CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.

Several genes involved in nitrogen metabolism are known to contribute to the virulence of pathogenic bacteria. Here, we studied the function of the nitrogen regulatory protein GlnR in the Gram-positive human pathogen Streptococcus pneumoniae. We demonstrate that GlnR mediates transcriptional repression of genes involved in glutamine synthesis and uptake (glnA and glnPQ), glutamate synthesis (gdhA), and the gene encoding the pentose phosphate pathway enzyme Zwf, which forms an operon with glnPQ. Moreover, the expression of gdhA is also repressed by the pleiotropic regulator CodY. The GlnR-dependent regulation occurs through a conserved operator sequence and is responsive to the concentration of glutamate, glutamine, and ammonium in the growth medium. By means of in vitro binding studies and transcriptional analyses, we show that the regulatory function of GlnR is dependent on GlnA. Mutants of glnA and glnP displayed significantly reduced adhesion to Detroit 562 human pharyngeal epithelial cells, suggesting a role for these genes in the colonization of the host by S. pneumoniae. Thus, our results provide a thorough insight into the regulation of glutamine and glutamate metabolism of S. pneumoniae mediated by both GlnR and GlnA.

The large-scale transcriptional program of two Clostridium acetobutylicum strains (SKO1 and M5) relative to that of the parent strain (wild type [WT]) was examined by using DNA microarrays. Glass DNA arrays containing a selected set of 1,019 genes (including all 178 pSOL1 genes) covering more than 25% of the whole genome were designed, constructed, and validated for data reliability. Strain SKO1, with an inactivated spo0A gene, displays an asporogenous, filamentous, and largely deficient solventogenic phenotype. SKO1 displays downregulation of all solvent formation genes, sigF, and carbohydrate metabolism genes (similar to genes expressed as part of the stationary-phase response in Bacillus subtilis) but also several electron transport genes. A major cluster of genes upregulated in SKO1 includes abrB, the genes from the major chemotaxis and motility operons, and glycosylation genes. Strain M5 displays an asporogenous and nonsolventogenic phenotype due to loss of the megaplasmid pSOL1, which contains all genes necessary for solvent formation. Therefore, M5 displays downregulation of all pSOL1 genes expressed in the WT. Notable among other genes expressed more highly in WT than in M5 were sigF, several two-component histidine kinases, spo0A, cheA, cheC, many stress response genes, fts family genes, DNA topoisomerase genes, and central-carbon metabolism genes. Genes expressed more highly in M5 include electron transport genes (but different from those downregulated in SKO1) and several motility and chemotaxis genes. Most of these expression patterns were consistent with phenotypic characteristics. Several of these expression patterns are new or different from what is known in B. subtilis and can be used to test a number of functional-genomic hypotheses.The recent completion and tentative annotation of numerous genome sequences, and the many to follow, create both a wealth of new information for comparative genomic studies and enormous challenges. The latter include the need to develop fast if not high-throughput strategies to understand the major cellular programs of the newly sequenced organisms and to begin to assign functions to groups of genes or individual genes. These challenges are made especially difficult for the majority of cases where the genetics of the organism are not well developed, few genes have been transcriptionally examined or otherwise functionally assigned, mutants are scarce, and the closest related organisms are also minimally understood at the genomic level. The spore-forming strict anaerobe Clostridium acetobutylicum is typical of this situation. Its genome has been sequenced and computer annotated (18) and its physiology has been extensively studied, but only a small number of genes have been studied and functionally identified. The number of available mutants is small, and chromosomal integration for genetic studies remains a major hurdle. None of the other closely or distantly related clostridia is understood genetically (let alone genomically) any better than C. acetobutylicum, and thu...

The transcriptional regulator GlnR of Streptococcus pneumoniae is involved in the regulation of glutamine and glutamate metabolism, controlling the expression of the glnRA and glnPQ-zwf operons, as well as the gdhA gene. To assess the contribution of the GlnR regulon to virulence, D39 wild-type and mutant strains lacking genes of this regulon were tested in an in vitro adherence assay and murine infection models. All of the mutants, except the ⌬glnR mutant, were attenuated in adherence to human pharyngeal epithelial Detroit 562 cells, suggesting a contribution of these genes to adherence during the colonization of humans. During murine colonization, only the ⌬glnA mutant and the glnP-glnA double mutant (⌬glnAP) were attenuated, in contrast to ⌬glnP, indicating that the effect is caused by the lack of GlnA expression. In our pneumonia model, only ⌬glnP and ⌬glnAP showed a significantly reduced number of bacteria in the lungs and blood, indicating that GlnP is required for survival in the lungs and possibly for dissemination to the blood. In intravenously infected mice, glnP and glnA were individually dispensable for survival in the blood whereas the ⌬glnAP mutant was avirulent. Finally, transcriptome analysis of the ⌬glnAP mutant showed that many genes involved in amino acid metabolism were upregulated. This signifies the importance of glutamine/glutamate uptake and synthesis for full bacterial fitness and virulence. In conclusion, several genes of the GlnR regulon are required at different sites during pathogenesis, with glnA contributing to colonization and survival in the blood and glnP important for survival in the lungs and, possibly, efficient transition from the lungs to the blood.

Recent murine studies have demonstrated that the role of response regulator 09 (RR09) of Streptococcus pneumoniae in virulence is different in different strains. In the present study, we used a murine pneumonia model of infection to assess the virulence of a TIGR4 rr09 mutant, and we found that TIGR4⌬rr09 was attenuated after intranasal infection. Furthermore, we investigated the in vitro transcriptional changes in pneumococcal rr09 mutants of two strains, D39 and TIGR4, by microarray analysis. The transcriptional profiles of the rr09 mutants of both strains had clear differences compared to the profiles of the parental wild-type strains. In D39⌬rr09, but not in TIGR4⌬rr09, genes involved in competence (e.g., comAB) were upregulated. In TIGR4, genes located on the rlrA pathogenicity islet, which are not present in the D39 genome, appeared to be regulated by RR09. Furthermore, several phosphotransferase systems (PTSs) believed to be involved in sugar uptake (e.g., the PTS encoded by sp0060 to sp0066) were strongly downregulated in D39⌬rr09, while they were not regulated by RR09 in TIGR4. To examine the role of one of these PTSs in virulence, D39⌬sp0063 was constructed and tested in a murine infection model. No difference between the virulence of this strain and the virulence of the wild type was found, indicating that downregulation of the sp0063 gene alone is not the cause of the avirulent phenotype of D39⌬rr09. Finally, expression of rr09 and expression of three of our identified RR09 targets during infection in mice were assessed. This in vivo experiment confirmed that there were differences between expression in wild-type strain TIGR4 and expression in the rr09 mutant, as well as differences between expression in wild-type strain D39 and expression in wild-type strain TIGR4. In conclusion, our results indicate that there is strain-specific regulation of pneumococcal gene expression by RR09.