that multiple compounds induce mar expression in whole cells ( Cohen et al. 1993. J. Bacteriol. 175:7856; Seoane and Levy. 1995. J. Bacteriol.
177:3414).
However, the mechanism by which induction was achieved was hitherto unknown.
Previous in vitro experiments demonstrated that salicylic acid bound MarR with a KD of 0.5 mM and sodium salicylate abolished MarR-mar0 complexes in gel shift assays (Martin, R.G., et al 1995. Proc. Natl. Acad. Sci. 92, 5456-5460). This finding was confirmed in whole cells (Table 1 ) and in vitro. Sodium benzoate had a lesser effect on this activity.

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 The oxidative stress agents, plumbagin and DNP, were the most effective compounds tested with visible deprotection occurring of 250 qM. Menadione reversed deprotection at 800 ~M. However, paraquat, another oxidative stress agent, did not show any effect, a finding which correlates well with its apparent preferred induction of the SoxRS regulon (reviewed in Demple. B. 1991. Ann. Rev. Genet. 25, 315-337).
In the in vitro assays, ampicillin (~ mM) was the only antibiotic that appeared to antagonize the DNA binding activity of MarR. Chloramphenicol, tetracycline and norfloxacin, at the same concentration, had no effect on this activity but slight deprotection was observed when the chloramphenicol concentration was increased to 10 mM. With respect to chloramphenicol and tetracycline it is probable that marRAB
expression in whole cells is induced through an indirect action. One possibility is that an unidentified cellular product generated upon exposure to any one of these compounds may function as the inducer (Alekshun, M.N., et al (1997) Antimicrob. Agents Chemother. 10, 2067-2075). Alternatively, both antibiotics may simply increase mRNA
stability (Lopez, F.J., et al 1998. Proc. Natl. Acad. Sci. 95, 6067-6072). In control experiments, in which both the inducing and non-inducing chemicals were added to the SspI restriction endonuclease, no altered activity or specificity of the enzyme was seen.
Experiments in which MarR was added to pSup-Test before inducer produced results identical to those described above. These findings suggest that compounds which induce marRAB expression can interact with MarR whether it is free or bound to DNA
and that this interaction alters the DNA binding activity of the repressor.
The low background of ethidium bromide staining seen with some samples is attributed to two factors: intrinsic fluorescence of the inducers under ultraviolet light and the formation of unique nucleoprotein complexes. Previous results indicated that purified MarR forms multimers (Seoane, A.S., et al 1995. J. Bacteriol. 177, and Martin, R.G., et al 1995. Proc. Natl. Acad. Sci. 92, 5456-5460). These oligomeric forms of the repressor were visualized on SDS-PAGE gels containing 8 M urea.
The background staining seen in samples containing MarR, but not with vector alone, may represent different multimeric forms of MarR complexed with DNA.

WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 These findings provide the first evidence that multiple structurally unrelated chemicals (inducers) interfere directly with MarR function in vitro. While the multidrug binding profiles of efflux proteins have been demonstrated (Paulsen, LT., et al 1996.
Microbiol. Rev. 60, 575-608; Lewis, K. 199-1. Trends Biochem. Sci 19, 119-1123 and Nikaido, H. 1996. J. Bacteriol. 178, 5853-~8~9), there are also examples of cytoplasmic proteins that bind multiple structurally unrelated substrates. In Bacillus subtilis, BmrR, the negative regulator of the Bmr mutidrug transporter, binds chemicals (rhodamine 6G
and tetraphenylphosphonium) that are substrates of the pump (Ahmed, M., et al 1994. J.
Biol. Chem. 269, 28506-28513). The gene product of fabI in E. cola, encoding enoyl reductase, binds natural fatty acid substrates and interacts with triclosan and, presumably, diazaborine, that inhibit function of the protein (McMurry, L.M., et al 1998.
Nature 394, 531-532; Hearth et al. 1998 J. Biol. Chem. 273:30316; Levy et al.
1999.
Nature. 398:383). With respect to the marR:4B operon of E. cola, it appears that the protein which negatively regulates expression of the locus is well adapted to control the cell's rapid response to multiple environmental hazards.
TABLEI
Inducer concentrations needed to kill E. cola DHScz bearing pSup-Test Inducer Concentration (mM)~SD
Plumbagin 0.01 ~ 0.01 2,4-Dinitrophenol 0.05 ~ 0.01 Salicylate (Sodium salt) 0.53 t 0.64 Benzoic Acid (Sodium salt) 1.41 ~ 0.157 Menadione (Sodium bisulfate) 2.8 ~ 1.41 WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 a As determined by gradient plates containing potential inducers at the concentrations indicated: plumbagin (0.4 mM), 2,4-dinitrophenol (0.4 mM), sodium salicylate (10 mM), sodium benzoate ( 10 mM), and menadione (5 mM).
Example 2 Characterization of negative complementing MarR mutants in vivo Negative complementing marR mutants (marR-d) were generated in order to identify the DNA binding domain of this repressor. Prior to their crystallization, the functional regions of the multimeric lac (LacR) and trp (TrpR) repressors were characterized by studying mutant proteins (Betz, 1987, J. Mol. Biol. 195: 495;
Hurlburt and Yanofsky, 1990, J. Biol. Chem. 26~: 7853; Kelley and Yanofsky, 1985, Proc.
Natl.
Acad. Sci. USA 82: 482; Klig and Yanofsky, 1988, J. Biol. Chem. 263: 243;
Miller, 1980, The operon. Cold Spring Harbor, NY. Cold Spring Harbor Laboratories;
O'Gorman et al., 1981, Biochim. Biophys. Acta 653: 237; Pfahl, 1979, J.
Bacteriol. 137:
137). Negative complementing trpR and particular lacl alleles encode proteins with impaired DNA binding properties but that are still able to form multimers, resulting in association with, and inactivation of (traps-dominance), wild type subunits (Adler et al., 1971, Nature 237: 322; Gilbert and Miiller-Hill, 1970, The lactose operon;
Kelley and Yanofsky, 1985, Proc. Natl. Acad. Sci. USA 82: 482; Miller, 1980, The operon Cold Spring Harbor, NY. Cold Spring Harbor Laboratories). Mutants that are able to bind operator DNA but either prevent the binding of wild type repressor or are defective in controlling steps subsequent to repressor binding, e.g mutants that are unable to prevent isomerization of RNA polymerase, would also be isolated in this scheme.
The marR-d mutants isolated in this study are clustered in two regions and lie within domains that show homology to HTH binding motifs of known crystal structure.
Particular mutations in each putative HTH yield proteins that are unable to form wild type complexes with operator DNA. Another class of mutants that are able to bind DNA but have limited intracellular functions have been identified and have novel implications. These experiments suggest that MarR interacts with operator DNA
through one or two HTH motifs and that the N-terminus of this protein has a role in protein-DNA
interactions.

