Summerfield, Andrew (2009) Role of co-factor biosynthesis in Listeria monocytogenes virulence. PhD thesis, University of Warwick.

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Abstract

Pathogenesis in L. monocytogenes is generally well characterised and understood, but
the mechanisms of intracellular growth and survival, and how L. monocytogenes
changes in the intracellular environment are less well understood. The study of L.
monocytogenes strains which display abnormal growth in tissue culture, yet display
wild type growth in broth, resulted in the identification of several genes important in
pathogenesis. Several of these genes were identified as known virulence factors such
as plcA and actA, however several strains were not defective for known virulence
genes, yet displayed an abnormal intracellular phenotype. Three such strains were
studied in this work, DP-L793 (aig3), DP-L2214 (aig4) and DP-L1107 (plq). These
strains were the result of transposon mutagenesis, but only in DP-L2214 was the
transposon known to be linked to the virulence phenotype (David Hodgson, personal
communication).
A transposon tagging approach revealed a region thought to contain the mutation
responsible for the Aig3 phenotype; however sequencing of this region did not reveal
any base changes in any open reading frame. A point mutation was identified in the
putative promoter of the para aminobenzoic acid operon, genes coding for an
important enzyme in folate biosynthesis. The expression of the pabA gene was shown
to be abolished in broth culture, and after starvation in minimal media, DP-L793 was
shown to be folate requiring. The DP-L1107 strain was previously shown to be a
threonine auxotroph, and also displayed a small plaque phenotype in tissue culture.
The potential link between these two phenotypes was investigated by phage
transduction. It was determined that no link exists between the two phenotypes of DPL1107.
The aig4 strain, DP-L2214 has the lipoate protein ligase gene lmo0931
disrupted by the transposon Tn917. A second putative copy of this gene was identified
by genome sequencing. Attempts were made to perform deletions of both copies of
the lipoate protein ligase genes however no deletions were successfully made.
Expression of these genes was monitored in broth, and as expected lmo0931
expression was not detected in the DP-L2214 strain, however lmo0764 expression
was detected. Yeast-Two-Hybrid assays were used to investigate the interactions
between the lipoate protein ligases and potential partners from the lipoate dependent
enzyme systems, the pyruvate dehydrogenase complex, the branched chain alpha keto
acid dehydrogenase and the glycine cleavage system. Expected interactions were
identified for the gene products of both copies of the lipoate protein ligase proteins, a
further interaction was noted for the gene product of lmo0764 and a protein of
unknown function, which is associated with the gene lmo0931 through proximity.
Phylogenetic analysis of the putative lipoate protein ligases, lmo931 and lmo0764
demonstrated that the two genes were divergent.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Listeria monocytogenes -- Genetics
Official Date:	August 2009
Dates:	Date Event August 2009 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Hodgson, D. A. (David A.)
Extent:	162 leaves : ill., charts
Language:	eng

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