Molecular characterization of bacteriocin-like activity in tomato race-three strains of Xanthomonas campestris pv. vesicatoria

Tomato race 3 (T3) strains of Xanthomonas campestris pv. vesicatoria are inhibitory to other strains within the species. Ten cosmid clones were isolated from a T3 strain genomic library, for their ability to inhibit a sensitive indicator strain in plate assays. Southern hybridization analysis grouped these into three subsets of overlapping activity, BCN-A+, BCN-B+, and BCN-C+ . Groups could be differentiated by variations in inhibitory spectrum and levels of activity in plate assays. Tn3-genetated mutants deficient in each were used to mutate individual wild-type loci. Marker-exchange mutants all retained inhibitory activity. Two N-methyl-N '-nitro-N-nitrosoguanidine (NTG) mutants deficient in wild-type BCN+ activity were obtained.;Restriction enzyme mapping, Tn3 mapping and subcloning were used to localize the genes responsible for expression of the BCN-A+ phenotype to a 8.0-kb KpnI/EcoRI fragment. Expression of this in a BCN-A- strain conferred both inhibitory activity and immunity to BCN-A+ (IMM-A+). Sequence analysis of the region revealed a cluster of 7 open reading frames (ORF). The largest ORF (bcnA), approximately 3.6-kb, was required for BCN-A+ activity. IMM-A+ was encoded by an ORF downstream of this. Homology searches with bcnA revealed no homology with any known genes involved in bacteriocin activity, however, it was homologous to wapA and Rhs elements. Both of these contain multiple copies of an almost identical ligand-binding motif. Seven copies of a similar motif were found in bcnA. BCN-A+ activity was found to be analogous to bacteriocin activity, and the name vesicacin A is proposed for the newly described bacteriocin. A bcnA-specific probe was homologous to genomic DNA of T3 strains and a bacteriocin-producing strain of X. c. pv. glycines. Tn3-gus::bcnA translational fusions were used to demonstrate expression of this gene in planta and in selected media types.;T3 strains were inhibitory to a sensitive indicator strain in planta, but this effect was only observed when T3 strains were applied in advance of the sensitive strain. NTG-mutant analysis showed that production of these antimicrobial compounds was important in mediating the observed antagonism. Further analysis revealed vesicacin A to be an essential and dominant component in the suppression of the sensitive indicator strain in tomato leaf tissues.