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 Experimental procedures Bacterial strains, plasmids, and genetic techniques SPC 105 (Cohen, et al., 1993, J. Bacteriol. 175: 7856) contains a wild type mar locus and SPC107 (MO100) has a 39-kb deletion that includes the mar locus (Seoane and Levy, 1995, J. Bacteriol. 177: 3414); both strains bear a chromosomal (AmpR) Pmarll-IacZ
fusion on 7~ at the attachment site (Cohen. et al., 1993, J. Bacteriol. 175:
7856; Seoane and Levy, 1995, J. Bacteriol. 177:3414). E. coli BL21 (DE3) (Novagen, Madison, WI) was the strain used for high level MarR expression. Mutant marR alleles were sequenced from plasmids purified using the Qiagen plasmid purification kit (Qiagen) or from PCR products generated using the mutant plasmids as template DNA and Platinum Taq DNA polymerise high fidelity according to the manufacturer's protocols (Life Technologies). DNA sequence analysis was performed using an ABI automated DNA
sequencer. Competent cells were prepared as previously described (Tang et al., 1994, Nuc. Acids Res. 22: 2857). Plasmid pAC-MarR (WT) (KanR) was constructed from pACT7 (Maneewannakul et al., 1992. Mol. Micorbiol. 6:2961 ), a pACYC 184 derivative, and is a low copy number wild type MarR expression vector which has a plSA origin of replication and has been previously described (Alekshun and Levy, 1999, J. Bacteriol. In press). In this plasmid, synthesis of MarR is governed by the lacPl promoter (IPTG inducible) and the wild type MarR ribosome binding site (AGGG) and start codon (GTG). To create high level MarR expression vectors, the wild type and marR-d mutant alleles were amplified by PCR from the mutant low copy number vectors [pAC-MarR (WT) derivatives] and subsequently cloned into pETl3a (Studier et al., 1990, Meth. Enzymol. 185: 60). In these plasmids, expression of marR is regulated by the T7 RNA polymerise promoter and a near consensus ribosome binding site.
Mutagenesis Hydroxylamine and nitrosoguanidine mutagenesis of pAC-MarR (WT) were performed according to established protocols (Miller, 1972, Experiments in molecular genetics Cold Spring Harbor, NY. Cold Spring Harbor Laboratories). In order to maximize the isolation of independent mutants, the entire transformation mix ( 1 ml) was divided into WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 ten equal portions prior to phenotypic expression and subsequent plating.
Subsequently, transformants were selected at either 30°C or 37°C and a maximum of four colonies per plate were retained for further analysis.
Isolation of mutants Mutant marR alleles which specify peptides that can interact with and inactivate wild type marR are defined as negative complementing or traps-dominant. A mixture of hydroxylamine and nitrosoguanidine mutagenized pAC-MarR (WT) plasmids were transformed into SPC105 and plated on MacConkey lactose agar containing ampicillin (100 pg/ml), kanamycin (30 ~g/ml), and IPTG (50 pM). The host alone or containing pACT7 (Maneewannakul, et al., 1992, Mol. ~~licrobiol. 6: 2961 ), the parent plasmid of pAC-MarR (WT), yielded weak Lac+ colonies, while cells containing pAC-MarR
(WT) were Lac-. Plasmids bearing negative complementing marR mutations were initially identified as strong Lac+ colonies.
/j- galactosidase assays for repressor activity Low-copy number plasmids bearing wild type or mutant marR were transformed into reporter strains E. coli SPC 1 OS (marR+) or SPC 107 (AmarR). Cells were grown at 30°C
or 37°C to mid-logarithmic phase in LB broth, without glucose, containing the appropriate antibiotics and IPTG (50 pM). (3- galactosidase assays were performed in cells permeabilized with chloroform/SDS as previously described (Miller, 1972, Experiments in molecular genetics Cold Spring Harbor, NY. Cold Spring Harbor Laboratories; Seoane and Levy, 1995, J. Bacteriol. 177: 3414).
Preparation of total cellular lysates and Western blot analysis SPC107 and BL21(DE3) bearing marR alleles in traps were grown in LB broth containing the appropriate antibiotics to mid-logarithmic phase and induced with IPTG.
Cells were collected by centrifugation, washed, resuspended in 50 mM sodium phosphate (pH 7.4), 2 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF, serine protease inhibitor) (Sigma), and 5 mM EDTA, and sonicated.
Insoluble WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 proteins were removed by centrifugation at 14,900 x g at 4°C and total soluble protein concentration determinations were performed using the Bio-Rad protein assay kit (Bio-Rad).
Equivalent amounts of protein from control and experimental cultures were separated by SDS-PAGE on 15% gels. Western blot analysis using anti-MarR
polyclonal antibodies was performed essentially as described (McDermott et al., 1998, J.
Bacteriol. 180: 2995).
Gel mobility shift assays An 163-by DNA fragment containing the entire Pmarll mar0 region was labeled at the 3' end by incubation with terminal transferase and digoxigenin-11-dUTP
(DIG-11-dUTP) according to the manufacturer's protocols (Roche Molecular Biochemicals). DNA binding reactions (20 ~1) contained 20 mM HEPES (pH 7.6), 1 mM EDTA, 10 mM (NH4)2504, 5 mM DTT, 0.2% Tween 20, 30 mM KCI, 50 fmol of DIG-11-dUTP labeled DNA fragment, 0.1 mg/ml BSA, 1 pg poly(dA-dT) (Roche Molecular Biochemicals), and either 1.25 ~ g or 2.5 pg total soluble protein from control (containing wild type MarR) or experimental cellular lysates, respectively.
The reaction mixtures were incubated at room temperature for 20 min and then subjected to electrophoresis in 6% polyacrylamide gels (22.3 mM Tris, 22.3 mM boric acid, 0.5 mM
EDTA, pH 8.0 (0.25X TBE)) at 200V for 2 hr at 4°C. The gels were processed and detection of DIG-11-dUTP labeled DNA were perfumed according to the manufacturer's protocols (Roche Molecular Biochemicals).
Results Characterization of negative complementing MarR mutants In order to identify marR alleles that encode negative complementing traps-dominant (marR-d) proteins, a low copy number wild type MarR expression vector (pAC-MarR (WT) (Alekshun and Levy, 1999, J. Bacteriol)) was mutagenized in vitro and the mutated plasmids were used in the transformation of a marR+ host (SPC

(Cohen, et ai., 1993. J. Bacteriol. 175:1484; Cohen et al. 1993. J. Bacteriol.
175:7856)) WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 -6~-bearing a chromosomal Pmarll-lacZ fusion (see Experimental procedures). This selection depends on the ability of MarR to form multimers ((Martin and Rosner, 1995, Proc. Natl. Acad. Sci. 92: 5456; Seoane and Levy, 1995, J. Bacteriol. 177:
3414) and to bind DNA (see e.g., Table 2). A number of different mutants were isolated.
Eleven negative complementing traps-dominant mutants were identified by their ability to interfere with the functioning of the wild type repressor, resulting in increased (3-galactosidase expression in this host (Table 3). Other mutants, with near wild-type or moderate repressor activity were also identified (Table 3 and see below). For many of these mutants, the negative complementing phenotype was presumably attributed to subunit mixing that would occur among the wild type and mutant proteins forming relatively inactive repressors. Among these mutants, the mutation at position 73 had the most profound negative complementing traps-dominant effect on the activity of wild type MarR (Table 3). Mutations identified between amino acids 94 and 116 (Fig.

4 and Table 2) also displayed negative complementing traps-dominant phenotypes (Table 3).
It was postulated that the MarR-d mutations identified in these two regions (aa 61-80 and 97-116) might reside in separate HTH DNA binding domains (see below). When comparing the single (G69E) and double (R16H/G69R) mutants, it is apparent that a single non-conservative change at position 69 is sufficient to confer the negative dominant phenotype (Table 3). That the latter is more strongly negative complementing suggests that in addition to affecting DNA binding this mutant may also function in a novel manner (see below).
The repressor activities of the MarR-d mutants were then determined in strain SPC107 (OmarR). All of the proteins except the M74I, L100F, and T101I mutants showed virtually no repressor activity in this host (Table 3). In context with the above results, these findings suggested that the lesion in each of the other mutations resided in a region critical for DNA binding, but did not interfere with the oligomerization of MarR.
With respect to the Q42Amber mutant, this allele would encode a 41 residue protein with the following properties: it is negative complementing and traps-dominant in the marR+ host and yet it has no repressor activity in the OmarR background (Table WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 3). This shows that the first 41 amino acids of MarR contain the majority of the contacts necessary to mediate protein-protein interactions.
Analysis of mutant protein expression in whole cells In order to determine the level of mutant protein expression in whole cells, total cell lysates of SPC 107 bearing the marR-d alleles on low copy number plasmids (pACYC184 derivatives; see Experimental procedures) were subjected to Western analysis using anti-MarR polyclonal antibodies. For western blot analysis of MarR
expressed in E. coli. each lane contained ~ ~g of a soluble cell lysate. A.

