CN103060224B - Space enterococcus faecium LCT-EF258 strain - Google Patents
Space enterococcus faecium LCT-EF258 strain Download PDFInfo
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Abstract
The invention relates to the biological characteristics, the genome DNA (deoxyribonucleic acid) sequence, the transcriptome and the differential proteomics of an enterococcus faecium LCT-EF258 strain in a space environment, and particularly relates to the effects of the functional sequence and molecules identified by performing comparative analysis with ground enterococcus faecium on micro-organisms in the predetermined space environment, and the applications of the functional sequence and molecules, wherein the comprehensive and accurate mutant genes, differential expression genes and proteins of the enterococcus faecium strain in the space environment can be obtained.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to faecium and biological characteristics, full-length genome order-checking, transcript profile order-checking and Discrepancy proteome Epidemiological Analysis.
Background technology
Along with mankind's solar-system operation is increasing and China's manned space program is implemented gradually, the health of spacefarer is the key issue that aerospace medicine ensures, wherein prevents bacteriological infection to be an importance.Studies have found that Salmonellas can strengthen the virulence of mouse infection under simulate microgravity, the immunity degradation of body in solar-system operation simultaneously, thus cause more easy infection.At present, having detected the various bacteria comprising faecalis in space station, is therefore an important topic of space microbe research for the research of space bacterial dissociation.At present, about the resource of space mutagenesis microorganism lacks very much, the development in this field is seriously hindered.
Faecium is considered to belong to D group streptococcus in early days and belongs to, and along with the development of the every technology of molecular biology, is detected, find that it belongs to enterococcus spp by the homology of gene DNA.Faecalis is a kind of normal microflora in human body, but can cause a disease equally when body immunity declines, therefore be a kind of conditionality pathogenic bacterium, multiple system often can be caused to infect, such as human reproductive system, urinary system, abdominal cavity infection, wound show to infect, and are also one of common floras causing ward infection simultaneously.In recent years, there is the enforcement of the inspection of wound property and treatment means and immunosuppressor with antibiotic a large amount of use along with various, enterococcal infection is got more and more.Due to this kind of normal natural drug resistance of flora, all have resistance to cynnematin, Macrolide, sulfamido, aminoglycoside, the faecalis strain in recent years tolerating vancomycin increases, and makes doctor usually at one's wit's end clinically.Research at present in intractable Enterococcus faecium infections has entered the bottleneck phase, does not well study point of penetration.
To the research that faecium related biological proterties (as virulence and resistance etc.) under steric requirements changes, the molecule mechanism analyzing its change is the effective means solving resistance problems in bacteriological infection and clinical treatment in solar-system operation.In recent years, the systems biology research meanses such as genome, transcript profile, protein science and information biology constantly occur, make to carry out the molecular mechanism research of faecium sudden change under space environment and more easily carry out.Can be space Biosafety assessment on the one hand to provide fundamental basis, the research that can be clinical Difficult infection on the other hand provides thinking.
Summary of the invention
One object of the present invention provides the faecium LCT-EF258 bacterial strain of a strain space environment
This bacterial strain provided by the invention, its preserving number is CGMCC NO.6525.
One Enterococcus faecalis, Gram-positive, in single, chain, agglomerate arrangement; Antibiotic resistance with ground contrast strain is compared, and more tolerates microbiotic amikacin (amikacin), Ciprofloxacin; Without the difference of the speed of growth; γ-aminobutyric acid and L MALIC ACID can be utilized to be carbon source, and can not p-hydroxyphenylaceticacid to be utilized.
Described faecium LCT-EF258 bacterial strain, carry out genome sequencing and Comparative genomic strategy Epidemiological Analysis draws mutator gene, in table 1, space environment causes the change of SNP in LCT-EF258 strain gene group, comprise the position of SNP on genome, mutational site and proteins encoded and the gene annotated out; Table 2 causes insertion in LCT-EF258 strain gene group and disappearance for space environment, comprises the region of InDel position and generation.
Table 1 space environment causes LCT-EF258 strain gene group is isozygotied the change of SNP
SNP position mutating alkali yl is with reference to gene I/D annotation database ID mutain Scaffold3_60853 C → A LCT-EF90GL001983 gi|293570275 S → I
Table 2 space environment causes insertion in LCT-EF258 strain gene group and disappearance
Described faecium LCT-EF258 bacterial strain, carry out transcript profile order-checking draw difference expression gene (FDR≤0.001 He | log
2ratio|>=2) see attached list 1.
