Molecular analysis of microcin 24 : Genetics, secretion and mode of action of a novel microcin
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Colicins and microcins are proteinaceous antimicrobial agents produced by members of the Enterobacteriaceae which are active against other members of this family. Colicin24 is a novel bacteriocin produced by a uropathogenic strain of Escherichia coli isolated at Christchurch Hospital. Through detailed genetic analysis of the DNA encoding this toxin and assaying the toxic activity, colicin 24 was re-classified as microcin 24 and has been shown to have a similar genetic organisation to that of colicin V and a novel mode of activity. The region of DNA encoding microcin 24 was subcloned from pGOB34 into pBR322 generating pGOB18 (5.44kb). Mutagenesis, DNA sequencing and transcomplementation identified two regions with high sequence similarity and functional homology to the ColV transporters CvaA and CvaB. The insert DNA of pGOB18 was sequenced in both directions and has been found to contain 5267bp encoding five open reading frames, mdbA, mtft, mtfS, mtfA and mtfB, forming three operons mdbA, mtfI/mtfS and mtfA/mtfB all of which were transcribed in the same direction. The predicted protein products of all the open reading frames except mtfB were confirmed by expressing the genes in minicells. Further mutagenesis and trans-complementation has identified mdbA as a cis acting positive regulatory gene with sequence similarity to the histone-like proteins. The mtfI and mtfS genes were confirmed as the Mcc24 immunity gene and the Mcc24 structural gene respectively. The genes mtfA and mtfB were found to encode the transport proteins homologous to CvaA and CvaB respectively, with mtfB encoding a protein which is a member of the ABC family of bacterial transporters. Transport also requires the TolC outer membrane protein. Analysis of the mtfS DNA sequence has identified a double glycine leader sequence, making MtfS the second microcin after ColV to belong to this class of peptide antibiotics. Experimental evidence suggested that unlike ColV, Mcc24 is inactive within the producing cell, however both toxins require the ABC transporter for post-translational modification of the pre-peptide. The regulation of Mcc24 synthesis is controlled by the interaction between σs, Fur, and MdbA, encoded by the mdbA gene. Analysis of the promoter sequences has identified putative regions of DNA bending which might facilitate the binding of σs and MdbA. A Fur-box with good sequence similarity to the consensus Fur-box has been identified in the mtfI/mtfS promoter and is the proposed site for Fur binding. The activity spectrum of Mcc24 is restricted to enteric bacteria and SernA, the MccE492 receptor, is also required as the receptor for Mcc24. Extracts of Mcc24 have been found to degrade both linearised and covalently closed circular DNA in vitro. The activity is absent in extracts from mtfS - strains, suggesting that Mcc24 inhibits the growth of sensitive cells by degrading DNA. The effect of Mcc24 expression on the virulence of E. coli was tested using the embryo lethality assay, however unlike ColV which increases the virulence of strains, the expression of Mcc24 did not appear to have a significant effect on E. coli virulence in this system.