title: Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI.
abstract: 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 (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). 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 K47G48N49 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 ΔompR 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 ΔdsbA and ΔdsbB 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.
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA drcall@wsu.edu.
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Eberhart
authors.affiliation: Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA.
authors.forename: Lisa H
authors.initials: LH
authors.lastname: Orfe
authors.affiliation: Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA.
authors.forename: Shao-Yeh
authors.initials: SY
authors.lastname: Lu
authors.affiliation: Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Besser
authors.affiliation: Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USA.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Call
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001430:Bacteriocins / Q000494:pharmacology*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics*; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D005820:Genetic Testing; D018272:Porins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D019779:Salmonella enterica / Q000187:drug effects / Q000235:genetics; D015008:Yersinia enterocolitica / Q000187:drug effects / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; C109237:OmpF protein; D018272:Porins; C020411:microcin