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.

FIG. 1.

Growth of the S. aureus A22223I parental wild-type strain (⧫) and its isogenic hemB mutant (▪) in TSB (with standard deviations). The times of sampling for transcriptional analysis are indicated by the symbols on the growth curves. Time points for RNA isolation were selected as previously described (12). Optical density was measured at 578 nm.

FIG. 2.

Clustered top-scoring reporter metabolites (P values < 0.05) which occurred at more than one time point. Black boxes denote time points when the respective metabolite that occurred was identified as significant. CoA, coenzyme A; FAD, flavin adenine dinucleotide; FADH₂, reduced flavin adenine dinucleotide.

FIG. 3.

Illustration of significant transcriptional changes in the context of metabolic pathways of central carbon metabolism based on the combined analysis of all time points. Metabolites in gray circles denote reporter metabolites (P values < 0.05). Gene names are given in italics. A gene's down-regulation in the hemB mutant compared to the parental strain is indicated by a red box around the gene locus (based on the S. aureus N315 sequence), and a gene's up-regulation is indicated by a green box. Dark colors denote strong down- and up-regulation. PEP, phosphoenolpyruvate; FAD, flavin adenine dinucleotide; FADH₂, reduced flavin adenine dinucleotide.