Strains of Escherichia coli causing enterohemorrhagic colitis belonging to the O157:H7 lineage are reported to be highly related. Fifteen strains of E. coli O157:H7 and 1 strain of E. coli O46:H ؊ (nonflagellated) were examined for the presence of potassium tellurite resistance (Te r ). Te r genes comprising terABCDEF were shown previously to be part of a pathogenicity island also containing integrase, phage, and urease genes. PCR analysis, both conventional and light cycler based, demonstrated that about one-half of the Te r E. coli O157:H7 strains (6 of 15), including the Sakai strain, which has been sequenced, carried a single copy of the Te r genes. Five of the strains, including EDL933, which has also been sequenced, contained two copies. Three other O157:H7 strains and the O46:H ؊ strain did not contain the Te r genes. In strains containing two copies, the Te r genes were associated with the serW and serX tRNA genes. Five O157:H7 strains resembled the O157 Sakai strain whose sequence contained one copy, close to serX, whereas in one isolate the single copy was associated with serW. There was no correlation between Te r and the ability to produce Shiga toxin ST1 or ST2. The Te r MIC for most strains, containing either one or two copies, was 1,024 g/ml, although for a few the MIC was intermediate, 64 to 128 g/ml, which could be increased to 512 g/ml by pregrowth of strains in subinhibitory concentrations of potassium tellurite. Reverse transcriptase PCR analysis confirmed that in most strains Te r was constitutive but that in the rest it was inducible and involved induction of terB and terC genes. Only the terB, -C, -D, and -E genes are required for Te r . The considerable degree of homology between the ter genes on IncH12 plasmid R478, which originated in Serratia marcescens, and pTE53, from an E. coli clinical isolate, suggests that the pathogenicity island was acquired from a plasmid. This work demonstrates diversity among E. coli O157:H7 isolates, at least as far as the presence of Te r genes is concerned.Escherichia coli O157:H7 is of major interest in clinical practice, food safety, and evolutionary biology. Although only recognized as a human pathogen in 1982 (16), it has rapidly become prominent as an important cause of food-related and waterborne outbreaks of hemorrhagic colitis throughout the world (13). In a proportion of patients infection progresses to hemolytic-uremic syndrome, characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia (13). Each of these secondary sequelae carries significant mortality rates (18).The genome sequences of two strains of enterohemorrhagic E. coli O157:H7 were recently completed (6,14). Comparisons between laboratory E. coli strain K-12 MG1655 (2) and EDL933, an E. coli O157:H7 strain (14), demonstrated that they have a complex relationship since their divergence 4.5 million years ago (14). Homology between the two is interrupted by the presence of hundreds of islands of inserted DNA. K islands are DNA segments present in MG1655 ...

Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation.

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Conjugal transfer of IncHI plasmid DNA between Gram‐negative bacteria is temperature sensitive, as mating is optimal between 22°C and 30°C but is inhibited at 37°C. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180 kbp that has been sequenced completely. The gene encoding green fluorescent protein (GFP) was inserted into R27 in frame with trhC. TrhC is a mating pair formation (Mpf) protein that is essential for plasmid transfer and H‐pilus production. Fluorescence microscopy allowed visualization of the TrhC–GFP fusion protein, and Escherichia coli cells were examined for the subcellular localization and temperature‐dependent production of TrhC–GFP. At 27°C, TrhC–GFP was found at the periphery of cells as discrete foci, indicating an association of TrhC within protein complexes in the bacterial cell membrane, whereas at 37°C, little fluorescence was detected. These foci probably represent the intracellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf proteins. During temperature shift experiments from 37°C to 27°C, a long lag period was required for generation of GFP foci. Conversely, during short shifts from 27°C to 37°C, the GFP foci remained stable. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC was verified by cellular fractionation. Expression patterns of TrhC–GFP were confirmed with immunoblot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR). These results allow us to propose mechanisms to explain the temperature‐sensitive transfer of R27.

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