White Rose University Consortium logo
University of Leeds logo University of Sheffield logo York University logo

The identification of genes important to the growth of Staphylococcus aureus in in vitro models mimicking infection.

Wiltshire, Michael David (2001) The identification of genes important to the growth of Staphylococcus aureus in in vitro models mimicking infection. PhD thesis, University of Sheffield.

[img] Text (339938.pdf)
339938.pdf

Download (25Mb)

Abstract

Staphylococcus aureus is a major pathogen, which causes a wide range of infections. Despite its obvious clinical importance, little is known about the mechanisms of pathogenesis. An in vitro model mimicking infection was developed in order to identify putative virulence determinants. The model involves the growth of S. aureus in serum under microaerobic conditions. All known virulence factors tested were shown not to be required for growth, or preferentially expressed, in serum. Tn917 transposon libraries of S. aureus were screened to identify genes preferentially expressed in serum, compared to a nutrient-rich growth medium. 73 clones were identified and the transposon insertion site was characterised for 23 of these clones. Analysis of sequence flanking the transposon insertion revealed the identity of the mutated loci. 10 out of 23 sequenced clones, contained transposons inserted within genes involved in the biosynthesis of the aspartate family of amino acids (lysine. threonine, methionine and isoleucine). These were: the two common pathway enzymes; aspartokinase (lysC) , and aspartate semi aldehyde dehydrogenase (asd) , along with; dihydrodipicolinate dehydrogenase (dapA), and cystathionine y-synthase (yjcf) , involved in the biosynthesis oflysine and methionine respectively. Analysis of methionine biosynthesis indicated that S. aureus possesses only a single pathway, which proceeds via cystathionine. Several genes encoding methionine biosynthetic enzymes were found clustered on the S. aureus chromosome. The genes lyse, asd and dapA were found to be encoded by the first three genes of an eight gene operon, which also contains three other genes involved in lysine biosynthesis. This operon named the dap operon, is the major lysine biosynthetic operon of S. aureus. lysC, asd and dapA were all found to be repressed at the transcriptional level primarily by lysine, although factors other than the availability of lysine may be responsible for the regulation of lysine biosynthetic gene expression in serum. lysC, asd and dapA were all found to be expressed in vivo, in a murine pyelonephritis model using both RT-PCR and TaqMan techniques. However, these genes were not found to be important in three murine pathogenicity models. Finally, in addition to the development of a model mimicking infection, and the identification of genes with a potentially important role in vivo, this thesis has enhanced our understanding of both methionine and lysine biosynthesis in S. aureus.

Item Type: Thesis (PhD)
Keywords: Pathogen; Pathogenesis; Virulence; Bacteria
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Molecular Biology and Biotechnology (Sheffield)
Identification Number/EthosID: uk.bl.ethos.339938
Depositing User: EThOS Import Sheffield
Date Deposited: 19 Jan 2017 15:29
Last Modified: 19 Jan 2017 15:29
URI: http://etheses.whiterose.ac.uk/id/eprint/14812

You do not need to contact us to get a copy of this thesis. Please use the 'Download' link(s) above to get a copy.
You can contact us about this thesis. If you need to make a general enquiry, please see the Contact us page.

Actions (repository staff only: login required)