Congratulations to Pratap Vanga for completing his PhD.
Supervisor: Prof Clive Ronson
Thesis tite: The molecular basis for mobility of HAI2 in Pectobacterium atrosepticum.
Abstract: Horizontally acquired island 2 (HAI2), one of seventeen putative HAIs identified on the chromosome of Pba SCRI1043, encodes an important phytotoxin, coronafacic acid (CFA), that is a major virulence determinant required for infection of potato. The island is highly similar to pathogenicity islands (PAIs) belonging to the clc family of integrative and conjugative elements (ICEs). A quantitative PCR assay developed to detect the presence of excised HAI2 in bacterial populations showed excision of HAI2 from the chromosome. Excision of HAI2 required the HAI2-encoded protein XerC/D recombinase (Int), which likely catalyses unidirectional site-specific recombination between the attachment sites present on excised HAI2 (attP) and the phe-tRNA gene (attB), respectively.
HAI2 encodes one (PbTopo IIIβ; locus tag: ECA0525) of two putative topoisomerase III enzymes predicted to be produced by Pba SCRI1043. Inactivation of PbTopo IIIβ caused a significant increase in the formation of the excised form of HAI2 both in vitro and in planta, reduced competitiveness in vitro and lowered virulence on potato stems. Microarray analysis of changes in Pba SCRI1043 gene expression upon inactivation of PbTopo IIIβ revealed that PbTopo IIIβ exerted a wide-ranging transcriptional influence across the genome of Pba SCRI1043 both in vitro and in planta. Overall, these results suggest that PbTopo IIIβ may control as yet unidentified genes involved in stable maintenance of HAI2 in the chromosome of Pba SCRI1043 as well as fitness and virulence of the bacterium on potato.
Stable maintenance of HAI2 in the host chromosome also required HAI2-encoded ECA0613 and non-HAI2-encoded quorum sensing (QS) gene expI. Inactivation of ECA0613 caused higher levels of the excised form of HAI2 of the island upon growth in planta but not in vitro. ECA0613 encodes a hypothetical protein with homology to putative relaxases associated with excision and conjugation of ICEs. Inactivation of the QS gene expI causes elevated levels of both attP and attB in planta, whereas formation of attP and attB was not induced in the mutant in vitro.
In summary, studies carried out in this thesis provided a novel insight into the molecular mechanisms involved in the stable maintenance of HAI2 and related ICEs in the chromosome of their host bacteria.
