A genomic island, termed high-pathogenicity island, is present in certain non-O157 Shiga toxin-producing Escherichia coli clonal lineages.

Shiga toxin-producing Escherichia coli (STEC) strains cause a wide spectrum of diseases in humans. In this study, we tested 206 STEC strains isolated from patients for potential virulence genes including stx, eae, and enterohemorrhagic E. coli hly. In addition, all strains were examined for the presence of another genetic element, the high-pathogenicity island (HPI). The HPI was first described in pathogenic Yersinia species and encodes the pesticin receptor FyuA and the siderophore yersiniabactin. The HPI was found in the genome of distinct clonal lineages of STEC, including all 31 eae-positive O26:H11/H(-) strains and 7 of 12 eae-negative O128:H2/H(-) strains. In total, the HPI was found in 56 (27.2%) of 206 STEC strains. However, it was absent from the genome of all 37 O157:H7/H(-), 14 O111:H(-), 13 O103:H2, and 13 O145:H(-) STEC isolates, all of which were positive for eae. Polypeptides encoded by the fyuA gene located on the HPI could be detected by using immunoblot analysis in most of the HPI-positive STEC strains, suggesting the presence of a functional yersiniabactin system. The HPI in STEC was located next to the tRNA gene asnT. In contrast to the HPI of other pathogenic enterobacteria, the HPI of O26 STEC strains shows a deletion at its left junction, leading to a truncated integrase gene int. We conclude from this study that the Yersinia HPI is disseminated among certain clonal subgroups of STEC strains. The hypothesis that the HPI in STEC contributes to the fitness of the strains in certain ecological niches rather than to their pathogenic potential is discussed.

Characterization of Escherichia coli strains isolated from environmental water habitats and from stool samples of healthy volunteers.

This study was undertaken to determine the frequency of pathogenic Escherichia coli strains among wild-type E. coli strain isolates from the microbial flora of healthy volunteers and from natural residential water habitats of a defined geographic area. In total, 131 stool and 95 water isolates as well as 14 E.coli K12 strains were examined for DNA sequences specific for 20 different genes encoding E. coli pathogenicity factors, including adherence factors, toxins, invasins, capsules and iron uptake systems. The expression of the corresponding pathogenicity factors was also investigated. No pathogenicity factors were found to be present in the tested E. coli K12 strains. In contrast, 41.0% of the water samples and 63.4% of the stool samples contained pathogenicity factors specific for extraintestinal E. coli pathogens. While no virulence determinants specific for intestinal E. coli pathogens were found among the investigated environmental water isolates, 4.5% of the stool samples contained either only intestinal or both intestinal and extraintestinal virulence genes. Both the prevalence of the virulence genes and the expression of the corresponding pathogenicity factors were, in general, higher in stool than in water samples. These findings might indicate the prevalence of different clonal types and/or differential regulation of pathogenicity factor expression in diverse ecological niches.

Regulation of the Shiga-like toxin II operon in Escherichia coli.

Investigations of the regulation of the bacteriophage-encoded Shiga-like toxin II (SLT-II) in Escherichia coli demonstrated that bacteriophages exhibit a regulatory impact on toxin production by two mechanisms. Firstly, replication of the toxin-converting bacteriophages brings about an increase in toxin production due to concomitant multiplication of toxin gene copies. Secondly, an influence of a phage-encoded regulatory molecule was demonstrated by using low-copy-number plasmid pADR-28, carrying a translational gene fusion between the promoter and proximal portion of slt-IIA and the structural gene for bacterial alkaline phosphatase (phoA). PhoA activity, reflecting the slt-II promoter activity, was significantly enhanced in E. coli strains which and been lysogenized with an SLT-I or SLT-II-converting bacteriophage (H-19B or 933W, respectively) or bacteriophage lambda. Both mechanisms are dependent on bacteriophage induction and hence are recA dependent. Moreover, the study revealed that the DNA-binding protein H-NS has a regulatory impact on both bacteriophage-mediated SLT-II synthesis and the activity of the slt-II promoter of plasmid pADR-28. While a slight impact of growth temperature on SLT-II expression was observed, no impact of either osmolarity, pH, oxygen tension, acetates, iron level, or utilized carbon source could be demonstrated.

Domains of colicin M involved in uptake and activity.

