CN102994414B - Klebsiella pneumoniae LCT-KP289 strain in space environment - Google Patents
Klebsiella pneumoniae LCT-KP289 strain in space environment Download PDFInfo
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- CN102994414B CN102994414B CN201210380139.4A CN201210380139A CN102994414B CN 102994414 B CN102994414 B CN 102994414B CN 201210380139 A CN201210380139 A CN 201210380139A CN 102994414 B CN102994414 B CN 102994414B
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Abstract
The invention relates to biological characteristics, genomic DNA sequences, transcriptome and differential proteomics of Klebsiella pneumoniae LCT-KP289 strain in space environment, and in particular relates to functions and application of functional sequences and molecules analyzed and identified through comparison with Klebsiella pneumoniae in ground to microorganisms in space environment. According to the invention, complete and accurate mutant genes are obtained through space mutation of Klebsiella pneumoniae strain, differential expression genes and proteins.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to Klebsiella pneumonia under space environment biological characteristics, genome sequencing information, transcribe group order-checking and Discrepancy proteome Epidemiological Analysis.
Background technology
The multiple organ physiological functions of human body in space flight process all can be affected, and the resident personnel's in space function of immune system declines, and is more easily subject to the infection of pathogenic bacteria; In addition, virulence and pathogenic may change of bacterium in space environment, environment resistibility strengthens and antibiotic susceptibility is declined to external world.Therefore, understanding space environment to the particularly impact of opportunistic pathogen of bacterium, and formulate on this basis effective safeguard procedures, is very important to the health that ensures the resident personnel in space.Meanwhile, the relevant achievement of feature that under space environment, bacterium changes can be applicable to ground.
Klebsiella Pneumoniae, claims again pneumobacillus, is that one is distributed widely in one of natural gram-negative, is the normal microflora of human body.In the patient who accepts invasive diagnosis and treatment or immunologic hypofunction, often can cause respiratory tract infection, urinary tract infections, abdominal cavity infection, septicemia, purulent meningitis, skin soft-tissue infection and wound infection etc., be to the pathogenic stronger significant opportunity pathogenic bacterium of people and nosocomial infection bacterium in enterobacteriaceae klebsiella spp.Along with the extensive application of extensive pedigree antibiotic clinically, its recall rate and resistant rate are in rising trend in recent years.
Genome based on information biology, to transcribe the research that the group such as group, protein groups learns more and more, are the better points of penetration that discloses various biological phenomena generation molecule mechanisms.The effect of method research space environment by group to Klebsiella pneumonia, lays a good foundation for bacterial dissociation mechanism under Study Sky environment, is conducive to carry out the Biosafety assessment in space, simultaneously for the research of intractable infection on ground provides new thinking.
Summary of the invention
One object of the present invention provides the Klebsiella pneumonia LCT-KP289 bacterial strain of space environment mutagenesis
This bacterial strain provided by the invention, its preserving number is CG MCC NO.6520.
One Klebsiella pneumoniae, its feature comprises: Gram-negative, is single, rod-short; Antibiotic resistance with ground contrast strain is compared, and trimethoprim-sulfamethoxazole is more tolerated; Without the difference of the speed of growth; Can not utilize N-acetyl-neuraminate and PEARLITOL 25C to carry out metabolism, and control group can utilize.
Described Klebsiella pneumonia, utilize genome sequencing and comparative genomics analysis to obtain transgenation, in table 1, space environment causes the variation of SNP on LCT-KP289 genome, comprise the position of SNP on genome, mutational site and proteins encoded and the gene annotating out.
Table 1: space environment causes the variation of SNP on LCT-KP289 genome
Described Klebsiella pneumonia LCT-KP289 bacterial strain, transcribes group order-checking qualification difference expression gene (FDR≤0.001 He | log2Ratio| >=2) and sees attached list 1.
Described Klebsiella pneumonia LCT-KP289 bacterial strain, carries out the differentially expressed protein of Differential Proteomic Analysis and Identification (when albumen abundance difference multiple exceedes more than 2 times; Check by statistics its p-value value to be less than 0.05; In repeating for three times, at least twice repetition, reach above-mentioned requirements) see attached list 2.