(OmarR) carrying marR alleles on low copy number (pACYC 184 derivatives) plasmids.
The contents of the lanes were: Lane 1: SPC 107 alone (negative control).
Lanes 2-12 were SPC107 containing: lane 2: wild type MarR; lane 3, R16H/G69R; lane 4:
Q42Amber; lane 5: G69E; lane 6, R73C; lane 7: M74I; lane 8: R77H; lane 9:
R94C; lane 10: L100F; lane 11: T101I; lane 12: Q1100chre; and lane 13: G116S. A blot was also done on lysates from cells BL21(DE3) (marR+) bearing marR alleles on the medium copy number, but high level expression, pETl3a (pBR322 derivative) vector. The contents of the lanes were: Lane l: SPC107 alone (negative control). Lanes 2-11 are BL21(DE3) containing: lane 2: wild type MarR; lane 3: Q42Amber; lane 4: G69E;
lane 5: R73C; lane 6: M74I; lane 7: R77H; lane 8: L100F; lane 9: T101I; lane 10:
Q1100chre; lane 11: G116S. The R16H/G69R, G69E, R73C, R77H, and G116S
mutants were easily detected and their intracellular levels were comparable to that of the host bearing pAC-MarR (WT). However, the expression of the M74I and R94C
mutants was considerably lower than that of wild type MarR and the Q42Amber, L 1 OOF, T 1 O 1 I, Q1100chre proteins were undetectable by this method.
In order to improve protein expression in vivo and to generate sufficient quantities of these peptides for further biochemical characterization (see below), the mutant alleles were cloned into pETl3a and overexpressed in E. coli BL21(DE3).
Total cell lysates were prepared from these strains and proteins were again subjected to Western analysis. In this system, expression of the M74I and L100F mutants was improved and synthesis of the T101I protein, albeit at a much lower level, was now detectable. However, even in this high level expression system, the Q42Amber and WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 Q110Ochre mutants remained undetectable. One possibility is that these proteins are very unstable in the cell. It is also possible that the MarR polyclonal antibodies recognize epitopes C-terminal to the amino acid 109.
Identification of a helix-turn-helix motif in ~l~IarR
The MarR-d mutants identified in this study are clustered within two general regions: residues 69-77 and 94-116 (Fig. 4 and Table 2). Since this class of mutants was anticipated to affect residues critical to DNA binding (see above), a search for a putative HTH in MarR among these mutations was conducted. Toward this end, initial efforts were designed to identify amino acid residues that are characteristic of proteins with a known HTH motif (Branden and Tooze, 1991, Introduction to protein structure New York, NY USA, London England, Garland Publishing, Inc.; Patio and Sauer, 1984, Ann.
Rev. Biochem. 53: 293; Patio and Sauer, 1992, Annu. Rev. Biochem. 61: 1053).
Most known HTH motifs are usually 20-21 residues in length and the following 1 S parameters generally apply: stereochemical requirements exist for residues 4-5, 8-10, and 15; positions 4 and 15 are most often completely buried and, thus, should be nonpolar with Val or Ile usually being found at position 15; position 5 is generally a small residue, such as alanine or glycine, and should not be a branched chain amino acid (Val, Leu, or Ile) since these larger residues would interfere with the conformation of the HTH motif; and residue 9 is a small amino acid residue, either glycine (most common) or alanine (Branden and Tooze, 1991, Introduction to protein structure New York, NY
USA, London England, Garland Publishing, Inc; Patio and Sauer, 1984, Ann. Rev.
Biochem. 53: 293; Patio and Sauer, 1992, Annu. Rev. Biochem. 61: 1053).
The clustering of the MarR-d mutations isolated suggested the presence of two potential HTH motifs in MarR among residues 61-80 and 97-116. When considered in context with the stereochemical requirements for particular residues of a HTH
motif, the best match is to residues 61-80 (MarR-M, Fig. 5). As illustrated for MarR-M, a hydrophobic residue (leucine) is present at positions 4 and 15 and residue 9 is glycine (Fig. 6). Unusually, serine is found at residue 5; however, MarA, TrpR, and y8 resolvase contain non-consensus amino acids (boldfaced residues in Fig. 5) at this position (Patio WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 and Sauer, 1984, Ann. Rev. Biocl~em. 53: 293; Rhee et al., 1998, Proc. Natl.
Acad. Sci.
USA 18: 10413 ).
The clustering of MarR-d mutations in the C-terminus of MarR and the traps-dominant negative complementing phenotype of the Q 11 OOchre mutant, considering that this allele should encode an 109 residue MarR with an intact HTH at MarR-M, suggested the presence of a second HTH. One was found between amino acids 97-116 (MarR-C, Fig. 5). The amino acids at positions 4 (hydrophobic, leucine) and 9 (small, alanine) within this putative HTH adhere to the consensus, while those at positions 5 (weakly polar, threonine) and 1 ~ (weakly polar, cysteine) do not (Fig. 5).
An amino acid with a large side chain (position 5), relative to glycine or alanine, and a weakly polar residue (position 15) at these positions were not anticipated.
However, the HTH of TrpR contains a lysine residue at position 5, which imposes a structural strain on the positioning of the helices within the motif (Harrison and Aggarwal, 1990, Ann. Rev.
Biochem. 59: 933), and the second HTH in MarA (Rhee, et al., 1998, Proc. Natl.
Acad.
Sci. USA 18: 10413) contains a moderately polar amino acid (threonine) at position 15 (Fig. 5). From these results, it is speculated that MarR requires both MarR-M
and l~IarR-C for efficient DNA binding (see below).
Within known HTH motifs, amino acids at positions 11-13, 16-17, and 20 make contacts with bases in the major groove (Branden and Tooze, 1991, Introduction to protein structure. New York, NY, USA: London, England: Garland Publishing, Inc:
1484; Patio and Sauer, 1992, Annu. Rev. Biochem. 61: 1053). This would explain why mutations R73C (position 13) and R77H (position 17) of MarR-M and G116S
(position 20) of MarR-C (Figs. 5 and 6) have a negative complementing traps-dominant phenotype. That the R73C mutation is especially active suggests that the wild type residue at this position makes a specific contacts) with a bases) in the operator.
Gly 69 of MarR would correspond to position 9 and occur in the turn of the MarR-M HTH motif (Fig. 6). Residues within this region of HTH motifs of known crystal structures are critical for the correct orientation of the two helices in the motif (Patio and Sauer, 1984, Ann. Rev. Biochem. 53: 293; Patio and Sauer, 1992, Annu. Rev.
Bioc~rem. 61: 1053). The size and charge of the glutamic acid or arginine side chains, relative to that of glycine or alanine, in the G69E and R16H/G69R mutants may distort WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 the helices and severely hinder DNA binding. Likewise, the L100F and TlOII
mutants found within MarR-C correspond to positions that are subject to stereochemical constraints (Figs. 5 and 6).
Particular MarR mutants have altered D~:4 complex stoichiometries Gel mobility shift assays demonstrated that wild type MarR, at various concentrations, was capable of forming three distinct stiochiometric complexes with operator DNA (Seoane and Levy, 1995. J. Bacteriol. 177: 3414). These complexes corresponded to protein:DNA ratios of 1:1, 2:1, and 3:1 (Seoane and Levy, 1995, J.
Bacteriol. 177: 3414) and in most instances, the 2:1 complex was the predominate species observed (Martin and Rosner, 199. Proc. Natl. Acad Sci. 92: 5456;
Seoane and Levy, 1995, J. Bacteriol. 177: 3414).
In order to confirm the genetic analysis of the negative complementing traps-dominant mutants, e.g. their anticipated reduced DNA binding affinities, gel mobility shift assays were performed using total cellular lysates prepared from E. coli BL21(DE3) bearing plasmid encoded mutant marR alleles. Gel mobility shift assays were run for wild type and MarR mutant repressors. Probe preparation, binding reactions , and electrophoresis methods are described in the Experimental procedures for this example. Free mar0 is the DIG-11-dUTP labeled 163-by target DNA and contains both the mar promoter (Pmarll) and operator regions (mar0). The protein:DNA
(1:1, 2:1, and 3:1) stoichiometries of the bands based on mobility comparisons (Seoane and Levy, 1995) to complexes of known stoichiometry were noted. The lanes contained:
Lane 1: Pmarlllmar0 only. Lanes 2-11 contain labeled DNA incubated with total proteins prepared from BL21(DE3) bearing wild type or mutant marR in traps.
Lane 2:
1.25 ~g wild type cell extract. Lanes 3-11: 2.5 ~g of cell extracts from cells containing MarR mutants; lane 3: Q42Amber; lane 4: G69E; lane 5: R73C; lane 6: M74I; lane 7:
R77H; lane 8: L100F; lane 9: T101I; lane 10: Q1100chre; and lane 11: G116S.
The formation of a normal 2:1 complex was observed when total proteins from a cell overexpressing wild type MarR was used. However, for the G69E, R73C, R77H
and Ql 100chre traps dominant mutants, the 1:1 (protein:DNA) stoichiometric species predominated. While the 1:1 (protein:DNA) species was not as pronounced with cell WO 00/65082 ~ 02370426 2001-10-22 extracts containing the M74I and T101I mutants, their reduced function in (OmarR) (Table 3) probably reflects lower DNA binding properties.
Some mutants, e.g. Q42Amber, L100F, and G116S, that displayed negative complementing traps-dominant phenotypes in SPC105 (marR+) (Table 3) were to some degree, able to bind operator DNA. This may suggest that their effect is subsequent to repressor binding, i.e., do not interfere with RNA polymerase isomerization, or prevent the binding of wild type MarR.
Characterization of other mutations within MarR
During the course of these experiments additional MarR mutants were identified (Fig. 4 and Table 3). The S34F/P35S, C47Y, and P88S MarR mutants exhibit near wild type activity and the A40T, V45M, A52T/V84M, and V84M alleles encode moderately active repressors with activities that are between 27-70% of the wild type MarR (Table 3). These results are not unexpected since these mutations reside either outside of the region presumed to participate in protein-protein interactions or between MarR-M and MarR-C (Fig. 5, 6). That the double (A52T/V84M) is slightly less active than the single (V84M) mutant suggests that mutations at both residues function cumulatively to lower MarR activity or that the former is less stable in the cell (see below).
The expression of all but one of these moderately active mutants, A52T/V84M, were easily detected from total cell lysates of SPC 107 bearing the mutants in traps (data not shown). This demonstrates that their lack of effect on the functioning of wild type MarR cannot be attributed to poor expression in whole cells.
Discussion A number of negative complementing traps-dominant mutations in MarR, identified in this study, are clustered in two areas of the full length protein and are presumed to form two DNA binding domains (Fig. 5,6). Two negative complementing marR alleles, marRl (Arg 77 Leu) and marR2 (Val 45 Glu), have been previously identified (Alekshun and Levy, 1997, Antimicrob. Agents Chemother. 10: 2067;
Cohen, et al., 1993, J. Bacteriol. 175: 1484; Seoane and Levy, 1995, J. Bacteriol.
177: 3414).
These alleles specify proteins incapable of repression in strains expressing wild type WO00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 MarR and, the presence of plasmids bearing the marRl and marR2 mutations resulted in increased antibiotic resistance and ~3- galactosidase expression from a Pmarl lacZ fusion (Cohen, et al., 1993, J. Bacteriol. 175: 1484: Seoane and Levy, 1995, J.
Bacteriol. 177:
3414). This finding suggested that the mutant peptides interacted with and decreased the activity (traps-dominance in a negative complementing manner) of wild type MarR.
Alternatively, in the case of the Val 45 Glu mutant, the mutant may prevent the binding of wild type repressor top mar0 or affect a point subsequent to this step (see below).
Two regions that resemble known HTH motifs (Branden and Tooze, 1991, Introduction to protein structure; Pabo and Sauer, 1984, Ann. Rev. Biochem. 53: 293; Pabo and Sauer, 1992, Annu. Rev. Biochem. 61: 1053) were identified in MarR (MarR-M, amino acids 61-80, and MarR-C, residues 97-116) (Fig. ~ and 6).
Four of the mutants isolated in this study, G69E, R73C, R77H, and Q1100chre, were unable to form wild type stoichiometric (2:1 ) complexes with operator DNA. Two mutants, M74I and T101I were able to form partially wild type protein:DNA
complexes but at a level less than that observed with native MarR. This may explain why the M74I
mutant lacks function in SPC107 (OmarR) while the T101I mutant retains some repressor activity in this host (Table 4). These data offer very sound proof for the presence of at least two regions in MarR that are required for proper DNA
binding. That mutations in the first putative HTH, MarR-M, were more detrimental to the fiznction of MarR both in whole cells (Table 4), especially for the R73C mutant, and in vitro suggests that MarR-M may be more critical to the DNA binding activity of the repressor.
Five of the traps-dominant mutations isolated in this study (R94C, L100F, T101I, Q1100chre, and G116S) were found very near to, or in, what appears to be a second HTH, in the C-terminus of MarR. The L100F and G116S mutants bind DNA in a manner similar to wild type MarR. It is possible that these mutants and others, e.g Q42Amber, prevent the binding of wild type MarR to mar0.
It is unknown whether MarR functions by preventing binding of RNA
polymerise to the mar promoter or interferes with the transcription complex subsequent to the binding of the repressor to mar0. The Arc repressor functions by inhibiting isornerization of RNA polymerise (Vershon et al., 1986, Prot. Str. Function Genet.

