Sensitivity of Escherichia coli to cloacin DF13 involves the major outer membrane protein OmpF

Abstract

Fourteen spontaneous cloacin DF13-insensitive mutants of an Escherichia coli strain expressing the aerobactin-cloacin DF13 receptor protein IutA were isolated. The mutants fell into three classes on the basis of outer membrane profiles analyzed by electrophoresis in denaturing polyacrylamide gels. The most frequent class lacked the lutA protein and was unable to bind cloacin DF13 or aerobactin. A second class of mutants had lost protein species corresponding in size to the porin proteins OmpF and OmpC. To determine which porin was required for the bactericidal activity of cloacin DF13, defined strains with mutations at the ompB (ompR envZ) locus were transformed with a recombinant plasmid carrying the iutA gene and screened for cloacin DF13 sensitivity. OmpF-strains, whether OmpC+ or OmpC-, were insensitive to cloacin DF13, indicating involvement of the OmpF protein in cloacin DF13 killing. An OmpC-OmpF+ strain, on the other hand, was more sensitive than the wild-type parent strain, probably because of compensatory overexpression of OmpF. The third class of cloacin DF13-insensitive mutant had lost an outer membrane protein of approximately 31 kDa. The nature and function of this protein are not yet known, but it is not the protease OmpT. Mutants of classes 2 and 3 bound cloacin DF13 and aerobactin as effectively as the cloacin DF13-sensitive parental strain, indicating that they remained IutA+. We propose that these mutants (more accurately described as cloacin DF13 tolerant) are defective in translocation of the active portion of cloacin DF13 across the bacterial membranes.Cloacin DF13 is a bacteriocin produced by strains of Enterobacter cloacae that harbor plasmid CloDF13 (21). Killing of sensitive enteric bacteria by cloacin DF13 involves three distinct stages (13, 25): binding to a specific surface receptor protein, transport of an active fragment of the bacteriocin molecule through the cell envelope, and endoribonucleolytic cleavage of 16S rRNA, resulting in defective protein synthesis. The cloacin DF13 receptor in several enterobacterial species also acts as a receptor for the hydroxamate siderophore aerobactin (2,14,25). Thus, strains of Escherichia coli that utilize aerobactin as a source of iron, a category that includes many pathogenic isolates (3,26), are sensitive to cloacin DF13 because they express the 74-kDa outer membrane aerobactin receptor protein lutA (2). Killing of E. coli by cloacin DF13, unlike many other bacteriocins, is not dependent on the cytoplasmic membrane protein TonB (20); the aerobactin receptor is therefore the only envelope-associated protein in E. coli reported to date to be required for cloacin DF13 activity. In this paper we show that the major outer membrane protein OmpF, and possibly an outer membrane protein of 31 kDa, are also essential for the lethal action of cloacin DF13. MATERIALS AND METHODSBacteria and culture conditions. The bacterial strains used in this study are described in Table 1. Plasmid pLG141 comprises a 6.5-kb BamHI fragment of plasmid that includes the...

Role of tol genes in cloacin DF13 susceptibility of Escherichia coli K-12 strains expressing the cloacin DF13-aerobactin receptor IutA

“…This strain was completely resistant to lysis by either bacteriophage K20 or colicin N, whereas the isogenic ompF+ strain MC4100 was susceptible to both. Therefore, our results do not support the conclusions by Wooldridge and Williams (36) and demonstrate that OmpF is not involved directly in cloacin DF13 susceptibility. Nevertheless, it is possible that OmpF may play a minor role in the cloacin susceptibility of E. coli because we observed that MH610 was slightly less sensitive to cloacin (two serial dilutions) than the parent strain MC4100 (Fig.…”

“…Wooldridge and Williams recently reported that the major outer membrane protein OmpF was essential for the lethal action of cloacin DF13 in E. coli (36). It has been shown previously that toL4 and toiC mutations reduce the expression of OmpF (13,20).…”

Survival of uropathogenic Escherichia coli in the murine urinary tract is dependent on OmpR

“…Changes in expression of the surface structures could also have an impact on the ability of UPEC to survive in a mouse or a human urinary tract. In addition, OmpR appears to regulate expression of enterochelin and the aerobactin receptor protein IutA (Oshima et al, 2002;Wooldridge & Williams, 1991). Iron acquisition is critical for UPEC survival in the urinary tract.…”

Monoclonal Antibodies Directed Against the Outer Membrane Protein of Bordetella avium

“…HM545299) was selected from preserved B. avium. (19) The aforementioned strain has a higher virulence and better cross-reactivity with other isolates of B. avium. The strains were cultured in bouillon medium at 37°C for 18 h. The bacteria were collected by centrifugation at 10,000 g for 15 min at 4°C, and then washed three times with chilled phosphatebuffered saline (PBS).…”