The microcin PDI inhibits a diverse group of pathogenic Escherichia coli strains. Coculture of a single-gene knockout library (BW25113; n ‫؍‬ 3,985 mutants) against a microcin PDI-producing strain (E. coli 25) identified six mutants that were not susceptible (⌬atpA, ⌬atpF, ⌬dsbA, ⌬dsbB, ⌬ompF, and ⌬ompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts in E. coli O157:H7 Sakai. Heterologous expression of E. coli ompF conferred susceptibility to Salmonella enterica and Yersinia enterocolitica strains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K 47 G 48 N 49 region within the first extracellular loop of E. coli OmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator for ompF, and consequently loss of susceptibility by the ⌬ompR strain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. In trans expression of ompF in the ⌬dsbA and ⌬dsbB strains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI. E scherichia coli strain 25 (E. coli 25; cattle origin) has an in vitro and in vivo competitive advantage against other E. coli strains that is linked to the production of the microcin PDI (MccPDI) (1-3). The inhibitory phenotype was first observed in vitro (4) and later called "proximity-dependent inhibition" (PDI), because inhibition occurred only when competing cells were in close proximity to sensitive cells (1). MccPDI appears to be most closely related to class IIa microcins, and the cluster of genes that encode MccPDI and associated immunity, activation, and export are located on a conjugative plasmid (2).E. coli produces various antimicrobial bacteriocins that are classified as colicins or microcins. Microcins are distinguished by their lower molecular mass (Ͻ10 kDa) and require active transport across the membrane of producing cells. Microcins typically have a narrow spectrum of activity that is mediated through specific receptors expressed on the surface of susceptible bacteria. To date, 16 microcins have been described, including MccPDI. The receptors for seven of these microcins have been identified and include the outer membrane proteins Cir, FepA, Fiu, FhuA, and OmpF, all of which normally function in iron and other nutrient uptake (5-7).While the gene encoding MccPDI has been identified and gene knockout and complementation studies have confirmed its inhibitory activity (2), little is known about how this microcin functi...

Microcin PDI inhibits a diversity of pathogenic Escherichia coli through the action of an effector protein, McpM. In this study we demonstrated that expression of the inhibitory phenotype is induced under low osmolarity conditions and expression is primarily controlled by the EnvZ/OmpR two-component regulatory system. Functional, mutagenesis and complementation experiments were used to empirically demonstrate that EnvZ is required for the inhibitory phenotype and that regulation of mcpM is dependent on binding of the phosphorylated OmpR to the mcpM promoter region. The phosphorylated OmpR may recognize three different binding sites within this promoter region. Site-directed mutagenesis revealed that the McpM precursor peptide includes two leader peptides that undergo sequential cleavage at positions G17/G18 and G35/A36 during export through the type I secretion system. Competition assays showed that both cleaved products are required for the PDI phenotype although we could not distinguish loss of function from loss of secretion in these assays. McpM has four cysteines within the mature peptide and site-directed mutagenesis experiments demonstrated that the first two cysteines are necessary for McpM to inhibit susceptible cells. Together these data combined with previous work indicate that MccPDI is unique amongst the microcins that have been described to date.

Background Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. Results In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing 1 species of Acetobacter, 8 species of Lactobacillus, 1 species of Pediococcus, 3 species of Streptococcus, and 1 species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5–7.5 up to at least 48 h. Maximum activity was observed at 50 °C up to at least 72 h. In addition, LysKB317 was stable in 30% ethanol up to at least 72 h. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 3-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with ≥ 0.01 µM LysKB317 was similar to uncontaminated controls. Conclusion Exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.

During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action.