Reporter Metabolite Analysis of Transcriptional Profiles of a Staphylococcus aureus Strain with Normal Phenotype and Its Isogenic hemB Mutant Displaying the Small-Colony-Variant Phenotype

Abstract

In this study, full-genome DNA microarrays based on the sequence of Staphylococcus aureus N315 were used to compare the transcriptome of a clinical S. aureus strain with a normal phenotype to that of its isogenic mutant with a stable small-colony-variant (SCV) phenotype (hemB::ermB). In addition to standard statistical analyses, systems biology advances were applied to identify reporter metabolites and to achieve a more detailed survey of genome-wide expression differences between the hemB mutant and its parental strain. Genes of enzymes involved in glycolytic and fermentative pathways were found to be up-regulated in the hemB mutant. Furthermore, our analyses allowed identification of additional differences between the normal-phenotype S. aureus and the SCV, most of which were related to metabolism. Profound differences were identified especially in purine biosynthesis as well as in arginine and proline metabolism. Of particular interest, a hypothetical gene of the Crp/Fnr family (SA2424) that is part of the arginine-deiminase (AD) pathway, whose homologue in Streptococcus suis is assumed to be involved in intracellular persistence, showed significantly increased transcription in the hemB mutant. The hemB mutant potentially uses the up-regulated AD pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. Moreover, genes involved in capsular polysaccharide and cell wall synthesis were found to be significantly up-regulated in the hemB mutant and therefore potentially responsible for the changed cell morphology of SCVs. In conclusion, the identified differences may be responsible for the SCV phenotype and its association with chronic and persistent infections.

The Mevalonate Pathway ofStaphylococcus aureus

“…This pathway converts arginine to carbamoyl phosphate, which is subsequently used to generate ATP. It has been postulated that this pathway acts as an ATP source in small colony variants lacking a functional respiratory chain (10,24), an idea that is supported by the fact that respiration is severely downregulated in uninduced RN4220spacmvaS, RN4220spacmvaA, and RN4220spacmvaK. Finally, further confirming that downregulation of the mevalonate pathway has a significant effect on cell wall biosynthesis, drp35, a stress lactonase shown to be upregulated in response to beta-lactams, bacitracin, vancomycin, and fosfomycin (17,18), was upregulated between 6 and 10-fold in all 3 downregulated mutants (see Table S2 in the supplemental material, p. 5).…”

Immunomodulation of Nasal Epithelial Cells by Staphylococcus aureus-Derived Serine Proteases

“…The sitedirected hemB mutants represent a stable and genetically defined SCV phenotype (18,20,21), and mutant III33 has been shown to produce almost 20 times more protease than its parental strain (16).…”

In Vivo Mutations of Thymidylate Synthase (Encoded by thyA ) Are Responsible for Thymidine Dependency in Clinical Small-Colony Variants of Staphylococcus aureus

“…The levels of mRNA expression of the different genes were normalized using the expression of the internal control gene yqiL (encoding acetyl-coenzyme A acetyltransferase), which is a housekeeping gene successfully used for quantitative RT-PCR (16,26). The levels of transcripts were expressed as increases (n-fold) relative to the values for the internal control (4).…”