The role of genomic islands in virulence of Pectobacterium carotovorum subspecies brasiliensis on potatoes

Pectobacterium carotovorum subsp. carotovorum is primarily responsible for soft rotting of potato tubers, although several strains have been shown to cause blackleg of potato stems. In contrast, P. atrosepticum is best known as a seedborne pathogen that causes blackleg. It is also responsible for soft rotting of tubers. Given the taxonomic distance between these two species and that only some strains of P. carotovorum subsp. carotovorum are able to cause blackleg, it was hypothesised that the capacity of P. carotovorum subsp. carotovorum to invade potato stems and elicit blackleg may have evolved through independent acquisition of genomic islands (GIs). GIs are large chromosomal regions in bacteria that are acquired by horizontal gene transfer and often encode virulence factors. To address this hypothesis, the genomes of P. carotovorum subsp. carotovorum strains associated with blackleg disease of potatoes were compared to those of non-blackleg causing strains, to identify GIs in blackleg causing strains and novel virulence factors encoded on these islands. First, the identity of a highly aggressive P. carotovorum subsp. carotovorum strain ICMP19477 and other New Zealand isolates known to cause blackleg, was re-assessed using molecular and phylogenetic assays. These assays identified the isolates as P. carotovorum subsp. brasiliensis. Comparative genomics using the genome sequence obtained for ICMP19477 and three other Pectobacterium in this study, as well as the genomes of 10 soft rot erwiniae (SRE) obtained from public databases, identified a total of 69 genomic islands and 10 gene islets in the genome of ICMP19477. Many of these islands and islets harboured genes predicted to be associated with the virulence of this pathogen (e.g. genes encoding putative plant cell wall degrading enzymes, phytotoxins, secretion systems, sugar utilisation, etc.), yet only one was present exclusively in the P. atrosepticum and P. carotovorum subsp. brasiliensis isolates historically known to cause blackleg. This islet (sim gene islet) encodes a sugar:phosphotransferase system, which is important in the pathogenicity of vascular plant pathogens such as Erwinia amylovora. The majority of other GIs and islets carrying putative virulence genes were either not present in the genomes of all blackleg causing strains or were present in strains that did not cause blackleg (e.g. GI PbN1_GI24, which encoded a putative non-ribosomal peptide with similarity to syringomycin). To confirm the role of several putative virulence factors in virulence of P. carotovorum subsp. brasiliensis ICMP19477, knockout mutants were constructed by allelic exchange mutagenesis. Mutations in the non-ribosomal peptide synthetase cluster of P. carotovorum subsp. brasiliensis (on PbN1_GI24) did not result in significant differences in virulence of the wild type and the mutant in pathogenicity assays on potato. In contrast, single crossover mutants in the islets encoding the sugar:phosphotransferase system and phenolic acid decarboxylase significantly reduced virulence of the pathogen. Complementation studies still need to be conducted to prove their role in disease. In summary, this study has provided new insights into the impact of GIs on the virulence of P. carotovorum subsp. braisliensis and also on the mechanisms by which SRE cause diseases. These data suggest that the accumulation of multiple virulence factors on these elements might play a larger role in virulence of Pectobacterium than the acquisition of a specific gene or gene cluster.