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.