In Gram-positive bacteria, the transcriptional regulator CcpA is at the core of catabolite control mechanisms. In the human pathogen Streptococcus pneumoniae, links between CcpA and virulence have been established, but its role as a master regulator in different nutritional environments remains to be elucidated. Thus, we performed whole-transcriptome and metabolic analyses of S. pneumoniae D39 and its isogenic ccpA mutant during growth on glucose or galactose, rapidly and slowly metabolized carbohydrates presumably encountered by the bacterium in different host niches. CcpA affected the expression of up to 19% of the genome covering multiple cellular processes, including virulence, regulatory networks and central metabolism. Its prevalent function as a repressor was observed on glucose, but unexpectedly also on galactose. Carbohydrate-dependent CcpA regulation was also observed, as for the tagatose 6-phosphate pathway genes, which were activated by galactose and repressed by glucose. Metabolite analyses revealed that two pathways for galactose catabolism are functionally active, despite repression of the Leloir genes by CcpA. Surprisingly, galactose-induced mixed-acid fermentation apparently required CcpA, since genes involved in this type of metabolism were mostly under CcpA-repression. These findings indicate that the role of CcpA extends beyond transcriptional regulation, which seemingly is overlaid by other regulatory mechanisms. In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation. Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels. Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall. Hence, CcpA may play a key role in mediating the interaction of S. pneumoniae with its host. Overall, our results support the hypothesis that S. pneumoniae optimizes basic metabolic processes, likely enhancing in vivo fitness, in a CcpA-mediated manner.

SummaryHigh levels of copper are toxic and therefore bacteria must limit free intracellular levels to prevent cellular damage. In this study, we show that a number of pneumococcal genes are differentially regulated by copper, including an operon encoding a CopY regulator, a protein of unknown function (CupA) and a P1-type ATPase, CopA, which is conserved in all sequenced Streptococcus pneumoniae strains. Transcriptional analysis demonstrated that the cop operon is induced by copper in vitro, repressed by the addition of zinc and is autoregulated by the copperresponsive CopY repressor protein. We also demonstrate that the CopA ATPase is a major pneumococcal copper resistance mechanism and provide the first evidence that the CupA protein plays a role in copper resistance. Our results also show that copper homeostasis is important for pneumococcal virulence as the expression of the cop operon is induced in the lungs and nasopharynx of intranasally infected mice, and a copA -mutant strain, which had decreased growth in high levels of copper in vitro, showed reduced virulence in a mouse model of pneumococcal pneumonia. Furthermore, using the copA -mutant we observed for the first time in any bacteria that copper homeostasis also appears to be required for survival in 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.

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.

Homeostasis of Zn2؉ and Mn 2؉ is important for the physiology and virulence of the human pathogen Streptococcus pneumoniae. Here, transcriptome analysis was used to determine the response of S. pneumoniae D39 to a high concentration of Zn 2؉ . Interestingly, virulence genes encoding the choline binding protein PcpA, the extracellular serine protease PrtA, and the Mn 2؉ uptake system PsaBC(A) were strongly upregulated in the presence of Zn 2؉ . Using random mutagenesis, a previously described Mn 2؉ -responsive transcriptional repressor, PsaR, was found to mediate the observed Zn 2؉ -dependent derepression. In addition, PsaR is also responsible for the Mn 2؉ -dependent repression of these genes. Subsequently, we investigated how these opposite effects are mediated by the same regulator. In vitro binding of purified PsaR to the prtA, pcpA, and psaB promoters was stimulated by Mn 2؉ , whereas Zn 2؉ destroyed the interaction of PsaR with its target promoters. Mutational analysis of the pcpA promoter demonstrated the presence of a PsaR operator that mediates the transcriptional effects. In conclusion, PsaR is responsible for the counteracting effects of Mn 2؉and Zn 2؉ on the expression of several virulence genes in S. pneumoniae, suggesting that the ratio of these metal ions exerts an important influence on pneumococcal pathogenesis.

In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous b-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.

To find the right conditions to isolate natively expressed antimicrobial peptides from a wide range of different microorganisms can be a challenge. Here, we exploited a heterologous expression system to produce and characterize several novel lantibiotics. We identified 54 novel putative class I and class II lantibiotics after inspecting all publicly available prokaryotic genomes using the in-house developed mining tool BAGEL3. The genes encoding these new lantibiotics fused to the nisin leader peptide gene sequence were synthesized, and the constructs were plugged into the nisin expression and modification system. Using this approach 30 peptides could be expressed, 27 of which were dehydrated by NisBC on at least 1 predicted position. Good antimicrobial activity against several pathogenic bacteria could be demonstrated for 5 novel heterologously modified lantibiotics. Lantibiotics from Corynebacterium lipophiloflavum DSM 44291 and Streptococcus agalactiae ATCC 13813, named flavucin and agalacticin, respectively, were fully modified and displayed high antimicrobial activity. The efficiency of functional expression was significantly enhanced when we made use of the native nisin leader cleavage site, instead of an artificial factor Xa site. Thus, we describe an efficient way for heterologous production of active lantibiotics, facilitating a rapid identification of promising molecules.

Zinc (Zn(2+)) is an important trace metal ion that has been shown to regulate the expression of several (virulence) genes in streptococci. Previously, we analyzed the genome-wide response of S. pneumoniae to Zn(2+)-stress. In this work, we have performed a transcriptomic analysis to identify genes that are differentially expressed under intracellular Zn(2+) limitation. This revealed a number of genes that are highly upregulated in the absence of extracellular Zn(2+), amongst which the genes belonging to the regulon of the Zn(2+)-responsive repressor AdcR, like adcBCA, encoding a Zn(2+)-dependent ABC-uptake system, adcAII, encoding a Zn(2+)-binding lipoprotein, and also virulence genes belonging to the Pht family (phtA, phtB, phtD and phtE). Using transcriptome analysis, lacZ-reporter studies, in vitro DNA binding experiments, and in silico operator predictions, we show that AdcR directly represses the promoters of adcRCBA, adcAII-phtD, phtA, phtB and phtE in the presence of Zn(2+). AdcR can also function as an activator, since in the presence of Zn(2+) it directly induces expression of adh that encodes a Zn(2+)-containing alcohol dehydrogenase. In conclusion, the genome-wide transcriptional response of S. pneumoniae to Zn(2+) limitation was established, which is mainly mediated via direct regulation by the Zn(2+)-dependent regulator AdcR.