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dc.contributor.advisorRonson, Clive
dc.contributor.advisorPitman, Andrew
dc.contributor.authorVanga, Bhanupratap Reddy
dc.date.available2013-08-01T04:29:33Z
dc.date.copyright2013
dc.identifier.citationVanga, B. R. (2013). The molecular basis for mobility of HAI2 in Pectobacterium atrosepticum (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/4186en
dc.identifier.urihttp://hdl.handle.net/10523/4186
dc.description.abstractHorizontally 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). Sequence characterisation revealed that HAI2 is a 97.87 kb chromosomal fragment integrated at the 3′ end of a phe-tRNA gene located adjacent to ECA0515. A quantitative PCR assay developed to detect the presence of excised HAI2 in bacterial populations showed excision of HAI2 from the chromosome, a process that is independent of growth phase and that results in the production of a circular intermediate both in vitro (in free-living culture) and in planta. Excision of HAI2 required the HAI2-encoded protein XerC/D recombinase (Int), which likely catalyses site-specific recombination between the direct repeats (DR) at each end of the island (attL and attR), and/or the attachment sites present on the excised form of HAI2 (attP) and the phe-tRNA gene (attB). 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 (103-104-fold) 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. Reduced virulence did not appear to be due to a reduction in transcription of the CFA biosynthetic gene cluster. However, 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. 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 ECA0521 also caused higher levels of the excised form of HAI2 both in vitro and in planta. ECA0521 encodes a hypothetical protein with no detectable conserved domains. However, the increased levels of the excised form of HAI2 could not be complemented upon transformation of the mutant with ECA0521 provided in trans. Quorum sensing (QS) moderated the formation of the excised form of HAI2 in planta. 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 addition, like topBβ and ECA0613, inactivation of expI altered transcription of numerous ORFs belonging to HAI2 in planta. 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.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectHorizontally acquired island 2 (HAI2)
dc.subjectpathogenicity islands (PAIs)
dc.subjectintegrative and conjugative elements (ICEs)
dc.subjectPectobacterium atrosepticum
dc.subjecttopoisomerase
dc.subjectExcision
dc.subjectputative relaxases
dc.subjectQuorum sensing (QS)
dc.titleThe molecular basis for mobility of HAI2 in Pectobacterium atrosepticum
dc.typeThesis
dc.date.updated2013-08-01T01:30:18Z
dc.language.rfc3066en
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanyes
otago.openaccessAbstract Only
otago.evidence.presentYes
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