核心多糖结构变异对大肠杆菌自凝集特性的影响
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摘要
以大肠杆菌K-12菌株为研究对象,通过对比分析野生型W3110及脂多糖分子核心多糖结构变异菌株的自凝集特性,对细胞膜上核心多糖结构与细菌自凝集的相关性进行分析。结果表明,核心多糖的结构变异会增加大肠杆菌的自凝集能力,在4℃下静置12 h,突变菌株自凝集率较野生型菌株提高近3倍。以核心多糖缺失突变菌株ΔwaaC和外核心糖缺失突变菌株ΔwaaG为研究对象,进行菌株自凝集动力曲线测定。结果表明,在静置2 h后,菌株自凝集率均可达80%以上。通过对不同温度和不同菌液浓度下菌株自凝集特性的分析,温度与菌株的自凝集特性无明显相关性,而较高的菌液浓度能够提高菌株的自凝集能力。为分析突变菌株自凝集形成机制,以野生型W3110为对照,对突变菌株自转运蛋白编码基因flu进行RT-PCR分析,突变菌株中flu基因的转录水平增加2~4倍。同时,利用表达质粒pWSK29构建自转运蛋白质表达载体,通过诱导表达,确定自转运蛋白质在大肠杆菌W3110自凝集中的作用。
Theautoaggregation level of E.coli K-12 W3110 was tested by the comparison with its mutants in core oligosaccharide(core OS),and the relationship between autoaggragation and the structure of core oligosaccharide was analyzed. The results showed that the mutation of core OS would induced autoaggregation. The autoaggregation ratio of mutants were three times better than wild type W3110 in 12 hours of standing at 4 ℃. The mutants of core OS deficiency(ΔwaaC) and outer core OS deficiency(ΔwaaG) were used to investigate the properties of autoaggregation. In this study,the autoaggregation ratios of ΔwaaC and ΔwaaG strains were 80% after 2 hours of static culture. The autoaggregation level with different temperature and cell concentration were also tested,the results indicated that the autoaggregation was not influenced by the temperature,but enhanced by the increase of cell concentration. In order to elucidate the mechanism of autoaggregation,the transcription level of flu(gene of Ag43) was analyzed between W3110 and the mutants by RT-PCR,and the results showed that the transcription of flu was raised 2~4 times in the mutants of core OS,and it may be the reason of the increase of autoaggregation ratio in mutants. Finally,the preliminary results of gene expression of flu proved the effect of Ag43 on autoaggregation in W3110.
Theautoaggregation level of E.coli K-12 W3110 was tested by the comparison with its mutants in core oligosaccharide(core OS),and the relationship between autoaggragation and the structure of core oligosaccharide was analyzed. The results showed that the mutation of core OS would induced autoaggregation. The autoaggregation ratio of mutants were three times better than wild type W3110 in 12 hours of standing at 4 ℃. The mutants of core OS deficiency(ΔwaaC) and outer core OS deficiency(ΔwaaG) were used to investigate the properties of autoaggregation. In this study,the autoaggregation ratios of ΔwaaC and ΔwaaG strains were 80% after 2 hours of static culture. The autoaggregation level with different temperature and cell concentration were also tested,the results indicated that the autoaggregation was not influenced by the temperature,but enhanced by the increase of cell concentration. In order to elucidate the mechanism of autoaggregation,the transcription level of flu(gene of Ag43) was analyzed between W3110 and the mutants by RT-PCR,and the results showed that the transcription of flu was raised 2~4 times in the mutants of core OS,and it may be the reason of the increase of autoaggregation ratio in mutants. Finally,the preliminary results of gene expression of flu proved the effect of Ag43 on autoaggregation in W3110.
引文
[1]RAETZ C R H,WHITFIELD C.Lipopolysaccharide endotoxins[J].Annual Review of Biochemistry,2002,71:635-700.
[2]WANG X Y,QUINN P J.Lipopolysaccharide:Biosynthetic pathway and structure modification[J].Progress in Lipid Research,2009,49(2):97-107.
[3]RONCERO C,CASADABAN M J.Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12:three operons in the rfa locus[J].Journal of Bacteriology,1992,174(10):3250-3260.
[4]KAWASAKI K.Complexity of lipopolysaccharide modifications in Salmonella enterica:Its effects on endotoxin activity,membrane permeability,and resistance to antimicrobial peptides[J].Food Research International,2011,45(2):493-501.
[5]PARK B S,SONG D H,KIM H M,et al.The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex[J].Nature,2009,458:1191-1195.
[6]CHANG P C,WANG C J,YOU C K,et al.Effects of a HP0859(rfa D)knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and t he bacterial adhesion on AGS cells Po-Chun[J].Biochemical and Biophysical Research Communications,2011,405:497-502.