Described faecium LCT-EF258 bacterial strain, carries out Discrepancy proteome Epidemiological Analysis and show that differentially expressed protein molecule is (when protein abundance fold differences is more than more than 2 times; 0.05 is less than through its p-value value of statistical test; Above-mentioned requirements is reached at least twice repetition in repeating for three times) see attached list 2.
Accompanying drawing explanation
Fig. 1 faecium LCT-EF258 bacterial strain gramstaining result
Fig. 2 faecium LCT-EF258 bacterial strain Physiology and biochemistry qualification result
Fig. 3 faecium LCT-EF258 strain growth curve
Fig. 4 faecium LCT-EF90 strain growth curve
The GC content of Fig. 5 LCT-EF258 bacterial strain and Depth association analysis figure
The two-dimentional collinearity figure of Fig. 6 LCT-EF258 bacterial strain and ground control group LCT-EF90
The transcript profile sequenced genes coverage figure of Fig. 7 LCT-EF258 bacterial strain
The difference expression gene quantity of Fig. 8 transcript profile order-checking qualification
The differential expression protein quantity of Fig. 9 Differential Proteomic qualification
Annex explanation
The difference expression gene information of annex 1 faecium LCT-EF258 bacterial strain transcript profile sequencing analysis
The differentially expressed protein information that annex 2 faecium LCT-EF258 bacterial strain quantitative proteomics is analyzed
Embodiment
Experimental technique used in following embodiment, if no special instructions, is ordinary method.Material used in following embodiment, reagent, if no special instructions, all can obtain from commercial channels.
Embodiment one: the gramstaining of faecium LCT-EF258 bacterial strain
Get 50ul sample centrifugal, abandon supernatant, add sterilized water 50ul, draw 20ul and be fixed on slide glass; First dye, draws gramstaining I (Viola crystallina) 2 and carries out just contaminating 1min in slide specimen, then use tap water dye liquor; Mordant dyeing, draws gramstaining II (iodine liquid) 2 covering painting face and contaminates about 1min, then use tap water dye liquor, draw the moisture on painting face with thieving paper; Decolouring, draws gramstaining III (alcohol) 2 and drops in about 30s on painting face, rinse slide glass with water, draw the moisture on painting face with thieving paper; Redye, draw gramstaining IV (sarranine) 2 and drop in about 1min on painting face, rinse slide glass with water, draw the moisture on painting face with thieving paper; Observe, first find the target visual field with microscope low power lens, and then observe with oily mirror (100 ×), judge and record result, take pictures, seeing Fig. 1, Gram-positive, in single, chain, agglomerate arranges.
Embodiment two: the antibiotics sensitivity of faecium LCT-EF258 bacterial strain detects
Preparation LB solid medium 25L, falls 1500 flat boards after bacterium of having gone out, dry, for subsequent use; The preparation of bacteria suspension, takes out the inclined-plane of these single bacterium, and prepares the 1.5ml centrifuge tube filling 1ml stroke-physiological saline solution, dips proper amount of strains in 1.5ml centrifuge tube, mixing, adjust bacteria concentration about 10 with the bamboo let of sterilizing from inclined-plane
7~ 10
8; Dilution spread, draws the bacteria suspension 100 μ l got ready and is coated with, and evenly, every strain list bacterium is coated with 6 flat boards, is often painted with a strain bacterium in coating as much as possible, just carries out next step (pasting susceptibility sheet); Paste susceptibility sheet, select microbiotic to carry out drug sensitive test, to filter out the bacterial strain of drug-resistant mutation; After having pasted susceptibility sheet, at flat board being placed on 37 DEG C, cultivate 18 ~ 24h; Observations, by slat chain conveyor 18 ~ 24h, take out the antibacterial situation of observing dull and stereotyped upper susceptibility sheet, and record the size (diameter cm) of inhibition zone, wherein susceptibility sheet diameter is about 0.6cm.To amikacin (amikacin) and Ciprofloxacin tolerance, without inhibition zone, and the inhibition zone of ground control group is respectively 1.2cm and 1.4cm.
