We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain-length determinant (CLD) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli O111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states: 'E' facilitating extension and 'T' facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cld gene.

In chickens, colibacillosis is caused by avian pathogenic Escherichia coli (APEC) via respiratory tract infection. Many virulence factors, including type 1 (F1A) and P (F11) fimbriae, curli, aerobactin, K1 capsule, and temperature-sensitive hemagglutinin (Tsh) and plasmid DNA regions have been associated with APEC. A strong correlation between serum resistance and virulence has been demonstrated, but roles of virulence factors in serum resistance have not been well elucidated. By using mutants of APEC strains TK3, MT78, and 7122, which belong to serogroups O1, O2, and O78, respectively, we investigated the role of virulence factors in resistance to serum and pathogenicity in chickens. Our results showed that serum resistance is one of the pathogenicity mechanisms of APEC strains. Virulence factors that increased bacterial resistance to serum and colonization of internal organs of infected chickens were O78 lipopolysaccharide of E. coli 7122 and the K1 capsule of E. coli MT78. In contrast, curli, type 1, and P fimbriae did not appear to contribute to serum resistance. We also showed that the iss gene, which was previously demonstrated to increase resistance to serum in certain E. coli strains, is located on plasmid pAPEC-1 of E. coli 7122 but does not play a major role in resistance to serum for strain 7122.Avian pathogenic Escherichia coli (APEC) belongs to the extraintestinal pathogenic group of E. coli. These bacteria cause airsacculitis, omphalitis, peritonitis, salpingitis, synovitis, and colisepticemia in poultry (17). APEC is also associated with cellulitis or necrotic dermatitis of the lower abdomen and thighs and with granuloma. APEC strains belong predominantly to three serogroups, O1, O2, and O78. Virulence factors associated with APEC strains include type 1 and P fimbriae, curli, aerobactin, K1 capsule, and temperature-sensitive hemagglutinin (Tsh) of the autotransporter family (9, 17). Serum resistance also appears to be an important virulence mechanism of APEC, and it may play a major role in the pathogenesis of avian colibacillosis. For instance, serum resistance has often been associated with isolates from septicemic turkeys and chickens (13,33), and a correlation between serum resistance and virulence and lethality in isolates from septicemic chickens and turkeys has been observed (13,15,17).At this time, it is not known if avian E. coli strains differ from mammalian isolates in their mechanisms of serum resistance and virulence. Studies carried out with mammalian E. coli showed that many virulence factors, such as capsules, lipopolysaccharide (LPS), and outer membrane proteins (OMPs), includingOmpA and the ColV plasmid-encoded proteins TraT and Iss, are associated with complement resistance of E. coli (17). TraT is a surface exclusion protein encoded by conjugative plasmids (32), and Iss is a plasmid-encoded OMP homologous to the Bor protein of bacteriophage (32). In APEC, the role of different virulence factors in serum resistance has generally been speculative. Nolan et al. (22) produced an a...

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coliχ7122 conferred mannose‐resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR‐like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain χ7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA–lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild‐type S. typhimurium SL1344 or SR‐11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low‐copy‐number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature‐ and RpoS‐independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS‐dependent curli and extracellular matrix production by E. coli and S. typhimurium.

The rfb (O antigen) gene cluster of group C2 Salmonella differs from that of group B in a central region of 12.4 kb: we report the sequencing of this region of strain M67 (group C2) and a subsequent comparison with the central region of strain LT2 (group B). We find a block of seven open reading frames unique to group C2 which encode the O antigen polymerase (rfc) and the transferases responsible for assembly of the group C2 O antigen. The remaining rfb genes are common to strains M67 and LT2, but rfbJ (CDP-abequose synthase) and rfbM and rfbK (GDP-mannose synthesis), which are immediately adjacent to the central region, are highly divergent. All these genes have a low G+C content and appear to have been recent additions to Salmonella enterica. We discuss the evolutionary significance of the arrangement and divergence of the genes in the polymorphism of the rfb cluster.