_72_ 1:302). Some of the mutants isolated in this study, Q42Amber, L100F, and G116S, may have lost the ability to interfere with transcriptional initiation.
The two HTH motif proposal is also supported by the phenotype (Table 3) of the nonsense mutation at residue 110 (Q 11 OOchre) and its behavior in vitro. The Q I I OOchre allele encodes a 109 residue MarR which would include an intact HTH
(MarR-M) between residues 61-80. Since this mutant displays a negative complementing traps-dominant phenotype. it is suggested that both regions (MarR-M
and MarR-C) are necessary for DNA binding. That it cannot form wild type protein:DNA complexes in vitro indicates that residues C-terminal to amino acid 109 are important for DNA binding. The T101I mutation may also alter the orientation of the helices in MarR-C resulting is a structurally unfavorable HTH thereby hindering DNA
binding.
Since the phenotype of the Q42Amber mutant is traps-dominant and negative complementing, the majority of the contacts needed to maintain protein-protein interactions between subunits of the multimer must reside within the first 41 amino acids of MarR. That this mutant has no activity by itself in the whole cell assay (Table 3) may reflect its instability in the cell.
Two putative HTH motifs were identified among the traps-dominant MarR
mutations. That MarR would contain two HTH motifs is unusual for prokaryotic transcription factors. A genetic and biochemical approach was originally used to suggest the existence of two HTH motifs in AraC (Francklyn and Lee, 1988, J. Biol.
Chem. 263:
4400) and this proposal was later confirmed (Niland et al., 1996, J. Mol.
Biol. 264: 667).
Moreover, the crystal structure of MarA, an AraC homolog, the activator of Mar expression in E. coli and encoded with the same operon as MarR, provides this first direct proof of a prokaryotic transcription factor which contains two HTH
motifs (Rhee, et al., 1998, Proc. Natl. Acad. Sci. USA 18: 104313).
Additional support for this model is based on the sequence and organization of the MarR binding sites. MarR protects both strands of two regions (sites I and II) within the mar operator fom nuclease cleavage in vitro (Martin and Rosner, 1995, Proc. Natl.
Acad. Sci. 92: 5456). Two inverted nucleotide sequence repeats (indicated by arrows in Fig. 7) that are separated by a dyad axis of symmetry (represented as broken lined boxes WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 7J _ in Fig. 7) are found in sites I and II (Fig. 7). This type of organization might be expected for a protein with a dual HTH motif and these data suggest that MarR and other members of this family may recognize DNA in a common manner.

WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 Table 2. Characteristics of marR mutants Mutant Phenotype Replacement Codon Change Number of independent isolates, n R16H/G69R - d rg 16 His CGC -~ CAC 1 Gly 69 Arg GGA -~ AGA

S34F/P35S m Ser 34 Phe CT ~ TTT 1 ro 35 Ser CCG -~ TCG

40T m la 40 Thr GCG ~ ACG 1 1Q42Amber - d Gln 42 AmberCC -~ ACT (S)11 CAG ~ TAG

45M m al 45 Met GTG ~ ATG 3 C47Y w Cys 47 Tyr GC ~TAC 1 52T/V84M m la 52 Thr GCG -~ACG 1 al 8-1 Met GTG ~ ATG

G69E - d Gly 69 Glu GGA ~ GAA 3 73C - d rg 73 Cys CGT -~ TGT 1 74I - d et 74 Ile TG ~ ATA 1 77H - d rg 77 His CGC ~ CAC 1 84M m al 84 Met GTG ~ ATG 1 88S w ro 88 Ser CCG ~ TCG 1 94C - d rg 94 Cys CGC ~ TGC 2 95D r Gly 95 Asp GGC -~ GAC 2 100F - d eu 100 Phe CTT ~ TTT 1 lOII - d hr 101 Ile CC ~ ATC 1 1 lOOchre - d Gln 110 OchreCAA -~ TAA 1 1165 - d Gly 116 Ser GGC -~ AGC 1 r, encodes a recessive marR; w, near wild type activity; -d, traps-dominant MarR; m, moderate repressor activity (27-70%).

WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 1 This allele contains a silent (S) and nonsense mutation.
Table 3. Effect of marR mutations on (3- galactosidase activity from a Pmarll-lacZ
fusion in marR+ or OmarR backgrounds.
(3- galactosidase activity marR allele on plasmid marR+ (SPC105) OmarR (SPC107) one 329 1005 AC-MarR (WT) (marRt) 1 37 traps-dominant mutants R16H, G69R 753 961 Q42Amber 489 1033 TlOII 420 313 Q 11 OOchre 63 3 963 utants with near wild type epressor activity WO 00/65082 ~ 02370426 2001-10-22 utants with moderate epressor activity S34F, P35S 310 200 A52T, V84M 267 582 ecessive Example 3. Characterization of MarR superrepressor mutants The chromosomal multiple antibiotic resistance (mar) locus of Escherichia coli controls an adaptational response to antibiotics and other environmental hazards (Alekshun, M.N., and Levy, S.B., 1997, Antimicrob. Agents Chemother. 10:
2067). The expression of multiple genes on the E. coli chromosome are regulated by MarA, a transcriptional activator encoded within the marRAB operon (Alekshun, M.N., and Levy, S.B., 1997, Antimicrob. Agents Chemother. 10: 2067).
MarR negatively regulates expression of the marRAB operon (Cohen, S.P., et al 1993, J. Bacteriol. 175: 1484; Martin, R.G. and Rosner, J.L., 1995, proc.
Natl. Acad.
Sci. 92: 5456, Seoane, A.S. and Levy, S.B., 1995, J. Bacteriol. 177: 3414).
DNA
footprinting experiments suggest that MarR dimerizes at two locations, sites I
and II, within the mar operator (mar0) (Martin, R.G. and Rosner, J.L. 1995, Proc.
Natl. Acad.
Sci. 92: 5456); site I is positioned among the -35 and -10 hexamers and site II spans the putative MarR ribosome binding site (Fig. 8A and reviewed in Alekshun, M.N.
and Levy, S.B., 1997 Antimicrob. Agents Chemother. 10: 2067). Many structurally dissimilar chemicals presumably affect MarR activity in whole cells (Ariza, R.R. et al.
1994, J. Bacteriol. 176: 143; Cohen, S.P. et al. 1993, J. Bacteriol. 175:
7856; Martin, R.G. and Rosner, J.L. Proc. Natl. Acad. Sci. 92: 5456; Seoane, A.S. and Levy, S.B., 1995, J. Bacteriol. 177: 3414). Experiments in vitro demonstrate that MarR
binds salicylic acid and, through the use of gel mobility shift assays, sodium salicylate inhibits WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 _77_ the formation of MarR-mar0 complexes (Martin, R.G. and Rosner, J.L. Proc.
Natl.
Acad. Sci. 92: 5456 ). These initial findings have been extended by demonstrating that the DNA binding activity of MarR in vitro is antagonized by several other chemicals.
Thus, MarR possesses DNA binding and effector molecule recognition properties.
Bacterial strains, plasmids, and genetic techniques. The bacterial strains and plasmids used are listed in Table 4. A low-copy number wild type MarR
expression vector was constructed using a modified version of pACT7 (Maneewannakul, K. et al.
1992, Mol. Microbiol. 6: 2961). marR was amplified by PCR from E. coli AG100 (George, A.M. and Levy, S.B. 1983, J. Bacteriol. 155: 531) chromosomal DNA
using Taq DNA polymerase according to the manufacturer's protocols (Life Technologies, Gaithersburg, MD). EcoRI and PstI restriction sites were incorporated into the forward and reverse primers to facilitate directional cloning into pACT7 in place of the T7-RNA
polymerase gene following digestion with EcoRI and PstI. In pAC-MarR (WT), transcription of marR is regulated by the IacPl promoter and protein synthesis is governed by the wild type MarR ribosome binding site (AGGG) and translational initiation (GTG) signals (Cohen, S.P. et al. 1993, J. Bacteriol. 175: 1484).
A high-copy number wild type MarR expression vector was constructed in pETl3a (Studier, F.W. et al. 1990, Meth. Enzymol. 185: 60), a kanamycin resistant version of pETI la (Novagen, Madison, WI). PCR amplification of marR was performed as described above using forward and reverse primers containing VspI
and BamHI restriction sites to facilitate directional cloning into NdellBamHI
digested pETl3a. In the resulting plasmid, pMarR-WT, expression of MarR is under the control of the T7-RNA polymerase promoter and a near consensus ribosome binding site.
For functional analysis of MarR in whole cells, a Pmarlllmar0-ccdB fusion was created in pETI ld (Novagen, Madison, WI). After digestion with EcoRV to remove the majority of lacl, pETl ld was purified using the Qiagen gel purification kit (Qiagen, Santa Clarita, CA), and religated. Pmarlllmar0, containing the marRAB promoter (Pmarll) and operator (mar0) sequences (Fig. 8A), was amplified by PCR with primers containing EagIlBsmI restriction sites and blunt-end cloned into EagIlBsmI
digested pETI ld (lacking lack creating plasmid pmar0. Subsequently, the lac0- ccdB
portion of pKIL 18 (Bernard, P. et al. 1994, Gene. 148: 71 ) was amplified by PCR so as to WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 - 78 _ exclude the tac promoter sequences. XhoI and BsmI restriction sites were incorporated into the forward and reverse primers to facilitate directional cloning downstream of Pmarlllmar0, into AvaI digested (mixed cohesive and blunt ends) pmar0. The resulting plasmid was designated pSup-Test (Fig. 8B).
DNA sequence analysis was performed using an ABI automated DNA sequencer.
Hydroxylamine and nitrosoguanidine mutagenesis of pAC-MarR (WT) were performed according to established protocols (Miller. J.H. 1972, Experiments in molecular genetics Cold Spring Harbor NY, Cold Spring Harbor Laboratories).
Selection of MarR superrepressors. In order to identify MarR superrepressors, expression of the lethal ccdB gene product on pSup-Test was exploited. Plasmid pAC-MarR (WT) was mutagenized in vitro and transformed into DHSa containing pSup-Test. Transformants were selected in the presence of sodium salicylate, a known marRAB operon inducer (Cohen, S.P. et al. 1993, J. Bacteriol. 175: 7856).
Growth of DHS a bearing pETl ld or pmar0 (pSup-Test lacking ccdB, Table 4) was unaffected by the highest concentration of this and other inducers tested (Table 5). DHS a cells containing pSup-Test in the absence or presence of plasmid-encoded wild type marR
were non-viable in the presence of sodium salicylate and other inducers (Table 5).
However, cells containing a putative MarR superrepressor survived higher concentrations of known marRAB operon inducers presumably by binding of the mutant protein to mar0 in front of ccdB on pSup-Test and preventing expression of the lethal gene product (Table 5). From a total of 276 transformants, twelve putative MarR
superrepressor mutants were independently isolated (Fig. 9).
Assay of repressor activity by Vii- galactosidase. The traps-dominant nature of the mutant plasmids was then independently retested in E. coli SPC105, a marR+
host which contains a chromosomally located Pmarll lacZ fusion at the ~, attachment site (Cohen, S.P. et al. 1993, J. Bacteriol. 75: 7856).
Cells were grown at 37°C to mid-logarithmic phase in LB broth, without glucose, containing the appropriate antibiotics and IPTG (50 mM) and sodium salicylate (5 mM) where appropriate. (3- galactosidase assays were performed in cells permeabilized with chloroform/SDS as previously described (Miller, J. H. 1972.

WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 Experiments in molecular genetics. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY.; Seoane, A. S., and S. B. Levy. 1995. J. Bacteriol. 177:3414-3419.).
In the absence of exogenously provided wild type MarR, SPC 1 OS exhibited an easily detectable basal level of LacZ expression (Fig. 10). LacZ expression in cells bearing wild type marR in traps was minimal and was virtually undetectable in cells containing any of the five putative MarRS mutants (Fig. 10). Sodium salicylate caused a >15-fold increase in LacZ expression in cells bearing plasmid-encoded wild type MarR
while those containing a MarRS mutant displayed greatly reduced responses to this inducer (Fig. 10). The Gly 95 Ser, Asp 26 Asn, Asp 26 Asn/Arg 27 His, and Val Met mutants showed little if any response while cells containing the Leu 135 Phe MarRS
mutation showed partial responsiveness to the inducer (Fig. 10).
Properties of MarRS mutants. DH~a bearing the D26N, D26N/R27H, G95S, or V 132M MarR mutant proteins displayed similar decreased susceptibilities to the chemically induced expression of the lethal ccdB gene product as assayed by gradient I S plates (Table 5). The inducer responsiveness of the L135F MarR mutant-containing cells was less than the wild type control cells. but greater than cells bearing the other superrepressor mutants (Table 5).
Western blot analysis using MarR polyclonal antibodies, generated in rabbits using purified MarR (Covance Research Products Inc., Denver, PA), showed that cells bearing the D26N, D26N/R27H, and L13~F mutants expressed 1.3 - 2.0-fold more protein than did those with the wild type MarR. The intracellular levels of the G95S and V 132M mutant proteins were less, 20% and 40%, respectively, of wild type MarR. This result demonstrated that superrepression was not likely attributable to overexpression of the mutant proteins. This point was addressed more clearly by an in vitro DNA
binding assay.
The wild type and marR superrepressor genes were cloned into pETl3a, transformed into E. coli BL21 (DE3) for overexpression, and the MarR proteins purified.
Cells, grown in LB at 37°C to mid-logarithmic phase, were induced for 3 hr with 1 mM
IPTG, collected, washed, and frozen at -70°C. The frozen cell pellet was resuspended in 10 mL buffer A [50 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 2.5 mM
4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF, serine protease inhibitor) (Sigma, St. Louis, MO)] and lysed using sonication. After removal of insoluble matter by centrifugation at 30.000 x g for 1 hour, the supernatant was loaded onto a SP-sepharose HiTrap column (Pharmacia Biotech, Piscataway, NJ) equilibrated with 50 mM Tris-HC1 (pH 7.4). Following a 50 mM Tris-HC1 (pH 7.4) wash, MarR
was eluted using a linear gradient of 0-1 M NaCI in 50 mM Tris-HCl (pH 7.4).
Eluting at 0.2-0.3 mM NaCI, MarR was dialyzed against 100 volumes of 50 mM Tris-HCI (pH
7.4), 100 mM NaCI, 10 % glycerol, and 1 mM phenylmethylsulfonyl fluoride (PMSF, serine protease inhibitor) overnight at 4°C. Judged to be >90% pure on a SDS-PAGE
Coomassie stained gel, MarR was stored in aliquots at -70°C until further use.
A unique SspI recognition sequence within one of two MarR binding sites in mar0 (Fig. 8A) formed the basis of a restriction enzyme site protection assay (Kelly and Yanofshy. 1985. Proc. Natl. Acad. Sci. L'S.-I 82:482; Melville and Gunsalus.
1996.
Proc. Natl. Acad. Sci. 92:5456; Smith and Somerville. 1997. J. Bacteriol.
179:5914) to assess MarR binding to mar0. Serial dilutions of purified wild type or MarR
superrepressor proteins were added to a final volume of 20 ~1 containing 0.2 ~
g pSup-Test (target DNA, 3.4 nM), 10 mM Tris-HCl (pH 7.5), 5 mM NaCI, 1 mM
MgCl2, and 0.0025% Triton X-100. After incubation at room temperature for 10 min, 5 units of SspI (New England Biolabs, Beverly, MA) was added and the reaction mixture was incubated for 30 min at 37°C. The incubation was terminated by the addition of 1.5 ~ 1 stop buffer (0.25M EDTA (pH 8.0), 1 % SDS) and 5 ~ 16X agarose gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol) and analyzed on 0.7%
agarose (Life Technologies, Gaithersburg, MD) gels. The point of 50%
protection, determined by visual inspection of ethidium bromide-stained DNA in these gels, was assigned a value of 5 units of activity. The specific activity was then calculated from this value as previously described (Joachimiak, A.J. et al. 1983, Proc. Natl.
Acad. Sci.
USA 80: 668).
The D26N, G95S, and L135F superrepressor mutant proteins displayed at least a 9-fold greater DNA binding activity than the wild type repressor (Table 6).
Although the inducer susceptibility profiles of these three mutants were similar in intact E. coli DHSa (Fig. 10 and Table 5), their in vitro DNA binding properties were quite different WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 (Table 6). The G95S MarRS mutant showed -2 and 3.5-fold greater DNA binding than did the D26N and L135F mutants (Table 6).
The G95S MarRS mutation occurred within a region that is conserved among all members of the MarR family of proteins and the mutant protein is 30-fold more active than wild type MarR. traps-dominant negative complementing MarR mutants that are in proximity to this residue ( Cohen, S.P. et al. 1993, J. Bacteriol. 175: 7856;
Seoane, A.S.
and Levy, S.B. 1995, J. Bacteriol. 177) suggest that it may play a more direct role in DNA binding.
The D26N superrepressor mutation results in a charged amino acid being substituted for an uncharged residue. A decrease in electrostatic interactions between the protein and the DNA backbone may be the basis for this superrepressor activity. It is also possible that new hydrogen bonds between the asparagine side chain and the DNA
backbone contribute to an increased affinity for DNA. Thus, non-specific DNA
binding is expected to form the basis of superrepression. In the D26N/R27H double mutant, the latter mutation is probably not required since this mutant produced data similar to that of the protein bearing the single D26N change (Fig. 10 and Table 5).
The V132M MarRS mutant showed inducer responses like D26N and G95S
mutants in whole cells (Table 5). Since both mutations are expected to lie outside of the putative DNA binding domain of it is speculated that each plays an accessory role in DNA binding. With respect to the L135F mutant, the phenylalanine residue may increase DNA binding through newly acquired interactions with the phosphate backbone (Schildbach, J.F. et al. 1999, Proc. Natl. Acad. Sci. USA 96: 811 ). The lesion in each mutant may also reside in a region required for proper protein folding, MarR
oligomer assembly, or an inducer recognition domain. It is also possible that the superrepressor mutation in these or the other proteins affects transmission of the signal to the DNA
binding domain following inducer recognition.
That the MarR superrepressor mutations are scattered throughout the protein suggests that amino acid changes in several regions can enhance the DNA
binding activity of the repressor (Table 6). Four interspersed missense mutations in TrpR
resulted in superrepressor proteins (Hurlburt, B.K. and Yanofsky, C. 1990, J.
Biol.
Chem. 265: 783; Kelley, R.L. and Yanofsky, C., 1985, Proc. Natl. Acad. Sci.
USA

