Bacteriocin 28b production is induced by mitomycin in wild-type Serratia marcescens 2170 but not in Escherichia coli harboring the bacteriocin 28b structural gene (bss). Studies with a bss-lacZ transcriptional fusion showed that mitomycin increased the level of bss gene transcription in S. marcescens but not in the E. coli background. A S. marcescens Tn5 insertion mutant was obtained (S. marcescens 2170 reg::Tn5) whose bacteriocin 28b production and bss gene transcription were not increased by mitomycin treatment. Cloning and DNA sequencing of the mutated region showed that the Tn5 insertion was flanked by an SOS box sequence and three genes that are probably cotranscribed (regA, regB, and regC). These three genes had homology to phage holins, phage lysozymes, and the Ogr transcriptional activator of P2 and related bacteriophages, respectively. Recombinant plasmid containing this wild-type DNA region complemented the reg::Tn5 regulatory mutant. A transcriptional fusion between a 157-bp DNA fragment, containing the apparent SOS box upstream of the regA gene, and the cat gene showed increased chloramphenicol acetyltransferase activity upon mitomycin treatment. Upstream of the bss gene, a sequence similar to the consensus sequence proposed to bind Ogr protein was found, but no sequence similar to an SOS box was detected. Our results suggest that transcriptional induction of bacteriocin 28b upon mitomycin treatment is mediated by the regC gene whose own transcription would be LexA dependent.Serratia marcescens has been shown to produce bacteriocins upon induction with DNA-damaging agents (50). These bacteriocins have been classified into two groups (23): fraction 1 bacteriocins are active against Escherichia coli but not against S. marcescens, and fraction 2 bacteriocins are active against S. marcescens but not against E. coli (50). Bacteriocins belonging to fraction 1 are simple polypeptides that resemble colicins (50). Only colicin-like bacteriocins L and 28b have been studied in some detail. Bacteriocin L from S. marcescens JF246 has been isolated and characterized, and the effects of this bacteriocin on the incorporation of labelled leucine and thymidine and on the cellular levels of ATP in E. coli were similar to those produced by pore-forming colicins (17,18,40). On the other hand, the bacteriocin 28b structural gene (bss) has been cloned and sequenced, and the predicted amino acid sequence of the C-terminal part of this bacteriocin has been shown to have a high degree of similarity to the C-terminal domains of pore-forming colicins (56). The two bacteriocins are very closely related, if not the same, and similar bacteriocins are produced by most S. marcescens biotypes (21).Colicin production is induced by mitomycin and other DNAdamaging agents (38). Determinants for colicin production studied so far are encoded by either small high-copy-number colicinogenic plasmids (type I) or large low-copy-number colicinogenic plasmids (type II) (38). An important feature of these plasmids is that they confer on their host...
Serratia marcescens N28b produces bacteriocin 28b, active against Escherichia coli. Bacteriocin sensitivity tests performed on a collection of E. coli envelope mutants, and isolation and characterization of E. coli bacteriocin-28b-insensitive mutants, showed that the core lipopolysaccharide, outer membrane proteins OmpA and OmpF, and TolQ, TolA, and TolB proteins are involved in bacteriocin 28b lethal activity. These mutants are assayed for bacteriocin 28b sensitivity under normal and bypass conditions, and their bacteriocin-binding ability was determined. The results obtained suggest that the core lipopolysaccaride and outer membrane proteins OmpA and OmpF are involved in bacteriocin 28b binding. Furthermore, bacteriocin 28b translocation requires proteins TolA, TolB, and TolQ.
Serratia marcescens N28b synthesized and secreted a bacteriocin, with a molecular mass of 45 kDa, which was capable of inhibiting the growth of Escherichia coli. The expression of this bacteriocin was negligible unless induced with mitomycin C. The genes encoding the bacteriocin were cloned in plasmid pBR328. E. coli harbouring recombinant plasmid pBA189 or pBA289 expressed the Serratia rnarcescens N28b bacteriocin. The nucleotide sequence of the bss gene (Serratia marcescens N28b bacteriocin structural gene) was determined. The predicted amino acid sequence of the carboxy-terminal part of the bacteriocin 28b had a high degree of similarity to the poreforming domains of colicins A, El, B, N, Ia and Ib.
A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli and clones were screened for a bacteriocin 28b insensitive phenotype. One clone was found that showed partial resistance to bacteriocin 28b. By using TnStacl insertions it was shown that this phenotype was due to the expression in E. coli of an outer-membrane protein of 17 kDa (Omp4). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 515 bp. The deduced amino acid sequence has 172 residues with a theoretical molecular mass of 184 kDa. The protein contains an Nterminal signal sequence of 24 amino acid residues and, when compared to other enterobacterial outer-membrane proteins, most closely resembles a family of small outer-membrane proteins of Entembacteriaceae whose known functions appear to be related with virulence. lmmunoblotting experiments showed that Omp4 is present in 15 biotypes of 5. marcescens. The bacteriocin 28b resistance phenotype conferred on E. coli by Omp4 appears to be pleiotropic since overexpression of the Om@-encoding gene leads to a decrease in the amount of OmpA, OmpF and/or OmpC; OmpA and OmpF are the receptors for bacteriocin 28b in E. coli.
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