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Staphylococcus aureus is a highly adaptable human pathogen in which differential gene expression is known to occur in response to environmental conditions, both in vitro (1, 8, 17) and in vivo (9). Previous reports have demonstrated that in vitro conditions can be used to mimic those in vivo, for example, the use of cell culture extracts and mammalian cell cultures (13, 17). In this study, a model system of growth in serum has been established and characterized.

Growth of S. aureus in serum. S. aureus 8325-4 (12) was grown in both brain heart infusion (BHI) broth and pig serum (Sigma) under aerobic and microaerobic conditions (8% O₂-5% CO₂-87% N₂). Microaerobic growth in serum resulted in a higher growth rate and yield than did aerobic growth (optical densities at 600 nm [OD₆₀₀], 6.1 and 4.2, respectively; 7 h) (results not shown). Growth in human serum produced trends identical to those seen in pig serum (results not shown). BHI gave a higher growth yield than did serum and, in contrast to the results for serum, in BHI aerobic growth was found to be optimal (OD_600, 7.9 [microaerobic] and 10.2 [aerobic]; 7 h) (results not shown).

Identification of serum-expressed genes (seg). Genes specifically induced in serum versus BHI were identified by replica plating Tn917 insertion libraries (19) on serum agar and BHI agar, both containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) (80 µg/ml). Following incubation at 37°C (microaerobic for serum and aerobic for BHI), colonies that were blue on serum and white on BHI were selected and rescreened. Twenty-three clones with increased LacZ activity on serum were selected and further characterized. No growth defects on serum or BHI were observed for any of the clones.

Identification of seg loci. Following marker rescue cloning and DNA sequencing (19), transposon insertion sites were identified. In total, nine different seg genes were identified (Table 1). The genes insertionally inactivated in mutants seg5, seg7, and seg37 are all likely to encode surface proteins. Thus, differentially expressed surface proteins that are produced by S. aureus in serum may have a role in virulence.

Analysis of lysC, asd, and dapA genes in S. aureus. Nine independent transposon insertions were identified in genes involved in the biosynthesis of lysine and the other aspartate family amino acids. The entire dap locus was sequenced from S. aureus strain 8325-4, leading to the identification of a putative eight-gene transcription unit termed the dap operon (Fig. 1). Protein homology suggests that the dap operon contains six genes involved in the biosynthesis of lysine. The dapB, ykuQ, and lysA genes encode dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, and diaminopimelate decarboxylase, respectively. Notably, the dap genes are in the same order as they appear in the biosynthetic pathway (Fig. 1) (17).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Diagrammatic representation of the dap operon of S. aureus, showing the sequenced regions cloned in pSEG10 and pMTOPO9 and those cloned to generate the lacZ transcriptional fusion strains. Shaded genes are putatively involved in lysine biosynthesis. Tn917 insertions from left to right: aspartokinase II (lysC) control-leader region (putative)seg22 (siblings seg30 and seg33); aspartokinase II (lysC)seg14, seg25 (bottom, sibling seg50), seg45 (top, sibling seg48), seg13, and seg1 (sibling seg21); aspartate semialdehyde dehydrogenase (asd)seg24 and seg26; and dihydrodipicolinate synthase (dapA)seg10. ABC, ATP-binding cassette.

Amino acid requirements of selected seg mutants. A chemically defined medium (19) was adapted to allow the analysis of potential aspartate family amino acid auxotrophy (results not shown). These studies revealed that a mutation in lysC, asd, or dapA leads to lysine auxotrophy. Interestingly, while a mutation within the common pathway enzyme gene asd leads to an additional requirement for methionine and threonine, a lysC mutation does not. The phenotype of the lysC mutant can be explained only by the presence of multiple aspartokinase isozymes (as in B. subtilis) able to rescue the methionine and threonine biosynthesis functions of the lysC-encoded isozyme, but not lysine biosynthesis. The presence in S. aureus of a likely second aspartokinase isozyme homologous to aspartokinase III (yclM) of B. subtilis (44% over 171 amino acids) was confirmed by BLAST analysis of S. aureus databases (results not shown). Additionally, due to the genetic organization of the dap operon, where a lysC mutation is polar on asd, it is likely that a further promoter upstream of asd drives transcription independently of lysC.

Analysis of the expression and regulation of lysC, asd, and dapA. Reporter gene fusions MDW41 (lysC::lacZ), MDW42 (asd::lacZ), and MDW43 (dapA::lacZ), containing the fragments shown in Fig. 1, were cloned as BamHI-EcoRI PCR fragments into similarly digested pAZ106 (6). Recombinant plasmids were introduced into S. aureus RN4220 by electroporation (14), and the resulting chromosomal fusions were then transduced into S. aureus 8325-4 by phage transduction (12) and verified by Southern blot analysis (results not shown). The fusion strains all contained an intact copy of the dap operon. LacZ activity was measured as previously described (5).

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Expression of lysC, asd, and dapA in different chemically defined media (CDM). Shown is analysis of transcription (closed symbols) of lysC::lacZ (MDW41) (diamonds), asd::lacZ (MDW42) (circles), and dapA::lacZ (MDW43) (triangles). Basal CDM was prepared without the aspartate family amino acids (lysine [Lys], methionine [Met], threonine [Thr], and isoleucine [Ile]). Supplements to 100 mg of Lys and Met per liter and to 150 mg of Thr and Ile per liter were added as follows: a, none; b, Lys, Met, Thr, and Ile; c, Lys; and d, Met, Thr, and Ile. A representative growth curve (MDW41) is shown on each graph (OD600 [open squares]). MUG, methylumbelliferyl--D-galactoside.

In vivo analysis of lysC, asd, and dapA. Using reverse transcription PCR and a murine pyelonephritis model (11), lysC, asd, and dapA were all shown to be expressed in vivo (results not shown). Three murine infection models (mouse abscess [2], pyelonephritis, and wound infection [11]) were used to investigate the role of lysC, asd, and dapA in vivo. In all three models, there was no significant difference between the number of cells recovered from the host following a 7-day infection for any of the mutant strains and the number recovered for the wild-type strain (results not shown). However, Lys biosynthetic components, including asd, have been shown to have roles in pathogenesis, as they have been identified during signature-tagged mutagenesis screening of S. aureus using bacteremia models of infection (3, 10).

Nucleotide sequence accession number. The sequence determined for the entire dap locus from S. aureus strain 8325-4 has been deposited in GenBank under accession number AF306669.