WO 00/65082 ~ 02370426 2001-10-22 82:482; Klig, L.S. and Yanofsky, C. 1988. J. Biol. Chem. 263: 243) and none displayed altered binding affinities for the co-repressor tryptophan (Hurlburt, B.K. and Yanofsky, C. 1990, J. Biol. Chem. 265: 7853). Whether enhanced DNA binding is attributable to an increased association or decreased dissociation rate of repressor-operator complex formation or altered DNA complex stoichiometry, as was demonstrated for particular TrpR superrepressors (Hurlburt, B.K. and Yanofsky, C. 1990, J. Biol. Chem.
265: 7853;
Liu, Y.-C and Matthews, K.S. 1994, J. biol. Chem. 269: 1692), is currently unknown. It is of interest to note that only one of the TrpR superrepressor mutations lay within the protein's helix-turn-helix DNA binding domain (Otwinowski, J. et al. 1988, Nature.
335: 321). This finding demonstrates that there is no a priori reason to suspect that a mutation must be confined to the DNA binding domain of MarR in order to result in a superrepressor phenotype. In one embodiment of the claimed screening assays, forms of MarR family members which are superrepressors (e.g., forms that have lost their ability to be regulated by test compounds), can be used as controls which are insensitive to the effects of such test compounds in the subject assays.
Table 4. Bacterial strains and plasmids used in this study.
Strain or plasmidGenotype or CharacteristicsSource/Reference A. Strains DHSa endAl hsdRl7 supE44 aboratory collection thi-I

ecAl gyrA relAl (lacZYA-argF~ U169 eoR(~80lacZ O M15) BL21(DE3) - ompT hsdSB (rg- mB-)ovagen gal cm (DE3) SPC 1 OS C4100 (0 lac U169 araD(7) rpsL

elA thi flbB) containing a chromosomal Pmarll lacZ

sion at the ~, attachment site d a wild type mar locus WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 _83_ B. Plasmids pACT7 ow copy number T7 RNA (14) olymerase expression vector pETI ld High level expression ovagen vector (bla, laclJ

pETl3a Kanamycin resistant (22) version of ETlla pAC-MarR (WT) ow copy number wild his study type arR expression vector derived from pACT7 pmar0 ETl ld derivative lackinghis study lacl and containing Pmarlllmar0 pSup-Test ETl ld derivative lackinghis study lacl and containing a Pmarlllmar0 ccdB fusion WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 o ~ o tin n, _' y ,-;.
i~

on , ~.

M O O N ~ N ~ ~ O '~

O~n >, ~ c~ O
N N o0 ~ .-~ j, ~
~ ~ ~ t~, O O O ~1 .-~M

U y,y'~ ' .o ~ o o ~ ~ r-; i:, 3 ~ ~ ., ~+, o ~ ~ ;~ ~
N O O ~~ ~ M E"' H
C1.O
i ~
f v . ~.
3 ..-, , , a>
c~ N N ~ ~n ~ V~ ,~
S3, ~ :.O
' ~ ~ O O I~ ~ M
~ N

i ~
~

O

.fl O [~~ O O~ v~ d: oo bA ~ ~ '~"
~C ' O O O ,-~N ,~ O
.~ ~

U ~ '~ N
c~ ~ ~ by bA
o ~ o ~ 'L3 U H .-., 0 0 0 ,~

_ ~ _~
onfy, 'O ~ 'r o "~ ,.~ Q., ~ ~ 3 O O ....M n ~ n n n n V "O ~ by O
~t d: O O ~
x ~ w p O ~' M ,~ 'd ~' n n n n n o v ..s", O" ~ -O b .
4~,.., O
O O +r O .t", 0 ~ ~ 0 ~n > O y ~ Q. ~ +, .~ .O O
U CJ N CH ~
O CG N ~ O ~ ~
_G7~ ~ ~ ~_ C/~ W '~~ .fl ~ ~ ~ O ~ y "~'b ~3 ~ ~ ~ ~ ~ ~ O O
~ "o ''~~ U .~ o E-~on ,~ 0..N vo~ va ~ ~ ~ 3 v ~
v~

..o WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 Table 6. Assay of MarR DNA binding activity by Ssp I restriction enzyme site protection.
Purified MarR specific Fold increase in DNA binding MarR b activity (unitslmg) a Wild Type 5, 495 1 Asp 26 Asn 89, 286 16.2 Gly 95 Ser 166, 666 30.3 Leu 135 Phe 48, 544 8.8 ~

a Specific activity was determined based on 50% protection of the SspI
recognition enzyme sequence in site I of the mar operator (designated as SU of activity).
Results are representative of experiments performed at least twice.
b Relative to the wild type protein.
Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific polypeptides, nucleic acids, methods, assays and reagents described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 SEQUENCE ~~STING
<110> Trustees of Tufts College <120> THE MAR REPRESSOR FAMILY OF PROTEINS AND METHODS OF
THEIR USE
<130> PKZ-029CPPC
<140>
<141>
<150> USSN 09/298,735 <151> 1999-04-23 <160> 15 <170> PatentIn Ver. 2.0 <210> 1 <211> 7878 <212> DNA
<213> Escherichia coli <400>

