Characterisation of phosphorylation-deficient mutants of enteropathogenic Escherichia coli

EPEC infection of cultured epithelial cells causes rearrangements of the cytoskeleton resulting in the effacement of microvilli and the formation of characteristic lesions involving the accretion of actin at the site of bacterial attachment. Attaching and effacing (AE) lesion formation involves the subversion of host cell signalling systems including tyrosine and serine/threonine phosphorylation of proteins, activation of phospholipase C-1, and increases in the second messengers IP3 and Ca2+.;In this study the transposon TnphoaA was used to isolate EPEC mutants which were unable to induce the serine/threonine phosphorylation of host proteins. The five phosphorylation-negative mutants identified were all found additionally to be deficient in the assembly of the EPEC plasmid-encoded type IV bundle-forming pilus (BFP). Two of the mutations were localised to a gene encoding an outer membrane protein component of BFP. The other mutations defined two chromosomal genes emtA and tag which encode a novel endo-specific lytic transglycosylase and an uncharacterised inner membrane protein.;Based upon its known enzymatic activity it was proposed that EmtA was required to facilitate the assembly of BFP through the peptidoglycan cell wall. Construction of a non-polar mutation within the emtA gene f EPEC strain E2348-69 demonstrated only minor effects upon BFP biosynthesis. It is proposed that the phosphorylation-negative/BFP-negative phenotype observed in the original EPEC emtA mutant is due to polar effects of TnphoA insertion upon the expression of one of the adjacent uncharacterised genes.;Determination of the DNA sequence adjacent to the EPEC emtA locus identified a region which appears to have been deleted from E. coli K-12 strains. This locus contains genes encoding the TonB-dependent outer membrane receptor (OmpX) and inner membrane components of a novel siderophore uptake system. Analysis of the distribution of ompX amongst pathogenic E. coli strains demonstrated inconsistencies with the expected phylogeny