大肠杆菌和志贺氏菌O-抗原糖基转移酶与聚合酶的生物信息学研究

利用生物信息学手段对大肠杆菌和志贺氏菌的 1 1 0个O 抗原糖基转移酶与 39个O 抗原聚合酶的序列进行分析 ,探讨这两种酶的序列和结构特点。统计了其序列一致性 ,密码子使用和 (G C) %含量的特点 ;讨论了O 抗原糖基转移酶和聚合酶对底物的特异性 ;推测了 6组糖基转移酶的功能 ;通过对蛋白拓扑结构的预测 ,发现O 抗原聚合酶中广泛存在一个位于细胞周质中的亲水环 (Loop) ,是可能的功能区域 ;通过对蛋白高级结构的预测 ,发现O 抗原糖基转移酶属于两个不同的蛋白超家族。     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

The effects of Escherichia coli capsule, O-antigen, host neutrophils, and complement in a rat model of Gram-negative pneumonia

Gram-negative enteric bacilli are agents of life-threatening pneumonia. The role of the bacterial capsule and O-antigen moiety of lipopolysaccharide in the pathogenesis of Gram-negative pneumonia was assessed. In a rat model of pneumonia the LD(50) of a wild-type extraintestinal pathogenic Escherichia coli strain (CP9) was significantly less than its isogenic derivatives deficient in capsule (CP9.137), O-antigen (CP921) or both capsule and O-antigen (CP923) (P< or =0.003). Studies using complement depleted or neutropenic animals established that both neutrophils and complement are important for the pulmonary clearance of E. coli. Data from these studies also support that capsule and O-antigen serve, at least in part, to counter the complement and neutrophil components of the pulmonary host defense response. Lastly, the contribution of E. coli versus neutrophils in causing lung injury was examined. Findings suggest that E. coli virulence factors and/or non-neutrophil host factors are more important mediators of lung injury than neutrophils. These findings extend our understanding of Gram-negative pneumonia and have treatment implications.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

口蹄疫病毒P1基因在大肠杆菌中的高效表达及其生物活性的初步分析

将口蹄疫病毒 (FMDV)结构蛋白基因P1的完整cDNA序列插入原核表达性载体pGEX KG中 ,使P1基因与GST融合 ,获得融合表达质粒pKG P1,转化E .coliBL₂₁ (DE3) ,经IPTG诱导 ,SDS PADE结果表明GST P1融合蛋白获得高效表达 ,Western blot检测证实表达的融合蛋白具有免疫学活性 ,表达产物主要存在于细菌裂解液上清中。进一步采用GST纯化试剂盒纯化P1蛋白并作为诊断抗原 ,建立了P1 ELISA诊断方法 ,与FMD间接血凝 (IHA)检测方法平行检测 86 4份血清样品 ,总的符合率达87%。     

Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction

Summary A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage gt11. P1 phage grown on cells lysogenized with the recombinant phage could transduce the mutant gene into the original site on the Escherichia coli chromosome.     

鸡细胞色素P450 1A5的原核表达与活性重组

旨在对鸡细胞色素P450 1A5(CYP1A5)蛋白进行体外功能研究,采用大肠杆菌系统进行CYP1A5的异源表达。以鸡的cDNA为模板,扩增出CYP1A5基因,将该基因的N端编码区进行修饰,并连接到pCW载体中构建His-CYP1A5,经IPTG诱导在大肠杆菌中表达。经CO-差示光谱检测,所获得的His-CYP1A5具有典型的P450吸收峰。该蛋白与细胞色素P450还原酶(CPR)进行体外重组,构成的重组酶系表现出乙氧基试卤灵-O-脱乙基酶活性。结果表明,所采用的表达策略可以成功产生出具有催化活性的鸡细胞色素P450 1A5(CYP1A5)蛋白。     