Embodiment three: the Physiology and biochemistry identification experiment of faecium LCT-EF258 bacterial strain
Collecting cells, takes out the inclined-plane of bacterial strain, dips strain inoculation (4ml system) in LB liquid nutrient medium with sterilizing bamboo let, cultivates 24h.Then get the centrifugal 5min of 2ml bacterium liquid 6000r/s, abandon supernatant substratum, leave centrifugal thalline, then the brine thalline of sterilizing is used, the centrifugal 5min of 6000r/s, supernatant discarded again, carry out suspension thalline by Biolog inoculation liquid again, finally go in the inoculation liquid of 15ml again.Be adjusted to concentration about 10
7~ 10
8; Inoculation, by above bacteria suspension down in inoculation ware, concussion is evenly, application of sample is carried out, each Biolog plate hole application of sample 100ul, totally 96 holes with 12 roads (or the 8 roads) volley of rifle fire, after adding sample, cover Biolog plate lid, cultivate at being placed on 37 DEG C, 24h and 48h observes, and record result, see Fig. 2 and table 3, can find out that from figure neutralization table essential difference is that LCT-EF258 can utilize γ-aminobutyric acid and L MALIC ACID to be carbon source, and can not p-hydroxyphenylaceticacid be utilized.
The Physiology and biochemistry qualification result of table 3:LCT-EF258 bacterial strain
| Numbering | Substrate | LCT-EF258 bacterial strain | Contrast LCT-EF90 bacterial strain |
| B8 | N-acetyl-D-galactosamine | +/- | - |
| C8 | L-rhamnosyl | +/- | - |
| D4 | Inositol | +/- | - |
| E9 | Serine | - | +/- |
| F3 | Galactonolactone | +/- | - |
| F4 | Maltonic acid | - | +/- |
| F6 | Glucuronamide | - | +/- |
| G1 | P-Hydroxy-phenyl-acetic acid | - | + |
| G3 | D-ALPHA-Hydroxypropionic acid methyl esters | +/- | - |
| G5 | Citric acid | - | +/- |
| G8 | L MALIC ACID | + | - |
| H2 | γ-aminobutyric acid | + | - |
Embodiment four: the growth curve of faecium LCT-EF258 bacterial strain
Activated strains, cultivates 1d, is then seeded in 100 hole perforated plates, carries out the mensuration of growth curve in Bioscreen in 15ml test tube (4ml substratum), measures 24h, reading of data; The calculating of the value that the OD value that every strain bacterium obtains is averaged, and then curve plotting, be shown in Fig. 3 and Fig. 4, as can be seen from Figure under space environment LCT-EF258 bacterial strain with ground LCT-EF90 strain growth curve indifference.
Embodiment five: the gene order-checking of faecium LCT-EF258 bacterial strain and icp gene group analysis
Cultivate faecium and extract genomic dna, first ultrasonic method is adopted the qualified DNA sample of detection to be interrupted at random, obtain a series of DNA fragmentation, after the process such as T4DNA polysaccharase, Klenow archaeal dna polymerase and T4 polynueleotide kinase, reclaim object fragment, obtain sequencing library, utilize the Genome Analyzer II of Illumina to check order; The reads obtained after order-checking terminates, carries out assembling and analyzing after process after filtration; Adopt Glimmer3.0 software annotation gene and analyze SNP, Indel, gene rearrangement and structure variation etc.The SNP sudden change of isozygotying found is in table 1, and Indel analyzes and do not find considerable change.Fig. 5 is shown in by Quality Control and GC content and the Depth association analysis statistical graph of order-checking, and wherein X-coordinate is GC content, and ordinate zou is mean depth.Take 500bp as window without calculating its GC content and mean depth of repeating, this scatter diagram is similar to Poisson's distribution, namely in sequencing procedure GC without obvious skewed popularity.The two-dimentional collinearity figure seeing Fig. 6 is analyzed in collinearity and rearrangement, in figure, transverse axis is reference sequences genome, the longitudinal axis is institute's cls gene group, the level that color is more shallow or vertical straight line represent the segmentation between each Scaffold, red lines represent that the nucleotide sequence of institute's cls gene group is in normal chain, and the nucleotide sequence of blue expression institute cls gene group is in minus strand.
Embodiment six: the transcript profile sequencing analysis of faecium LCT-EF258 bacterial strain
After extracting faecium LCT-EF258 bacterial strain total serum IgE, after removing rRNA with test kit.Add fragmentation buffer and mRNA is broken into short-movie section, take mRNA as template, Article 1 cDNA chain is synthesized with hexabasic base random primer (random hexamers), then add damping fluid, dNTPs, RNase H and DNA polymerase I synthesizes Article 2 cDNA chain, doing end reparation after adding EB buffer solution elution through QiaQuick PCR kit purifying, add polyA and connect sequence measuring joints, then clip size selection is carried out with agarose gel electrophoresis, finally carry out pcr amplification, the sequencing library Illumina HiSeq built up
tM2000 check order.Add up rRNA statistics after original sequence data being removed impurity data, evaluate the distribution gene coverage of Reads on genome, add up gene expression amount difference, analyze difference expression gene etc., in table 3, table 4, Fig. 7 and Fig. 8, difference expression gene for details see attached table 1, down-regulated gene 497, up-regulated gene 20.Fig. 7 is that gene sequencing coverage refers to the per-cent that each gene is covered by reads, and its value equals the ratio of base number with all base numbers in gene coding region of unique mapping reads covering in gene.
