The expression of many virulence-associated genes in Streptococcus pyogenes is controlled in a growth phase-dependent manner. Unlike the model organisms Escherichia coli and Bacillus subtilis, such regulation is apparently not dependent upon alternative sigma factors but appears to rely on complex interactions among several transcriptional regulators, including Rgg. The purpose of this study was to identify changes in gene expression associated with inactivation of the rgg gene in S. pyogenes strain NZ131 (serotype M49). To this end, the transcriptomes of wild-type and rgg mutant strains were analyzed during both the exponential and postexponential phases of growth using Affymetrix NimbleExpress gene chips. Genomewide differences in transcript levels were identified in both phases of growth. Inactivation of rgg disrupted coordinate expression of genes associated with the metabolism of nonglucose carbon sources, such as fructose, mannose, and sucrose. The changes were associated with an inability of the mutant strain to grow using these compounds as the primary carbon source. Bacteriophage transcript levels were also altered in the mutant strain and were associated with decreased induction of at least one prophage. In order to survive, bacteria must be able to respond to changes in the environment and tolerate a variety of stressors. Such adaptation typically involves genomewide changes in transcription, as well as posttranscriptional and posttranslational changes. Escherichia coli and Bacillus subtilis use alternative sigma factors, such as RpoS and SigH, respectively, to coordinate changes in transcription upon entry into the stationary phase of growth. The mechanism facilitates rapid changes in expression without the need to assemble transcriptional complexes de novo. In contrast, the human pathogen Streptococcus pyogenes does not require alternate sigma factors to adapt to the stationary phase of growth (20,21). Rather, S. pyogenes is thought to rely on interactions among multiple transcriptional factors, including Mga, RofA-like proteins (RALP), two-component regulators (CovRS/CsrRS, FasBCAX, and Ihk/Irr), and Rgg (12). Importantly, these regulators also control the expression of many virulence factors, which are typically expressed in a growth phase-dependent manner. Identifying how such regulatory networks function is important in understanding the regulation of gene expression in S. pyogenes and in designing therapeutic strategies aimed at inhibiting virulence factor expression.

The transcriptional regulatory protein Rgg coordinates amino acid catabolism and virulence factor expression in Streptococcus pyogenes. We used a proteomic approach to compare cytoplasmic proteins isolated from S. pyogenes wild-type strain NZ131 (serotype M49) to proteins isolated from an rgg mutant strain during the exponential and stationary phases of growth. Proteins were separated by two-dimensional gel electrophoresis, and 125 protein spots of interest were identified by tandem mass spectrometry. Comparative analysis of proteins isolated from the isogenic strains revealed that growth phase-associated regulation of enzymes involved in the metabolism of arginine (ArcABC), histidine (HutI), and serine (SdhA) was abrogated in the rgg mutant strain, which synthesized the proteins in the exponential phase of growth. In contrast, the enzymes were detected only among wild-type proteins isolated from organisms in the stationary phase of growth. The differences in protein composition were correlated with previously described metabolic changes. In addition, proteins associated with thermal and oxidative stress responses, including ClpE and ClpL, were present in samples isolated from the rgg mutant strain but not in samples isolated from the wild-type strain. The rgg mutant strain was more tolerant to elevated temperature and puromycin than the wild-type strain; however, the mutant was less tolerant to paraquat. We concluded that Rgg is a global regulatory factor that contributes to growth phase-dependent synthesis of proteins associated with secondary metabolism and oxidative and thermal stress responses.

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10 3 CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.

Streptococcus pyogenes is responsible for approximately 500,000 deaths each year worldwide. Many of the associated virulence factors are expressed in a growth phase-dependent manner. To identify growth phase-associated changes in expression on a genomescale, the exponential and stationary phase transcriptomes and proteomes of S. pyogenes strain NZ131 (serotype M49) were compared by using Affymetrix NimbleExpress gene chips and two-dimensional gel electrophoresis. At the transcript level, the expression of 689 genes, representing approximately 40% of the chromosome, differed by twofold or more between the two growth phases. The majority of transcripts that were more abundant in the early-stationary phase encoded proteins involved in energy conversion, transport, and metabolism. At the protein level, an average of 527 and 403 protein spots were detected in the exponential and stationary phases of growth, respectively. Tandem mass spectrometry was used to identify 172 protein spots, 128 of which were growth phase regulated. Enzymes involved in glycolysis and pyruvate metabolism and several stress-responsive proteins were more abundant in the stationary phase of growth. Overall, the results identified growth phase-regulated genes in strain NZ131 and revealed significant post-transcriptional complexity associated with pathogen adaptation to the stationary phase of growth.

Group A Streptococcus is characterized by the ability to cause a diverse number of human infections including pharyngitis, necrotizing fasciitis, toxic shock syndrome, and acute rheumatic fever, yet the regulation of streptococcal genes involved in disease processes and survival in the host is not completely understood. Genome scale analysis has revealed a complex regulatory network including 13 two-component regulatory systems and more than 100 additional putative regulators, the majority of which remain uncharacterized. Among these is the streptococcal regulator of virulence, Srv, the first Group A Streptococcus member of the Crp/Fnr family of transcriptional regulators. Previous work demonstrated that the loss of srv resulted in a significant decrease in Group A Streptococcus virulence. To begin to define the gene products influenced by Srv, we combined microarray and two-dimensional gel electrophoresis analysis. Loss of srv results in a chromosome wide reduction of gene transcription and changes in the production of the extracellular virulence factors Sic (streptococcal inhibitor of complement) and SpeB (cysteine proteinase). Sic levels are reduced in the srv mutant, whereas the extracellular concentration and activity of SpeB is increased. These data link Srv to the increasingly complex GAS regulatory network.