gttaactgtggtggttgtcaccgcccattacacggcatacagctatatcgagccttttgt60 acaaaacattgcgggattcagcgccaactttgccacggcattactgttattactcggtgg120 tgcgggcattattggcagcgtgattttcggtaaactgggtaatcagtatgcgtctgcgtt180 ggtgagtacggcgattgcgctgttgctggtgtgcctggcattgctgttacctgcggcgaa240 cagtgaaatacacctcggggtgctgagtattttctgggggatcgcgatgatgatcatcgg300 gcttggtatgcaggttaaagtgctggcgctggcaccagatgctaccgacgtcgcgatggc360 gctattctccggcatatttaatattggaatcggggcgggtgcgttggtaggtaatcaggt420 gagtttgcactggtcaatgtcgatgattggttatgtgggcgcggtgcctgcttttgccgc480 gttaatttggtcaatcattatatttcgccgctggccagtgacactcgaagaacagacgca540 atagttgaaaggcccattcgggccttttttaatggtacgttttaatgatttccaggatgc600 cgttaataataaactgcacacccatacataccagcaggaatcccatcagacgggagatcg660 cttcaatgccacccttgcccaccagccgcataattgcgccggagctgcgtaggcttcccc720 acaaaataaccgccaccaggaaaaagatcagcggcggcgcaaccatcagtacccaatcag780 cgaaggttgaactctgacgcactgtggacgccgagctaataatcatcgctatggttcccg840 gaccggcagtacttggcattgccagcggcacaaaggcaatattggcactgggttcatctt900 ccagctcttccgacttgcttttcgcctccggtgaatcaatcgctttctgttgcggaaaga960 gcatccgaaaaccgataaacgcgacgattaagccgcctgcaattcgcagaccgggaatcg1020 aaatgccaaatgtatccatcaccagttgcccggcgtaatacgccaccatcatgatggcaa1080 atacgtacaccgaggccatcaacgactgacgattacgttcggcactgttcatgttgcctg1140 ccaggccaagaaataacgcgacagttgttaatgggttagctaacggcagcaacaccacca1200 gccccaggccaattgctttaaacaaatctaacattggtggttgttatcctgtgtatctgg1260 gttatcagcgaaaagtataaggggtaaacaaggataaagtgtcactctttagctagcctt1320 gcatcgcattgaacaaaacttgaaccgatttagcaaaacgtggcatcggtcaattcattc1380 atttgacttatacttgcctgggcaatattatcccctgcaactaattacttgccagggcaa1440 ctaatgtgaaaagtaccagcgatctgttcaatgaaattattccattgggtcgcttaatcc1500 atatggttaatcagaagaaagatcgcctgcttaacgagtatctgtctccgctggatatta1560 ccgcggcacagtttaaggtgctctgctctatccgctgcgcggcgtgtattactccggttg1620 aactgaaaaaggtattgtcggtcgacctgggagcactgacccgtatgctggatcgcctgg1680 tctgtaaaggctgggtggaaaggttgccgaacccgaatgacaagcgcggcgtactggtaa1740 aacttaccaccggcggcgcggcaatatgtgaacaatgccatcaattagttggccaggacc1800 tgcaccaagaattaacaaaaaacctgacggcggacgaagtggcaacacttgagtatttgc1860 ttaagaaagtcctgccgtaaacaaaaaagaggtatgacgatgtccagacgcaatactgac1920 gctattaccattcatagcattttggactggatcgaggacaacctggaatcgccactgtca1980 ctggagaaagtgtcagagcgttcgggttactccaaatggcacctgcaacggatgtttaaa2040 WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 aaagaaaccggtcattcattaggccaatacatccgcag~cgtaagatgacggaaatcgcg2100 caaaagctgaaggaaagtaacgagccgatactc~atc~lgcagaacgatatggcttcgag2160 tcgcaacaaactctgacccgaaccttcaaaaat~ac~_~gatgttccgccgcataaatac2220 cggatgaccaatatgcagggcgaatcgcgcttt~taca~ccattaaatcattacaacagc2280 tagttgaaaacgtgacaacgtcactgaggcaatcatgaaaccactttcatccgcaatagc2340 agctgcgcttattctcttttccgcgcagggcgt-~gca;acaaaccacgcagccagttgt2400 tacttcttgtgccaatgtcgtggttgttcccccatcgcaggaacacccaccgtttgattt2460 aaatcacatgggtactggcagtgataagtcggatgccc~cggcgtgccctattataatca2520 acacgctatgtagtttgttctggccccgacatc~cgg,;cttattaacttcccaccttta2580 10ccgctttacgccaccgcaagccaaatacattgatata~agcccggtcataatgagcaccg2640 cacctaaaaattgcagacccgttaagcgttcatccaacaatagtgccgcacttgccagtc2700 ctactacgggcaccagtaacgataacggtgcaacccg~~aggtttcatagcgtcccagta2760 acgtcccccagatcccataaccaacaattgtcgccacaaacgccagatacatcagagaca2820 agatggtggtcatatcgatagtaaccagactgtgaatcatggttgcggaaccatcgagaa2880 15tcagcgaggcaacaaagaagggaatgattgggattaaa~cgctccagattaccagcgaca2940 tcaccgccggacgcgttgagtgcgacatgatcttttta~~gaagatgttgccacacgccc3000 aactaaatgctgccgccagggtcaacataaagccgagcatcgccacatgctgaccgttca3060 gactatcttcgattaacaccagtacgccaaaaatcgc~aaggcgatccccgccaattgtt3120 tgccatgcagtcgctccccgaaagtaaacgcgccaagca~gatagtaaaaaacgcctgtg3180 20cctgtaacaccagcgaagccagtccagcaggcataccqaagttaatggcacaaaaaagaa3240 aagcaaactgcgcaaaactgatggttaatccataccccagcagcaaattcagtggtactt3300 tcggtcgtgcgacaaaaaagatagccggaaaagcgaccagcataaagcgcaaaccggcca3360 gcatcagcgtggcatgttatgaagccccactttgatgaccacaaaatttagcccccatac3420 gaccactaccagtagcgccaacaccccatcttttcgcgacattctaccgcctctgaattt3480 25catcttttgtaagcaatcaacttagctgaatttacttttctttaacagttgattcgttag3540 tcgccggttacgacggcattaatgcgcaaataagtcgctatacttcggatttttgccatg3600 ctatttctttacatctctaaaacaaaacataacgaaacgcactgccggacagacaaatga3660 acttatccctacgacgctctaccagcgcccttcttgcctcgtcgttgttattaaccatcg3720 gacgcggcgctaccgtgccatttatgaccatttacttgagtcgccagtacagcctgagtg3780 30tcgatctaatcggttatgcgatgacaattgcgctcactattggcgtcgtttttagcctcg3840 gttttggtatcctggcggataagttcgacaagaaacgctatatgttactggcaattaccg3900 ccttcgccagcggttttattgccattactttagtgaa~aacgtgacgctggttgtgctct3960 tttttgccctcattaactgcgcctattctgtttttgc~accgtgctgaaagcctggtttg4020 ccgacaatctttcgtccaccagcaaaacgaaaatcttctcaatcaactacaccatgctaa4080 35acattggctgaccatcggtccgccgctcggcacgctgttggtaatgcagagcatcaatct4140 gcccttctggctggcagctatctgttccgcgtttcccatgcttttcattcaaatttgggt4200 aaagcgcagcgagaaaatcatcgccacggaaacaggcagtgtctggtcgccgaaagtttt4260 attacaagataaagcactgttgtggtttacctgctctggttttctggcttcttttgtaag4320 cggcgcatttgcttcatgcatttcacaatatgtgatggtgattgctgatggggattttgc4380 40cgaaaaggtggtcgcggttgttcttccggtgaatgctgccatggtggttacgttgcaata4440 ttccgtgggccgccgacttaacccggctaacatccgcgcgctgatgacagcaggcaccct4500 ctgtttcgtcatcggtctggtcggttttattttttccggcaacagcctgctattgtgggg4560 tatgtcagctgcggtatttactgtcggtgaaatcatttatgcgccgggcgagtatatgtt4620 gattgaccatattgcgccgccagaaatgaaagccagctatttttccgcccagtctttagg4680 45ctggcttggtgccgcgattaacccattagtgagtggcgtagtgctaaccagcctgccgcc4740 ttcctcgctgtttgtcatcttagcgttggtgatcattgctgcgtgggtgctgatgttaaa4800 agggattcgagcaagaccgtgggggcagcccgcgctttgttgatttaagtcgaacacaat4860 aaagatttaattcagccttcgtttaggttacctctgctaatatctttctcattgagatga4920 aaattaaggtaagcgaggaaacacaccacaccataaacggaggcaaataatgctgggtaa4980 50tatgaatgtttttatggccgtactgggaataattttattttctggttttctggccgcgta5040 tttcagccacaaatgggatgactaatgaacggagataatccctcacctaaccggcccctt5100 gttacagttgtgtacaaggggcctgatttttatgacggcgaaaaaaaaccgccagtaaac5160 cggcggtgaatgcttgcatggatagatttgtgttttgcttttacgctaacaggcattttc5220 ctgcactgataacgaatcgttgacacagtagcatcagttttctcaatgaatgttaaacgg5280 55agcttaaactcggttaatcacattttgttcgtcaataaacatgcagcgatttcttccggt5340 ttgcttaccctcatacattgcccggtccgctcttccaatgaccacatccagaggctcttc5400 aggaaatgcgcgactcacacctgctgtcacggtaatgttgatatgcccttcagaatgtgt5460 gatggcatggttatcgactaactggcaaattctgacacctgcacgacatgcttcttcatc5520 attagccgctttgacaataatgataaattcttcgcccccgtagcgataaaccgtttcgta5580 WO 00/65082 ~ 02370426 2001-10-22 PCT/US00/10829 _;_ atcacgcgtccaactggctaagtaagttgccagggtg aatactacatcgccgattaa5640 ~t atgcccgtagtatcattaaccaatttaaatcggtcaa~atccaacaacattaaataaaga5700 ttcagaggctcagcgttgcgtaactgatgatcaaaggattcatcaagaacccgacgaccc5760 ggcaatcccgtcaaaacatccatattgctacggatcg=cagcaaataaattttgtaatcg5820 S gttaatgccgcagtaaaagaaagcaacccctcctgaaaggcgtcgaaatgcgcgtcctgc5880 cagtgattttcaacaatagccagcattaattcccgaccacagttatgcatatgttgatgg5940 gcagaatccattagccgaacgtaaggtaattcatcgt_atcgagtggccccagatgatca6000 atccaccgaccaaactggcacagtccataagaatggt~atccgttatttctggcttactg6060 gcatctctcgcgaccacgctgtgaaacatactcacca4ccactggtagtgggcatcgata6120 gccttattgagatttaacaagatggcatcaatttccg=tgtcttcttgatcattgccact6180 cctttttcacagttccttgtgcgcgctattctaacga.;agaaaagcaaaattacgtcaat6240 attttcatagaaatccgaagttatgagtcatctctg~~ataacattgtgatttaaaacaa6300 aatcagcggataaaaaagtgtttaattctgtaaattacctctgcattatcgtaaataaaa6360 ggatgacaaatagcataacccaataccctaatggcccagtagttcaggccatcaggctaa6420 tttatttttatttctgcaaatgagtgacccgaacgacggccggcgcgcttttcttatcca6480 gactgccactaatgttgatcatctggtccggctgaac~:ctcgtccatcaaagacggccg6540 caggaataacgacattaatttcaccgctcttatcgcgaaaaacgtaacggtcctctcctt6600 tgtgagaaatcaaattaccgcgtagtgaaaccgaagcgccatcgtgcatggtttttgcga6660 aatcaacggtcattttttttgcatcatcggttccgcca-agccatcttctattgcatgag6720 gcggcggtggcgctgcatcctgttttaaaccgccctg:tcatctgccaacgcataaggca6780 tgacaagaaaacttgctaatacaatggcctgaaatttcatactaactccttaattgcgtt6840 tggtttgacttattaagtctggttgctatttttataa=tgccaaataagaatattgccaa6900 ttgttataaggcatttaaaatcagccaactagctgtcaaatatacagagaatttaactca6960 ctaaagttaagaagattgaaaagtcttaaacatattt-cagaataatcggatttatatgt7020 ttgaaaattattatattggacgagcatacagaaaaagcaaatcacctttacatataaaag7080 cgtggacaaaaaacagtgaacattaatagagataaaattgtacaacttgtagataccgat7140 actattgaaaacctgacatccgcgttgagtcaaagacttatcgcggatcaattacgctta7200 actaccgccgaatcatgcaccggcggtaagttggctagcgccctgtgtgcagctgaagat7260 acacccaaattttacggtgcaggctttgttactttcaccgatcaggcaaagatgaaaatc7320 ctcagcgtaagccagcaatctcttgaacgatattctgcggtgagtgagaaagtggcagca7380 gaaatggcaaccggtgccatagagcgtgcggatgctgatgtcagtattgccattaccggc7440 tacggcggaccggagggcggtgaagatggtacgccagcgggtaccgtctggtttgcgtgg7500 catattaaaggccagaactacactgcggttatgcattttgctggcgactgcgaaacggta7560 ttagctttagcggtgaggtttgccctcgcccagctgc~gcaattactgctataaccaggc7620 tggcctggcgatatctcaggccagccattggtggtgtttatatgttcaagccacgatgtt7680 gcagcatcggcataatcttaggtgccttaccgcgccattgtcgatacaggcgttccagat7740 cttcgctgttacctctggaaaggatcgcctcgcgaaaacgcagcccattttcacgcgtta7800 atccgccctgctcaacaaaccactgataaccatcatcggccaacatttgcgtccacagat7860 aagcgtaataacctgcag 7878 <210> 2 <211> 125 <212> PRT
<213> Escherichia coli <400> 2 Met Val Asn Gln Lys Lys Asp Arg Leu Leu Asn Glu Tyr Leu Ser Pro Leu Asp Ile Thr Ala Ala Gln Phe Lys Val Leu Cys Ser Ile Arg Cys Ala Ala Cys Ile Thr Pro Val Glu Leu Lys Lys Val Leu Ser Val Asp Leu Gly Ala Leu Thr Arg Met Leu Asp Arg Leu Val Cys Lys Gly Trp Val Glu Arg Leu Pro Asn Pro Asn Asp Lys Arg Gly Val Leu Val Lys WO 00/65082 ~ 02370426 2001-10-22 Leu Thr Thr Gly Gly Ala Ala Ile Cys Glu Gln Cys His Gln Leu Val Gly Gln Asp Leu His Gln Glu Leu Thr Lys Asn Leu Thr Ala Asp Glu Val Ala Thr Leu Glu Tyr Leu Leu Lys Lys Val Leu Pro <210> 3 <211> 58 <212> DNA
<213> Escherichia coli <400> 3 taatcaacgg gaccgttcat taatcaacgt cccctattat aacgggtccg ttcatatt 58 <210> 4 <211> 20 <212> PRT
<213> Escherichia coli <400> 4 Lys Lys Val Leu Ser Val Asp Leu Gly Ala Leu Thr Arg Met Leu Asp Arg Leu Val Cys <210> 5 35 <211> 20 <212> PRT
<213> Escherichia coli <400> 5 40 Leu Val Lys Leu Thr Thr Gly Gly Ala Ala Ile Cys Glu Gln Cys His Gln Leu Val Gly <210> 6 <211> 22 <212> PRT
50 <213> Escherichia coli <400> 6 Glu Lys Val Ser Glu Arg Ser Gly Tyr Ser Lys Trp His Leu Gln Arg Met Phe Lys Lys Glu Thr WO 00/65082 ~ 02370426 2001-10-22 pCT/US00/10829 <210> 7 <211> 24 <212> PRT
<213> Escherichia coli <400> 7 Ile Leu Tyr Leu Ala Glu Arg Tyr Gly Phe Glu Ser Gln Gln Thr Leu Thr Arg Thr Phe Leu Asn Tyr Phe <210> 8 15 <211> 20 <212> PRT
<213> Escherichia coli <400> 8 20 Ala Ser His Ile Ser Lys Thr Met Asn Ile Ala Arg Ser Thr Val Tyr Lys Val Ile Asn <210> 9 <211> 20 <212> PRT
<213> Escherichia coli <400> 9 Thr Arg Lys Leu Ala Gln Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys 40 <210> 10 <211> 20 <212> PRT
<213> Escherichia coli 45 <400> 10 Gln Thr Arg Ala Ala Leu Met Met Gly Ile Asn Arg Gly Thr Leu Arg Lys Lys Leu Lys <210> 11 <211> 20 55 <212> PRT
<213> Escherichia coli <400> 11 Gly Ala Glu Leu Lys Asn Glu Leu Gly Ala Gly Ile Ala Thr Ile Thr WO 00/65082 ~ 02370426 2001-10-22 pCT~S00/10829 Arg Gly Ser Asn <210> 12 <211> 22 <212> PRT
10 <213> Escherichia coli <400> 12 Thr Pro Val Glu Leu Lys Lys Val Leu Ser Val Asp Leu Gly Ala Leu Thr Arg Met Leu Asp Arg 20 <210> 13 <211> 30 <212> DNA
<213> Escherichia coli <400> 13 tgcctcccgt atattacggt gggcacccgt 3C
<210> 14 <211> 30 <212> DNA
<213> Escherichia coli <400> 14 acggagggca tataatgcca cccgtactaa 30 <210> 15 <211> 63 <212> DNA
<213> Escherichia coli <400> 15 ttgacttata cttgcctggg caatattatc ccctgcaact aattacttgc cagggcaact 60 aat 63