Table 3: sample LCT-EF258 and the statistics with reference to genome alignment
Table 4: sample LCT-EF258 and the statistics with reference to gene comparison
Above table each row implication is as follows:
Embodiment seven: the Discrepancy proteome Epidemiological Analysis of faecium LCT-EF258 bacterial strain
Albumen is extracted from faecium LCT-EF258 sample; Carry out reductive alkylation process to the protein sample after extracting, opened disulfide bond is so that follow-up abundant enzymolysis protein; The concentration determination of protein is carried out by the 2D quant kit method of GE company; Equal-volume carries out SDS (sodium laurylsulfonate) electrophoresis; Enzymolysis protein; By iTRAQ reagent mark peptide section; Peptide section after mark is carried out balanced mix; Strong cation exchange chromatography (Strong Cation Exchange Choematography, SCX) is used to carry out pre-separation to mixed peptide section; Carry out liquid phase tandem mass spectrum (liquid chromatography coupled with tandem mass spectrometry, LC-MS/MS) analysis.To the source document of machine under mass spectrum, carry out peak identification, obtain peak list; Set up reference database, carry out the qualification of peptide section and protein; Finally the relation of the relative content of more each albumen between each sample is (when protein abundance fold differences is more than more than 2 times; 0.05 is less than through its p-value value of statistical test; Above-mentioned requirements is reached at least twice repetition in repeating for three times), thus obtain some interested important albumen, differential protein for details see attached table 2, raises down-regulation protein statistics and sees Fig. 9, upregulated protein 25, down-regulation protein 57.
Described faecium LCT-EF258 bacterial strain, according to the change of antibiotic resistance and molecule mechanism, can be applied to screening and identification clinical antibiotics resistance target spot, for the diagnosis and treatment product developing clinical intractable Enterococcus faecium infections provides reference.
About the explanation of the LCT-EF258 bacterial strain of preservation
A. depositary institution's title of bacterial classification and address
Title: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica
B. the date of Culture Collection is handed over
On September 5th, 2012
C. the preserving number that gives of preservation mechanism
CGMCC No.6525
D. Classification And Nomenclature faecium Enterococcus Faecium
Annex 1
Annex 2
Claims (1)
1. a space faecium LCT-EF258, its preserving number is: CGMCC NO.6525, is characterized in that: Gram-positive, in single, chain, agglomerate arrangement; Antibiotic resistance with ground contrast strain is compared, and more tolerates microbiotic amikacin, Ciprofloxacin; Without the difference of the speed of growth; γ-aminobutyric acid and L MALIC ACID can be utilized to be carbon source, and can not p-hydroxyphenylaceticacid to be utilized.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0508701A2 (en) * | 1991-04-08 | 1992-10-14 | Quest International B.V. | Probiotic |
| CN101629155A (en) * | 2009-04-30 | 2010-01-20 | 邵峰 | Enterococcus faecium |
| CN102031253A (en) * | 2010-12-16 | 2011-04-27 | 复旦大学 | Molecular marker method for identifying indica type rice and japonica rice by using rice grain |
| US20110104134A1 (en) * | 2008-07-04 | 2011-05-05 | Nippon Meat Packers, Inc. | Novel lactic acid bacterium having high immunoglobulin-a-inducing ability |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0508701A2 (en) * | 1991-04-08 | 1992-10-14 | Quest International B.V. | Probiotic |
| US20110104134A1 (en) * | 2008-07-04 | 2011-05-05 | Nippon Meat Packers, Inc. | Novel lactic acid bacterium having high immunoglobulin-a-inducing ability |
| CN101629155A (en) * | 2009-04-30 | 2010-01-20 | 邵峰 | Enterococcus faecium |
| CN102031253A (en) * | 2010-12-16 | 2011-04-27 | 复旦大学 | Molecular marker method for identifying indica type rice and japonica rice by using rice grain |
Non-Patent Citations (1)
| Title |
|---|
| 屎肠球菌活菌制剂的研究;袁杰利 等;《中国微生态学杂志》;20030230;第15卷(第1期);全文 * |
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