[7]KIM C H.A Salmonella typhimuriumrfa emutant recovers invasiveness for human epithelial cells when complemented by wild type rfa E(controlling biosynthesis of ADP-L-glycero-D-manno-heptose-containing lipopolysaccharide)[J].Molecules and Cells,2003,15(2):226-232.
[8]MA P,HU X Q,CHEN J Z,et al.The effect of the structure of lipopolysaccharide on the permeability of Escherichia coli cell membranes[J].Microbiology China,2011,38(8):1307-1315.
[9]LAU P C Y,LINDHOUT T,BEVERIDGE T J,et al.Differential lipopolysaccharide core capping leads to quantitative and correlated modifications of mechanical and structural properties in Pseudomonas aeruginosabio films[J].Journal of Bacteriology,2009,191(21):6618-6631.
[10]KHEMALEELAKUL S,BAUMGARTNER J C,PRUKSAKOM S.Autoaggregation and coaggregation of bacteria associated with acute endodontic infections[J].Journal of Endodontics,2006,32(4):312-318.
[11]FELEK S,LAWRENZ M B,KRUKONIS E S.The Yersinia pestisautotransporter Yap C mediates host cell binding,autoaggregation and biofilm formation[J].Microbiology,2008,154:1802-1812.
[12]MERRITT J,NIU G Q,OKINAGA T,et al.Autoaggregationresponse of Fusobacterium nucleatum[J].Applied and Environment Microbiology,2009,75(24):7725-7733.
[13]PODLADCHIKOVAA O,ANTONENKAB U,HEESEMANN J,et al.Yersinia pestis autoagglutination factor is a component of the type six secretion system[J].International Journal of Medical Microbiology,2011,301:562-569.
[14]LIU Yuehua,ZHOU Guanglin.The selection of agglutinating nature yeast on alcohol fermentation[J].Liquor Making,2007,3(2):58-61.(in Chinese)
[15]RACHMAN M A,NAKASHIMADA Y,KAKIAONO T,et al.Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor[J].Applied Microbiology and Biotechnology,1998,49:450-454.
[16]JANKOVIC I,VENTURA M,MEYLAN V,et al.Contribution of aggregation-promoting factor to maintenance of cell shape in Lactobacillus gasseri 4B2[J].Journal of Bacteriology,2003,185(11):3288-3296.
[17]HASMAN H,CHAKRABORTY T,KLEMM P.Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation[J].Journal of Bacteriology,1999,181(16):4834-4841.
[18]SHERLOCK O,VEJBORG R M,KLEMM P.The Tib Aadhesin/invasin from enterotoxigenic Escherichia coli is self-recognizing and induces bacterial aggregation and biofilm formation[J].Infection and Immunity,2005,73(4):1954-1963.
[19]ULETT G C,WEBB R I,SCHEMBRI M A.Antigen-43-mediated autoaggregation impairs motility in Escherichia coli[J].Microbiology,2006,152:2101-2110.
[20]BENZ I M,SCHMIDT A.Structures and functions of autotransporter proteins in microbial pathogens[J].International Journal of Medical Microbiology,2011,301(6):461-468.
[21]GIRARD V,COTE J P,CHARBONNEAU M E.Conformation change in a self-recognizing autotransporter modulates bacterial cell-cell interaction[J].The Journal of Biological Chemistry,2010,285(14):10616-10626.
[22]WANG Z,WANG J L,REN G,et al.Influence of core oligosaccharide of lipopolysaccharide to outer membrane behavior of Escherichia coli[J].Marine Drugs,2015,13:3325-3339.
[23]WANG R F,KUSHNER S R.Construction of versatile low-copy-number vectors for cloning,sequencing and gene expression in Escherichia coli[J].Gene,1991,100:195-199.
[24]ROUX A,BELOIN C,GHIGO J M.Combined inactivation/expression strategy to study gene function in physiological conditions:application to the identification of new adhesins in Escherichia coli[J].Journal of Bacteriology,2005,187:1001-1013.
[25]DANESE P N,PRATT L A,DOVE S L,et al.The outer membrane protein,Antigen 43,mediatescell-to-cell interactions within Escherichia coli biofilms[J].Molecular Microbiology,2000,37(2):424-432.
[26]VYAZMENSKY M,SELLA C,BARAK Z,et al.Isolation and characterization of subunits of acetohydroxy acid synthase isozyme III and reconstitution of the holoenzyme[J].Biochemistry,1996,35(32):10339-10346.
[27]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,2001,25(4):402-408.
[2]WANG X Y,QUINN P J.Lipopolysaccharide:Biosynthetic pathway and structure modification[J].Progress in Lipid Research,2009,49(2):97-107.
[3]RONCERO C,CASADABAN M J.Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12:three operons in the rfa locus[J].Journal of Bacteriology,1992,174(10):3250-3260.
[4]KAWASAKI K.Complexity of lipopolysaccharide modifications in Salmonella enterica:Its effects on endotoxin activity,membrane permeability,and resistance to antimicrobial peptides[J].Food Research International,2011,45(2):493-501.