密码子优化的肺炎支原体P1蛋白在大肠杆菌中的克隆表达

目的:获得密码子优化的肺炎支原体P1黏附蛋白优势表位抗原基因,并在大肠杆菌中表达,为临床诊断试剂和疫苗研制打下基础。方法:采用生物信息学分析肺炎支原体P1蛋白的抗原表位,筛选特异性P1蛋白优势表位区;采用大肠杆菌优势密码子,设计上述P1蛋白优势表位基因序列;采用退火PCR技术合成上述基因,并利用载体pGEX-4T-2实现P1优势表位抗原在大肠杆菌中的表达;采用ELISA法对纯化的P1抗原活性进行测定。结果:肺炎支原体P1蛋白特异性抗原表位主要位于1154~1521 aa,获得的P1优化密码子基因平行突变37个稀有密码子和2个终止密码子;在大肠杆菌中表达的GST-P1融合蛋白的相对分子质量为65.9×103,纯化后重组抗原能与肺炎支原体感染者血清发生特异性的免疫反应。结论:采用密码子优化基因合成技术实现了肺炎支原体P1优势表位抗原在大肠杆菌中的高效表达,为肺炎支原体感染的诊断试剂研究提供了重要参考。     

Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.     

Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time‐dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 µM . Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time‐dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration‐dependent, which correlates to the concentration‐dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration‐dependent killing is driven by the extent of the immediate permeabilizing action of the peptide. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Comparative sequence analysis of acid sensitive/resistance proteins in Escherichia coli and Shigella flexneri

The molecular basis for the survival of bacteria under extreme conditions in which growth is inhibited is a question of great current interest. A preliminary study was carried out to determine residue pattern conservation among the antiporters of enteric bacteria, responsible for extreme acid sensitivity especially in Escherichia coli and Shigella flexneri. Here we found the molecular evidence that proved the relationship between E. coli and S. flexneri. Multiple sequence alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns at regular intervals at the N-terminal region. It was observed that as the alignment approaches towards the C-terminal, the number of conserved residues decreases, indicating that the N-terminal region of this protein has much active role when compared to the carboxyl terminal. The motif, FHLVFFLLLGG, is well conserved within the entire gadC coded protein at the amino terminal. The motif is also partially conserved among other antiporters (which are not coded by gadC) but involved in acid sensitive/resistance mechanism. Phylogenetic cluster analysis proves the relationship of Escherichia coli and Shigella flexneri. The gadC coded proteins are converged as a clade and diverged from other antiporters belongs to the amino acid-polyamine-organocation (APC) superfamily.     

The cyclization of linear DNA in Escherichia coli by site-specific recombination

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox² plasmids) are cyclized in Cre⁺ E. coli strains after introduction either by transformation or by mini-Mu transduction, Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox² plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox² plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre⁺ E. coli recBC strains allow the efficient recovery of lox² plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.     

The cell wall receptor for bacteriophage Mu G(−) in Erwinia and Escherichia coli C

Abstract Adsorption of bacteriophage Mu with its invertible DNA segment in the G(−) orientation requires a terminal glucose residue for binding to the core lipopolysaccharide (LPS) of Gram-negative bacteria. Analysis of a Mu-resistant mutant shows that the receptor for Mu G(−) in Erwinia B374 is a Glc-β1,6-Glc disaccharide. A spontaneously occurring host-range mutant, Mu G(−)h101, grows on Escherichia coli C. The loss of the terminal β1,3-linked glucose from the LPS of E. coli C leads to resistance to the phage Mu. These mutants are also resistant to phage P1 and D108 which have largely homologous G segments. This shows that Mu G(+) and G(−) phage particles differ with respect to their cell-wall receptors in the type of glycosidic linkage of a terminal glucose residue: α1, 2 for G(+) and β1,6 for G(−).     

Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes

The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S. flexneri genome. Genomic subtraction identified a large unique region of DNA which was present in S. flexneri serotype 2a but absent from E. coli K-12. This 42-kb DNA segment was localised to the S. flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR). These findings indicate that this region may form a new PAI in the S. flexneri genome.     

