Analysis of electrophoretically heterogeneous lipopolysaccharides of Escherichia coli by immunoblotting

Abstract The purified lipopolysaccharide (LPS) from each of four Escherichia coli serogroups which was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by periodic acid-silver staining, exhibited a different distribution of bands. The same serogroup-specific banding patterns were evident on immunoblots developed with homologous but not heterologous O-antisera. Thus, the electrophoretic heterogeneity of LPS was confirmed and the multiple bands seen on SDS-PAGE further shown to contain O-serogroup-specific antigenic determinants.     

Structural studies of the Escherichia coli O-antigen 6

The structure of the Escherichia coli O-antigen 6 has been investigated using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the O-antigen is composed of pentasaccharide repeating-units having the following structure. (Formula: see text)     

Structural studies of the Escherichia coli O-antigen 25

The structure of the Escherichia coli O-antigen 25 has been investigated using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the O-antigen is composed of pentasaccharide repeating-units having the following structure.     

Structural studies of the Escherichia coli O78 O-antigen polysaccharide

The structure of the O-antigen polysaccharide from Escherichia coli O78 has been investigated; methylation analysis, partial solvolysis with liquid hydrogen fluoride, and 2D-n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure.----3)-beta-D-GlcpNAc-(1----4)-beta-D-GlcpNAc- (1----4)-beta-D-Manp-(1----4)-alpha-D-Manp-(1----     

Structural studies of the Escherichia coli O-149 O-antigen polysaccharide

The structure of the O-antigen polysaccharide from Escherichia coli O-149 has been investigated; methylation analysis, partial hydrolysis with acid, and n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of trisaccharide repeating-units having the following structure. (Formula: see text). The absolute configuration at the acetalic carbon atom of the pyruvic acid residue is S.     

Preparation of tritiated lipopolysaccharides from Escherichia coli K12

Tritiated lipopolysaccharide (LPS) from E. coli K12 was prepared by coupling [3H]ethanolamine to the LPS core residue ketodeoxyoctonate (KDO) via activation of its carboxylic function with N-hydroxysuccinimide or N-hydroxy-sulfosuccinimide. Specific activities of 1.5 microCi/mg and 9 microCi/mg were obtained, respectively. Experiments comparing the activity of native and derivatized LPS suggested that the preparation of the radiolabelled LPS did not alter the structural properties of E. coli K12 LPS. This probe will be useful for studying the interactions between LPS and proteins.     

Escherichia coli lipopolysaccharides produce serotype-specific hypothermic response in biotelemetered rats

We investigated whether LPS-induced hypothermia develops in a serotype-specific manner in biotelemetered conscious rats. Two different Escherichia coli serotypes of LPSs were injected at a dose of 250 mug/kg ip. E. coli O55:B5 LPS elicited an initial hypothermia and subsequent fever, but E. coli O111:B4 LPS caused more potent monophasic hypothermia. Serum tumor necrosis factor (TNF)-alpha levels were dramatically elevated at the initial phase of the hypothermia induced by both LPSs. This elevation tended to subside at the nadir of E. coli O55:B5 LPS-induced response but progressively increased at the nadir of E. coli O111:B4 LPS hypothermia. Serum IL-10 levels were moderately elevated at the initial phase of the hypothermia and persisted at the same level at the nadir of each LPS-induced response. No change was observed at the serum IL-18 levels. A selective cyclooxygenase (COX)-1 enzyme inhibitor, valeryl salicylate (20 mg/kg sc), abolished the hypothermia without any effect on the elevated cytokine levels. Another COX-1-selective inhibitor, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560; 1 mg/kg sc) inhibited hypothermic responses as well. Meanwhile, cytokine levels were also reduced by SC-560 treatment. These findings suggest that LPS-induced hypothermia may have serotype-specific characteristics in rats. E. coli O111:B4 LPS has more potent hypothermic activity than E. coli O55:B5 LPS; that may presumably be related to its higher or sustained capability to release antipyretic cytokines, such as TNF-alpha. COX-1 enzyme may be involved in the generation of the hypothermia, regardless of the type of LPS administered.     

Structure and genetics of the O-antigen of Escherichia coli O158

A structure of the O-polysaccharide (O-antigen) of Escherichia coli O158 has been reported (Datta, A. K.; Basu, S.; Roy, N. Carbohydr. Res.1999, 322, 219–227). In this work, we reinvestigated the O158 polysaccharide using sugar analyses, Smith degradation, and ¹H and ¹³C NMR spectroscopy and established the following structure, which is at variance with the structure established earlier:This structure is in agreement with the predicted functions of genes found in the O-antigen gene cluster of E. coli O158.     