Brief description of the drawings
Fig. 1 Klebsiella pneumonia LCT-KP289 bacterial strain gramstaining
The qualification of Fig. 2 Klebsiella pneumonia LCT-KP289 bacterial strain Physiology and biochemistry
Fig. 3 Klebsiella pneumonia LCT-KP289 strain growth curve
Fig. 4 Klebsiella pneumonia LCT-KP214 strain growth curve
The GC content of Fig. 5 LCT-KP289 bacterial strain and Depth association analysis figure
The two-dimentional collinearity figure of Fig. 6 LCT-KP289 bacterial strain and ground control group LCT-KP214
Sequenced genes coverage figure is organized in transcribing of Fig. 7 LCT-KP289 bacterial strain
Fig. 8 transcribes the difference expression gene of group order-checking qualification
The differential expression protein of Fig. 9 Differential Proteomic qualification
Annex explanation
Annex 1LCT-KP289 bacterial strain is transcribed the difference expression gene information of group sequencing analysis
The differentially expressed protein information that annex 2LCT-KP289 bacterial strain quantitative proteomics is analyzed
Embodiment
Experimental technique used in following embodiment, if no special instructions, is ordinary method.
Material, reagent used in following embodiment, if no special instructions, all can obtain from commercial channels.
Embodiment one: the gramstaining of Klebsiella pneumonia LCT-KP289 bacterial strain
Get 50ul sample centrifugal, abandon supernatant, add sterilized water 50ul, draw 20ul and be fixed on slide glass; Just dye, draw gramstaining I (Viola crystallina) 2 and in slide specimen, carry out just dying 1min, then rinse dye liquor with tap water; Mordant dyeing, draws 2 covering painting faces of gramstaining II (iodine liquid) and dyes about 1min, then rinses dye liquor with tap water, with the moisture on thieving paper absorption painting face; Decolouring, draws gramstaining III (alcohol) 2 and drops in about 30s on painting face, and water rinses slide glass, with the moisture on thieving paper absorption painting face; Redye, draw gramstaining IV (sarranine) 2 and drop in about 1min on painting face, water rinses slide glass, with the moisture on thieving paper absorption painting face; 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, see Fig. 1, LCT-KP289 bacterial strain Gram-negative as can be seen from Figure, is single, rod-short form.
Embodiment two: the antibiotics sensitivity of Klebsiella pneumonia LCT-KP289 bacterial strain detects
Preparation LB solid medium 25L, falls 1500 flat boards after the 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 to fill the 1.5ml centrifuge tube of 1ml stroke-physiological saline solution, dips proper amount of strains in 1.5ml centrifuge tube with the bamboo let of sterilizing from inclined-plane, mixes tune bacteria concentration approximately 107～108; Dilution spread, draws the bacteria suspension 100 μ l that get 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; Paste after susceptibility sheet, flat board is placed at 37 DEG C and cultivates 18～24h; Observations, cultivates 18～24h by flat board, takes out the antibacterial situation of observing dull and stereotyped upper susceptibility sheet, and records the size (diameter cm) of inhibition zone, and wherein susceptibility sheet diameter is about 0.6cm.Klebsiella pneumonia LCT-KP289 compares with ground contrast LCT-KP214 bacterial strain, and trimethoprim-sulfamethoxazole is more tolerated, and LCT-KP289 is without the formation of inhibition zone, and LCT-KP214 bacterial strain inhibition zone reaches 1.8cm.