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the TonB, ExbB and ExbD proteins. These colicins contain a sequence, called the TonB box, which has been implicated in transport via TonB. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with TonB may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193-197 and 223-252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the TonB box at the N-terminal end of colicin M must be involved in colicin uptake via TonB across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.

The biology of colicin M.

This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma, has been sequenced and the protein isolated and purified. With a deduced molecular size of 29,453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13,890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.

Binding of the immunity protein inactivates colicin M.

Colicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma-producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N-terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.

A colicin M derivative containing the lipoprotein signal sequence is secreted and renders the colicin M target accessible from inside the cells.

Colicin M is only released in very low amounts by cells harbouring this plasmid encoded colicin, due to the lack of a release (lysis) protein. A fusion gene (lpp'cma) was constructed which determined two proteins: Lpp'-Cma composed of the signal sequence of the murein lipoprotein (Lpp) and colicin M (Cma), and unaltered colicin M. Cells expressing the fusion gene released 50% of the total colicin M into the culture medium, compared to 1% found in the medium of cells synthesizing only colicin M. The release resulted from partial cell lysis caused by colicin M since a colicin M tolerant strain remained unaffected. Lpp'-Cma thus mimics phenotypically the action of colicin release proteins but displays a different lysis mechanism. In strains defective in components of the colicin M uptake system, Lpp'-Cma caused lysis as effectively as in uptake proficient strains. Apparently, Lpp'-Cma renders the colicin M target site accessible from inside the cell which stands in contrast to the action of colicin M which is only bactericidal when provided from outside.

Plasmid pColBM-Cl139 does not encode a colicin lysis protein but contains sequences highly homologous to the D protein (resolvase) and the oriV region of the miniF plasmid.

Colicins are usually released from producing cells by so-called lysis proteins. No sequence homologous to the structurally very similar colicin lysis genes was found in the gene cluster cmi cma cbi cba, which determines the activity and immunity proteins of colicin B and M on pColBM-Cl139. Instead, the region upstream of cmi contained sequences that showed 91% homology to the structural gene of protein D (resolvase) and 75.5% homology to the rfsF sequence of the Escherichia coli miniF plasmid. It is concluded that colicins B and M are not released via the activity of lysis proteins and that the highly homologous regions encode a resolvase and its target respectively.

Sequence, expression and localization of the immunity protein for colicin B.

Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20,000 was close to the calculated molecular weight of 20,185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.

Sequence, expression, and localization of the immunity protein for colicin M.

Escherichia coli strains carrying the cmi locus on plasmids are immune against colicin M, which primarily inhibits murein biosynthesis, followed by lysis of cells. The nucleotide sequence of the cmi region was determined. It contains an open reading frame for a polypeptide with a molecular weight of 19,227. However, the major protein band observed on polyacrylamide gels after transcription and translation in an in vitro system or in minicells had an apparent molecular weight between 15,000 and 16,000. The nucleotide sequence contained internal ATG codons, two of which could serve for the synthesis of polypeptides with molecular weights of 15,349 and 15,996, respectively. A subclone with a DNA fragment that encoded these two shorter polypeptides exhibited full immunity. The colicin M immunity protein was found in the cytoplasmic membrane. The colicin M activity and immunity genes were transcribed in opposite directions. Both properties are typical of the channel-forming colicins and are in contrast to the colicins with endonuclease activities. However, colicin M does not form channels and exhibits no structural similarity to channel-forming colicins.

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.

Cloning and expression of the activity and immunity genes of colicins B and M on ColBM plasmids.

The activity and immunity genes for colicins B and M on two conjugative ColBM plasmids, pCl139 and pF166, were cloned into pBR322 and pACYC184, respectively. The colicin regions on both recombinant plasmids were identical with regard to restriction endonuclease sites and the arrangement of the genes. They map close to each other in the order cmi cma cbi cba, where cmi denotes the locus that determines immunity to colicin M, cma the structural gene for colicin M, cbi immunity to colicin B, and cba the structural gene for colicin B. With the use of mutants obtained by insertion of the transposon Tn5, and by translation in minicells, the transcriptional polarity of cma and cba was found to be from right to left. cma and cba code for polypeptides with molecular weights of 27,000 and 58,000, respectively. No evidence of biosynthetic precursors was obtained.