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.

SummaryMaintenance of the intracellular homeostasis of metal ions is important for the virulence of many bacterial pathogens. Here, we demonstrate that the czcD gene of the human pathogen Streptococcus pneumoniae is involved in resistance against Zn 2+ , and that its transcription is induced by the transition-metal ions Zn 2+ , Co 2+ and Ni 2+ . Upstream of czcD a gene was identified, encoding a novel TetR family regulator, SczA, that is responsible for the metal ion-dependent activation of czcD expression. Transcriptome analyses revealed that in a sczA mutant expression of czcD, a gene encoding a MerR-family transcriptional regulator and a gene encoding a zinc-containing alcohol dehydrogenase (adhB) were downregulated. Activation of the czcD promoter by SczA is shown to proceed by Zn 2+ -dependent binding of SczA to a conserved DNA motif. In the absence of Zn 2+ , SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. This is the first example of a metalloregulatory protein belonging to the TetR family that has been described. The presence in S. pneumoniae of the Zn 2+ -resistance system characterized in this study might reflect the need for adjustment to a fluctuating Zn 2+ pool encountered by this pathogen during infection of the human body.

SummarySpi/Ssa is a unique type three secretion system that functions exclusively when Salmonella enterica is inside eukaryotic cells. Expression of the Spi/Ssa system and its secreted effectors is dependent on SsrB/ SpiR, a two-component regulatory system encoded in the SPI-2 pathogenicity island that also harbours the spi/ssa genes. Here we determine that the PhoP/PhoQ two-component system controls the intramacrophage expression of spi/ssa genes by regulating the SsrB/ SpiR system. We establish that PhoP regulates transcription of the response regulator SsrB and demonstrate binding of the PhoP protein to the ssrB promoter both in vivo using chromatin immunoprecipitation in Salmonella -infected macrophages, and in vitro using purified PhoP protein. We show that PhoP controls the SpiR sensor post-transcriptionally and identify a region in the 5 ¢ ¢ ¢ ¢ untranslated region of the spiR message that is required for this effect. The demonstration that the PhoP/PhoQ system is directly involved in the regulation of the SPI-2 pathogenicity island highlights PhoP/PhoQ's central role in Salmonella virulence. We suggest that different regulatory systems convey distinct signals over time to produce the SsrB/SpiR system, which then modulates expression of the Spi/Ssa apparatus and secreted effectors.

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comG promoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of ∼65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addAB promoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN 5 TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK-dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK.The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.