Embodiment three: the Physiology and biochemistry identification experiment of Klebsiella pneumonia LCT-KP289 bacterial strain
Collecting cells, the inclined-plane of taking-up bacterial strain, dips bacterial classification with sterilizing bamboo let and is seeded in (4ml system) in LB liquid nutrient medium, cultivates 24h.Then get the centrifugal 5min of 2ml bacterium liquid 6000r/s, abandon supernatant substratum, leave centrifugal thalline, then wash thalline, the centrifugal 5min of 6000r/s, supernatant discarded again with the physiological saline of sterilizing, with the Biolog inoculation liquid thalline that suspends, finally go to again in the inoculation liquid of 15ml again.Be adjusted to concentration approximately 10
7～10
8; Inoculation, by above bacteria suspension, to inoculating in ware, concussion is evenly, Yong12 road (or the 8 roads) volley of rifle fire carries out application of sample, each Biolog plate hole application of sample 100ul, totally 96 holes, add after sample, cover Biolog plate lid, be placed at 37 DEG C and cultivate, 24h and 48h observe, and record result, see Fig. 2 and table 2, can find out that LCT-KP289 bacterial strain can not utilize N-acetyl-neuraminate and PEARLITOL 25C to carry out metabolism.
The Physiology and biochemistry qualification result of table 2:LCT-KP289 bacterial strain
| Numbering | Substrate | LCT-KP289 bacterial strain | Ground control strain |
| A4 | D-trehalose | +/- | + |
| B9 | N-acetyl-neuraminate- | + | |
| D2 | PEARLITOL 25C- | + | |
| D4 | Inositol | +/- | + |
| F7 | Tetrahydroxyadipic acid | + | +/- |
| F9 | D-saccharic acid | + | +/- |
| G6 | α-ketoglutaric acid | +/- - |
Embodiment four: the growth curve of Klebsiella pneumonia LCT-KP289 bacterial strain
Activation bacterial strain is cultivated 1d in 15ml test tube (4ml substratum), is then seeded in 100 hole perforated plates, carries out the mensuration of growth curve in Bioscreen, measures 24h, reading of data; The calculating of OD value value of averaging that every strain bacterium is obtained, and then do curve, see Fig. 3 and Fig. 4, LCT-KP289 bacterial strain is compared indifference with control strain LCT-KP214.
Embodiment five: the hemolytic experiment of Klebsiella pneumonia LCT-KP289 bacterial strain
Employing dibbling method on blood agar culture-medium, comprises LCT-KP289 bacterial strain and ground control strain under space condition by the dibbling of LCT-KP289 bacterial strain simultaneously on each blood agar; 37 DEG C, inversion is observed haemolysis result after cultivating 24-48h, and result shows that LCT-KP289 bacterial strain does not all occur haemolysis with contrast LCT-KP214.
Embodiment six: the gene order-checking of LCT-KP289 bacterial strain and icp gene group analysis
Cultivate Klebsiella pneumonia and extract genomic dna, first adopting ultrasonic method to interrupt at random detecting qualified DNA sample, obtaining a series of DNA fragmentations, through T4DNA polysaccharase, Klenow
After archaeal dna polymerase and T4 polynueleotide kinase etc. are processed, reclaim object fragment, obtain sequencing library, utilize the Genome Analyzer ll order-checking of Illumina; The reads obtaining after order-checking finishes, assembles and analyzes after processing after filtration; Adopt Glimmer3.0 software annotation gene and analyze SNP, Indel, gene rearrangement and structure variation etc.The SNP finding changes gene in table 1, and Indel analyzes and do not find considerable change.The Quality Control of order-checking is that Fig. 5 is shown in by GC content and Depth association analysis statistical graph, and wherein X-coordinate is GC content, and ordinate zou is mean depth.Taking 500bp as window, without calculating its GC content and mean depth of repeating, this scatter diagram is similar to Poisson's distribution, i.e. in order-checking process, GC is without obvious skewed popularity.The two-dimentional collinearity figure that sees 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 cutting apart between each Scaffold, the nucleotide sequence that red lines represent institute's cls gene group is in normal chain, and the blue nucleotide sequence that represents institute's cls gene group is in minus strand.