鞘氨醇激酶-磷酸鞘氨醇轴在血管生成相关性疾病中的作用

血管生成是指在原有血管的基础上形成新血管的过程.病理性血管生成是癌症、心血管类疾病和视网膜病变等一系列疾病的标志.1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)是一种信号脂质,由鞘氨醇激酶(sphingosine kinases,SPHK)合成,通过5种G蛋白偶联受体(sphingosine-...     

The phage P.E1 isolated from hospital sewage reduces the growth of Escherichia coli

This study involves partial characterisation of a lytic bacteriophage P.E₁ against a multi drug-resistant clinical isolate of Escherichia coli, isolated from hospital sewage supply. The phage P.E₁ has showed a narrow host range suitable for its use in phage therapy. Phage showed lytic activity up to 70°C and at alkaline conditions, but at higher acidic conditions its activity decreased. Latent period and burst size of P.E₁ estimated from single-step growth curve was 40 min and 185 plaque-forming units per cell, respectively. The phage P.E₁ reduced the growth of host bacteria during the initial 12?h of infection; however, the host bacteria developed resistance afterwards. During the 24-hour observation period, the bacteriophage could still reduce the growth of its host bacteria evident by lower optical density in the phage-treated samples compared with control. The phage genome was double-stranded DNA and larger than 12?kb in size. Further manipulations of genome and proteins may help to unveil the unique aspects of this phage, to use it in phage therapy against E. coli.     

Transduction of Various R Factors by Phage P1 in Escherichia coli and by Phage P22 in Salmonella typhimurium

R factors fi(+) and fi(-), with various combinations of drug-resistance markers and isolated from independent sources, were transduced by phage P1kc in Escherichia coli and by phage P22 in Salmonella typhimurium. Usually the entire R factor was transduced by P1kc in E. coli, as indicated by the absence of segregation of the drug-resistance markers from their conjugal transferability. In contrast, the patterns of segregation of the drug-resistance markers and their conjugal transferability differed considerably among various R factors after transduction by P22 in S. typhimurium. Transduction frequencies varied among R factors in both transduction systems.     

The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2,4-dicarboxylic acid

Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor sigmas and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog L-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.     

Mutational analysis of the uracil DNA glycosylase inhibitor protein and its interaction with Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase inhibitor (Ugi), a protein of 9.4 kDa consists of a five-stranded antiparallel beta sheet flanked on either side by single alpha helices, forms an exclusive complex with uracil DNA glycosylases (UDGs) that is stable in 8M urea. We report on the mutational analysis of various structural elements in Ugi, two of which (hydrophobic pocket and the beta1 edge) establish key interactions with Escherichia coli UDG. The point mutations in helix alpha1 (amino acid residues 3-14) do not affect the stability of the UDG-Ugi complexes in urea. And, while the complex of the deltaN13 mutant with UDG is stable in only approximately 4M urea, its overall structure and thermostability are maintained. The identity of P37, stacked between P26 and W68, was not important for the maintenance of the hydrophobic pocket or for the stability of the complex. However, the M24K mutation at the rim of the hydrophobic pocket lowered the stability of the complex in 6M urea. On the other hand, non-conservative mutations E49G, D61G (cancels the only ionic interaction with UDG) and N76K, in three of the loops connecting the beta strands, conferred no such phenotype. The L23R and S21P mutations (beta1 edge) at the UDG-Ugi interface, and the N35D mutation far from the interface resulted in poor stability of the complex. However, the stability of the complexes was restored in the L23A, S21T and N35A mutations. These analyses and the studies on the exchange of Ugi mutants in preformed complexes with the substrate or the native Ugi have provided insights into the two-step mechanism of UDG-Ugi complex formation. Finally, we discuss the application of the Ugi isolates in overproduction of UDG mutants, toxic to cells.