[5]PARK B S,SONG D H,KIM H M,et al.The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex[J].Nature,2009,458:1191-1195.
[6]CHANG P C,WANG C J,YOU C K,et al.Effects of a HP0859(rfa D)knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and t he bacterial adhesion on AGS cells Po-Chun[J].Biochemical and Biophysical Research Communications,2011,405:497-502.
[7]KIM C H.A Salmonella typhimuriumrfa emutant recovers invasiveness for human epithelial cells when complemented by wild type rfa E(controlling biosynthesis of ADP-L-glycero-D-manno-heptose-containing lipopolysaccharide)[J].Molecules and Cells,2003,15(2):226-232.
[8]MA P,HU X Q,CHEN J Z,et al.The effect of the structure of lipopolysaccharide on the permeability of Escherichia coli cell membranes[J].Microbiology China,2011,38(8):1307-1315.
[9]LAU P C Y,LINDHOUT T,BEVERIDGE T J,et al.Differential lipopolysaccharide core capping leads to quantitative and correlated modifications of mechanical and structural properties in Pseudomonas aeruginosabio films[J].Journal of Bacteriology,2009,191(21):6618-6631.
[10]KHEMALEELAKUL S,BAUMGARTNER J C,PRUKSAKOM S.Autoaggregation and coaggregation of bacteria associated with acute endodontic infections[J].Journal of Endodontics,2006,32(4):312-318.
[11]FELEK S,LAWRENZ M B,KRUKONIS E S.The Yersinia pestisautotransporter Yap C mediates host cell binding,autoaggregation and biofilm formation[J].Microbiology,2008,154:1802-1812.
[12]MERRITT J,NIU G Q,OKINAGA T,et al.Autoaggregationresponse of Fusobacterium nucleatum[J].Applied and Environment Microbiology,2009,75(24):7725-7733.
[13]PODLADCHIKOVAA O,ANTONENKAB U,HEESEMANN J,et al.Yersinia pestis autoagglutination factor is a component of the type six secretion system[J].International Journal of Medical Microbiology,2011,301:562-569.
[14]LIU Yuehua,ZHOU Guanglin.The selection of agglutinating nature yeast on alcohol fermentation[J].Liquor Making,2007,3(2):58-61.(in Chinese)
[15]RACHMAN M A,NAKASHIMADA Y,KAKIAONO T,et al.Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor[J].Applied Microbiology and Biotechnology,1998,49:450-454.
[16]JANKOVIC I,VENTURA M,MEYLAN V,et al.Contribution of aggregation-promoting factor to maintenance of cell shape in Lactobacillus gasseri 4B2[J].Journal of Bacteriology,2003,185(11):3288-3296.
[17]HASMAN H,CHAKRABORTY T,KLEMM P.Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation[J].Journal of Bacteriology,1999,181(16):4834-4841.
[18]SHERLOCK O,VEJBORG R M,KLEMM P.The Tib Aadhesin/invasin from enterotoxigenic Escherichia coli is self-recognizing and induces bacterial aggregation and biofilm formation[J].Infection and Immunity,2005,73(4):1954-1963.
[19]ULETT G C,WEBB R I,SCHEMBRI M A.Antigen-43-mediated autoaggregation impairs motility in Escherichia coli[J].Microbiology,2006,152:2101-2110.
[20]BENZ I M,SCHMIDT A.Structures and functions of autotransporter proteins in microbial pathogens[J].International Journal of Medical Microbiology,2011,301(6):461-468.
[21]GIRARD V,COTE J P,CHARBONNEAU M E.Conformation change in a self-recognizing autotransporter modulates bacterial cell-cell interaction[J].The Journal of Biological Chemistry,2010,285(14):10616-10626.
[22]WANG Z,WANG J L,REN G,et al.Influence of core oligosaccharide of lipopolysaccharide to outer membrane behavior of Escherichia coli[J].Marine Drugs,2015,13:3325-3339.
[23]WANG R F,KUSHNER S R.Construction of versatile low-copy-number vectors for cloning,sequencing and gene expression in Escherichia coli[J].Gene,1991,100:195-199.
[24]ROUX A,BELOIN C,GHIGO J M.Combined inactivation/expression strategy to study gene function in physiological conditions:application to the identification of new adhesins in Escherichia coli[J].Journal of Bacteriology,2005,187:1001-1013.
[25]DANESE P N,PRATT L A,DOVE S L,et al.The outer membrane protein,Antigen 43,mediatescell-to-cell interactions within Escherichia coli biofilms[J].Molecular Microbiology,2000,37(2):424-432.
[26]VYAZMENSKY M,SELLA C,BARAK Z,et al.Isolation and characterization of subunits of acetohydroxy acid synthase isozyme III and reconstitution of the holoenzyme[J].Biochemistry,1996,35(32):10339-10346.
[27]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,2001,25(4):402-408.