Embodiment seven: Klebsiella pneumonia LCT-KP289 bacterial strain transcribe group sequencing analysis
Extract after the total RNA of Klebsiella pneumonia LCT-KP289 bacterial strain, with after test kit removal rRNA.Add fragmentation buffer that mRNA is broken into short-movie section, taking mRNA as template, with the synthetic Article 1 cDNA chain of hexabasic base random primer (random hexamers), then add damping fluid, dNTPs, RNase H and DNA polymerase l to synthesize Article 2 cDNA chain, do end reparation, add polyA and connect sequence measuring joints at process QiaQuick PCR test kit purifying and after adding EB buffer solution elution, then carry out clip size selection with agarose gel electrophoresis, finally carry out pcr amplification, the sequencing library of building up Illumina HiSeq
tM2000 check order.After original series data are removed to impurity data, add up rRNA statistics, evaluate distribution gene coverage, the statistics gene expression amount difference of Reads on genome, analyze difference expression gene etc., in table 3, table 4, Fig. 7 and Fig. 8, difference expression gene for details see attached table 1, meet FDR≤0.001 He | 99 of the up-regulated genes of log2Ratio| >=2 condition, 1939 of down-regulated genes.Fig. 7 is that gene sequencing coverage refers to the per-cent that each gene is covered by reads, and its value equals the base number of unique mapping reads covering in gene with the ratio of all base numbers in gene coding region.
Table 3: sample LCT-KP289 and the statistics with reference to genome alignment
Table 4: sample LCT-KP289 and the statistics with reference to gene comparison
The each row implication of above table is as follows:
Embodiment eight: the Discrepancy proteome Epidemiological Analysis of Klebsiella pneumonia LCT-KP289 bacterial strain
From Klebsiella pneumonia LCT-KP289 sample, extract albumen; Protein sample after extracting is carried out to reductive alkylation processing, and opened disulfide bond is so that follow-up abundant enzymolysis protein; Carry out the concentration determination of protein 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 to balanced mix; Use strong cation exchange chromatography (Strong Cation ExchangeChoematography, SCX) to carry out pre-separation to mixed peptide section; Carry out liquid phase tandem mass spectrum (liquidchromatography 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; The finally relation of the relative content of more each albumen between each sample, thereby obtain some interested important albumen, the protein molecular that upper mediation is lowered is shown in lower subordinate list 2, raise down-regulation protein statistics and see Fig. 9, statistics when exceeding more than 2 times for albumen abundance difference multiple; Check by statistics its p-value value to be less than 0.05; In repeating for three times, at least twice repetition, meet above-mentioned requirements, result shows that 35 protein expressions increase, and 114 expressing quantities decline.
About the explanation of the LCT-KP289 bacterial strain of preservation
A. depositary institution's title and the address of bacterial classification
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. hand over the date of mechanism's preservation
On September 5th, 2012
C. the preserving number that preservation mechanism gives
CGMCC No.6520
D. strain classification name: Klebsiella pneumonia Klebsiella pneumoniae
Annex 1
Annex 2
Claims (1)
1. a Klebsiella pneumonia LCT-KP289, its preserving number is: CGMCC NO.6520, is characterized in that: Gram-negative, is single, rod-short; Antibiotic resistance with ground contrast strain is compared, and trimethoprim-sulfamethoxazole is more tolerated; Without the difference of the speed of growth; Can not utilize N-acetyl-neuraminate and PEARLITOL 25C to carry out metabolism, and control group can utilize.
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Non-Patent Citations (6)
| Title |
|---|
| Antibiotic efficacy and microbial virulence during space flight;Klaus D.M.等;《Trends in Biotechnology》;20060207;第24卷(第3期);第131-136页 * |
| Klaus D.M.等.Antibiotic efficacy and microbial virulence during space flight.《Trends in Biotechnology》.2006,第24卷(第3期),第131-136页. * |
| 失重或模拟失重对微生物的生物学效应研究进展;薛小平等;《中华航空航天医学杂志》;20100830;第21卷(第2期);第144-148页 * |
| 空间环境对13株微生物生物学特性的影响;谢琼等;《中华医院感染学杂志》;20041120;第14卷(第11期);第1221-1224页 * |
| 薛小平等.失重或模拟失重对微生物的生物学效应研究进展.《中华航空航天医学杂志》.2010,第21卷(第2期),第144-148页. * |
| 谢琼等.空间环境对13株微生物生物学特性的影响.《中华医院感染学杂志》.2004,第14卷(第11期),第1221-1224页. * |
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