Claims (29)

What is claimed is:

1. A method for identifying a compound that modulates MarR family polypeptide activity or expression, comprising:
contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate the activity or expression of the MarR
family polypeptide to thereby identify a compound that modulates MarR family polypeptide activity or expression.

2. The method of claim 1, wherein the ability of the compound to modulate MarR
family polypeptide activity is detected.

3. The method of claim 1, wherein the ability of the compound to modulate MarR
family polypeptide expression is detected.

4. A method for identifying a compound that modulates the ability of a compound to modulate the ability of a MarR family polypeptide to interact with a MarR
binding partner, comprising:
contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate the ability of the MarR
family polypeptide to interact with a MarR binding partner to thereby identify a compound that modulates the ability of a MarR family polypeptide to interact with a MarR
binding partner.

5. The method of claim 4, wherein the MarR binding partner is a DNA molecule.

6. The method of claim 4, wherein the MarR binding partner is a polypeptide.

7. A method for identifying a compound that modulates MDR, comprising:
contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate MDR to thereby identify a compound that modulates MDR.

8. The method of claim 4, wherein said MarR family polypeptide is expressed in a cell.

9. The method of claim 4, wherein said MarR family polypeptide is an isolated polypeptide.

10. The method of claim 4, wherein the DNA binding activity of the MarR family polypeptide is measured by detecting transcription from a gene locus regulated by a MarR family polypeptide.

11. The method of claim 4, wherein the MarR family polypeptide is derived from a protein selected from the group consisting of: MarR, Ec17kd, MprA(EmrR), and MexR.

12. The method of claim 11, wherein the MarR family polypeptide is an E. coli MarR polypeptide.

13. The method of claim 4, wherein the MarR family polypeptide comprises a MarR
family polypeptide helix-turn-helix domain corresponding to about amino acids 61-80 or about 97-116 of MarR.

14. The method of claim 4, wherein the polypeptide comprises an amino acid sequence corresponding to about amino acid 1 to about amino acid 41 of MarR.

15. The method of claim 4, wherein the polypeptide comprises an amino acid sequence corresponding to about amino acid 41 to about amino acid 144 of MarR.

16. The method of claim 4, wherein the step of detecting the MarR family polypeptide activity comprises detecting transcription from a marRAB
responsive promoter.

17. The method of claim 11, wherein the step of detecting comprises detecting the ability of the compound to modulate the binding of MarR to marO.

18. The method of claim 16, wherein the marRAB responsive promoter is PmarII.

19. The method of claim 16, wherein the marRAB responsive promoter is linked to a reporter gene.

20. The method of claim 19, wherein the reporter gene is selected from the group consisting of lacZ, phoA, or green fluorescence protein.

21. The method of claim 19, wherein the step of detecting comprises detecting the amount of reporter gene product produced by the cell.

22. The method of claim 16, wherein the step of detecting comprises detecting the amount of RNA produced by the cell.

23. The method of claim 16, wherein the step of detecting comprises detecting the activity of the reporter gene product.

24. The method of claim 16, wherein the step of detecting comprises detecting the ability of an antibody to bind to the reporter gene product.

25. A method for identifying a compound that modulates MDR, comprising:
screening a library of bacteriophage displaying on their surface a MarR
polypeptide, the polypeptide sequence being specified by a nucleic acid molecule contained within the bacteriophage, for the ability to bind a compound to obtain those compounds having affinity for the MarR polypeptide; said method comprising:
contacting the phage which displays the MarR polypeptide with a compound so that the polypeptide can form a complex with a compound having an affinity for the polypeptide;
contacting the complex of the polypeptide and bound compound with an agent that dissociates the bacteriophage from the compound; and identifying the compounds that bound to the polypeptide to thereby identify a compound that modulates MDR.

26. The method of claim 4, wherein the compound is an antibiotic compound.

27. The method of claim 4, wherein the compound is non-antibiotic compound.

28. The method of claim 27, wherein the compound is a candidate disinfectant or antiseptic compound.

29. The method of claim 4, wherein the compound is derived from a library of compounds.