Structural analysis of the O-antigen polysaccharide from Escherichia coli O152

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined. Component analysis together with 1H, 13C and 31P NMR spectroscopy were used to elucidate the structure. Inter-residue correlations were determined by 1H,31P COSY, 1H,1H NOESY and 1H,13C heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. The structure is similar to that of the O-antigen polysaccharide from E. coli O173. The cross-reactivity between E. coli O152 and E. coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.     

Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase

The gene for firefly luciferase (luc) can be used as a generalized genetic probe. A method that aids in the analysis of shuttle vectors containing luc by allowing verification in Escherichia coli of a functional coding sequence is presented here. Colonies containing a functional form of luc are detected on film after luciferin is added to initiate the luminescent reaction. Two conditions, lowering the pH of the environment and maintaining aerobic conditions, were found to greatly improve the sensitivity of the assay. This technique may be useful in the development of genetic constructs that alter the natural coding sequence of luc, such as in gene fusions.     

Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.     

Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.     

Identification of lipopolysaccharides and phospholipids of Escherichia coli in polyacrylamide gels.

Polyacrylamide gel electrophoresis of unfractionated lysates of radioactively labeled cells resolves not only proteins and polynucleotides into discrete bands but also cellular lipopolysaccharides and phospholipids. This allows a determination of the intracellular amounts of all of these macromolecules. In addition, this technique is sensitive enough to detect mutational alterations in lipopolysaccharide structure. Polyacrylamide gel electrophoresis is herein shown to be a useful tool for investigations into the structure of lipopolysaccharides and the synthesis of lipopolysaccharides and phospholipids.     

大肠杆菌O54 O-抗原基因簇的破译及进化分析

破译了大肠杆菌O5 4O 抗原基因簇的序列 ,序列全长 1 4 0 6 2bp。用生物信息学方法分析序列并鉴定基因 ,共确定 1 0个基因 ,包括鼠李糖合成酶基因BDA和C(rmlBDA和rmlC) ,糖基转移酶基因 ,O 抗原转运酶基因 ,O 抗原聚合酶基因和合成磷酸丝氨酸侧链的基因及 1个不能确定功能的开放阅读框。对rmlC的 (G C) %含量 ,稀有密码子含量及进化分析都表明大肠杆菌O5 4O 抗原基因簇是在近期通过rmlC介导的重组形成 ,而且大肠杆菌O5 4和鲍氏志贺氏菌 9型的亲缘关系很近。对UTP 葡萄糖 1 磷酸 尿苷转移酶基因 (galF)和 6 磷酸葡萄糖脱氢酶基因(gnd)的进化分析揭示志贺氏菌属与大肠杆菌属在进化上属于同一个属。用PCR方法筛选出了针对大肠杆菌O5 4的特异基因 ,用于基因芯片或PCR方法对大肠杆菌O5 4的快速检测。     

Detection of submicrogram quantities of Escherichia coli lipopolysaccharides by agarose-gel electrophoresis

A sensitive agarose-gel electrophoresis method has been developed for the visualization of lipopolysaccharide (LPS) samples from several Escherichia coli serotypes, such as 026:B6, 055:B5, 0128:B12, 0111:B4, 0127:B8, and K235. This method can detect as little as 0.5-6 microg of LPS depending on the serotype by treatment of the plates with toluidine blue, and it is able to measure sub-microgram amounts of samples, approx. 0.05-0.5 microg, when the staining with toluidine blue followed by Stains-All procedure is adopted. Treatment of LPS with alkali under anhydrous conditions removes the ester-linked fatty acids and the phosphate groups of the Lipid A component, producing the detoxified LPS. The carbohydrate neutral moiety obtained from the hydrolysate does not migrate during electrophoresis. This was utilized to monitor quantitatively the removal process of the Lipid A component for the formation of the detoxified LPS.     

Loss of O-antigen increases cell shape abnormalities in penicillin-binding protein mutants of Escherichia coli

Escherichia coli mutants lacking multiple penicillin-binding proteins (PBPs) produce aberrantly shaped cells. However, most of these experiments have been performed in E. coli K12 strains, which do not attach a complete O-antigen to their outer membrane lipopolysaccharide. We constructed mutants in different genetic backgrounds and found that the frequency of morphological deformities was higher in strains lacking the O-antigen. Also, complementing O-negative mutants with a heterologous O-antigen from Klebsiella returned a substantial fraction of misshapen cells to a normal morphology. Thus, the O-antigen contributes to cell shape in E. coli, perhaps by reducing the number of ectopic poles, which may be the proximal cause of shape abnormalities.