In the Streptococcus pneumoniae genome, stkP, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of comCDE upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of comCDE transcription in balance with its repression by CiaRH.The human pathogen Streptococcus pneumoniae is highly versatile. Its response to environment determines the bacterium's virulence and transformability as shown by mutational studies targeting metabolic (1, 16), regulatory (4, 11), transport (5, 19), and signaling (6, 8) functions. The factors so far identified which influence the fate of the bacterium include mainly the concentrations of divalent cations (5,19,20) and H ϩ and dioxygen (6). Signaling involves two-component signal-transducing systems (6,8,13), an oxidase (7), and a global regulator (4). In the present work, we provide evidence for the role of the serine/threonine kinase StkP in competence signaling and in experimental virulence. StkP is membrane associated, carries the PASTA signature (22), and has been shown to avoid LytA-dependent autolysis induced by growth at alkaline pH and by low concentrations of cell wall-directed antibiotics. It is proposed that virulence expression and competence development represent population responses to cell wall stress induced in specific growth conditions. The rough S. pneumoniae RX derivatives (19) carrying the lytA mutation (17) and the smooth serotype 2 (2) strain D39 and serotype 6 strain 23477 (1) were used for competence tests and virulence studies, respectively. Bacterial growth and storage were as previously described (12,17,19). An insertion mutation in stkP was obtained in vitro with the pBluescript derivative (15) plasmid pPHK29 carrying a 2.96-kbp EcoRI/ SalI amplimer containing stkP from the RX chromosome. The 1.3-kbp BamHI fragment from pPJ1 (14) containing the aphA3 cassette was inserted into a BglII site of stkP to give the mutagenic plasmid pPKB3. The mutated stkP::aphA3 allele was introduced by genetic transformation into the relevant genetic backgrounds, and strains carrying the allelic exchange were selected on kanamycin (50 mg/liter) plates as described previously (6). Recombinant clones were verified by PCR, and it has been verified that the stkP::aphA3 mutation did not affect bacterial growth of the different strains in vitro and also that the insertion mutation was very unlikely to impact the expression of downstream genes in the region. Indeed, with the software described in reference 9, it is predicted that genes SP1731 and SP1732 following the stkP stop codon show no relationship with stkP. Moreover, in the 118-bp intergenic region between stkP ...

Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic‐type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese‐dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild‐type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP‐N‐acetylglucosamine, an essential common precursor to cell envelope components.

SummaryA soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O 2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox þ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O 2 . Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O 2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O 2 in the regulation of pneumococcal transformability and virulence.

Anaerobic aerotolerant Streptococcus pneumoniae modulates its genetic transformability and its virulence in response to the oxygen concentration. The activity of a single protein encoded by nox and showing NADH oxidase activity is involved in these adaptive responses to O2. Northern blot analysis of wild‐type cultures grown under aerobic and microaerobic conditions indicated transcriptional control of comCDE by O2. An O2‐independent mutant strain carrying the gain‐of‐function mutation comE38KE was isolated and its analysis showed that ComE is a key point in competence stimulation by O2. Plasmid insertion mutations in ciaRH revealed that this two component signal‐transducing system negatively regulates comCDE transcription. The level of comCDE transcripts appears as a major control point in competence regulation by O2 and also by growth phase and cell density.

In Streptococcus pneumoniae, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in micA was obtained, expression of the mutated allele micA59DA did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO 4 in vitro. Thus, mutations which either reduced the stability of MicA-PO 4 or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO 4 is involved in competence repression when oxygen becomes limiting. The micAB genes are flanked by mutY and orfC. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in orfC, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.In the catalase-negative pathogen Streptococcus pneumoniae, which has essentially fermentative metabolism, oxygen limitation in a microaerobic atmosphere abolishes developmental competence. Nox, an NADH oxidase that produces water by reducing O 2 as it recycles NADH, has been shown to contribute to competence regulation by oxygen (3,8). Studies of oxygenindependent mutant strains demonstrated the involvement of the two-component systems (TCSs) CiaRH and ComDE in this regulation (7,8). To characterize in more detail the regulatory network facilitating bacterial adaptation to oxygen availability, we searched for amino acid sequences corresponding to motifs putatively involved in O 2 and redox sensing, in the publicly available pneumococcal genome sequence (http://www .tigr.org).The PAS domain may perceive cell energetic status by sensing oxygen, redox potential, ligands, proton motive force, and light (22; for review, see reference 25). PAS domains have been found in bacterial, archaeal, and eukaryotic proteins. Redox sensing and the corresponding signal transduction via twocomponent systems carrying PAS domains is one strategy used by bacteria and archaea for adaptation to variations in ambient oxygen concentration (4). PAS domains are frequently found upstream from the kinase transmitter domain. A heme-containing domain...

The complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The AmiA, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R-factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.

had an 85% reduction in a-galactosidase activity and showed virtually no transport of galactose into the cells, which can explain these phenotypic changes. The DLDH-negative bacteria produced only 50% of normal capsular polysaccharide, a phenotype that may be associated with impaired carbohydrate metabolism. IntroductionDihydrolipoamide dehydrogenases (DLDH; EC 1.8.1.4) are homodimeric flavoproteins that catalyse the NAD + -dependent reoxidation of dihydrolipoamide (DLA) in a number of multienzyme complexes (Perham et al., 1987;Carothers et al., 1989;Williams, 1992;de Kok et al., 1998). These complexes are primarily involved in the conversion of 2-oxo acids to their corresponding acyl-CoA derivative and are involved in important steps in aerobic and anaerobic metabolism (Carothers et al., 1989;de Kok et al., 1998). DLDH makes up the E3 component of the pyruvate dehydrogenase, 2-oxo glutarate dehydrogenase and branchedchain 2-oxo acid dehydrogenase complexes. Additionally, DLDH functions in the glycine cleavage multienzyme complex (where it is referred to as the L protein), as well as in the acetoin dehydrogenase complex in bacteria such as Bacillus subtilis, Clostridium magnum and Pelobacter carbinolicus (Wieland, 1983;Dietrichs and Andreesen, 1990;Kruger et al., 1994;Oppermann and Steinbuchel, 1994;Berg and de Kok, 1997;de Kok et al., 1998;Aevarsson et al., 1999;Huang et al., 1999).Even though the main function of DLDH is associated with its role in 2-oxo acid dehydrogenase complexes, the fact that DLDH is present in organisms that lack these complexes suggests that the enzyme may have additional functions (Danson et al., 1987;Danson, 1988a). Trypanosoma brucei as well as various archeabacteria exhibit simpler ways of converting 2-oxo acids, while still containing a DLDH enzyme (Danson et al., 1987;Danson, 1988b). This suggests that the DLDH must have a separate function in these organisms, and that this function is possibly retained in species in which the DLDH is also part of a 2-oxo acid dehydrogenase complex. Richarme and Heine (1986) and Richarme (1989) proposed that DLDH may be involved in regulation of the transport of galactose, maltose and ribose across the membrane of SummaryIn the present study, we have characterized the dihydrolipoamide dehydrogenase (DLDH) of Streptococcus pneumoniae and its role during pneumococcal infection. We have also demonstrated that a lack of DLDH results in a deficiency in a-galactoside metabolism and galactose transport. DLDH is an enzyme that is classically involved in the three-step conversion of 2-oxo acids to their respective acyl-CoA derivatives, but DLDH has also been shown to have other functions. The dldh gene was virtually identical in three pneumococcal strains examined. Besides the functional domains and motifs associated with this enzyme, analysis of the pneumococcal dldh gene sequence revealed the presence of an N-terminal lipoyl domain. DLDH-negative bacteria totally lacked DLDH activity, indicating that this gene encodes the only DLDH in S. pneumoniae. The...

In Streptococcus pneumoniae, stkP and phpP, encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called "soluble" protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone "competence stimulating peptide." We show that PhpP activity is essential in a stkP Signal transduction via the transfer of phosphoryl groups and transient protein phosphorylation involves the concerted activities of kinases and phosphatases and controls various cellular functions (for a review, see reference 18). In eubacteria, the histidine kinases of two-component systems (TCSs) and the phosphatases with which they interact are involved in cellular adaptation to environmental conditions (for a review, see reference 44). Serine-threonine kinases and PP2C phosphatases also contribute to regulatory and developmental processes, as described in Bacillus subtilis (1,2,13,23,29,33,43). In mycobacteria, a membrane-associated PP2C phosphatase controls the activity of several serine-threonine kinases (4, 6, 52), and the transcriptional activator EmbR has been identified as a phosphorylation target for PknH kinase (3, 4, 42). In Streptococcus agalactiae, the signaling pair Stk1-Stp1 plays a role in virulence, by modifying cytotoxin production and purine metabolism (34,35,36). In Streptococcus pyogenes, a histonelike protein is phosphorylated by the serine-threonine kinase, generating a substrate for the kinase-related phosphatase (21). The human pathogen Streptococcus pneumoniae has only one gene encoding a PP2C-type phosphatase, PhpP, located upstream from the stkP gene, which encodes the only membrane-associated serine-threonine kinase, StkP (17). The phpP and stkP genes overlap by 4 bp and form an operon (31). In a mouse model of infection, null mutations affecting StkP greatly attenuate tissue and bloodstream invasion (17). In cultures, these mutations are highly pleiotropic, presenting a notably important impact on competence development for genetic transformation (17, 40). In vitro studies have shown that autophosphorylated recombinant StkP (StkP-P) is a substrate for recombinant PhpP, suggesting that StkP and PhpP may function in a coordinated manner (31). However, the regulators controlling the cellular level of StkP-P and the network involving StkP in growing cultures remain ill defined. Recent studies of strain TIGR4 revealed that the transcriptional regulator RitR (45) is phosphorylated by StkP and dephosphorylated by PhpP (50). In order to get better insight into StkP signaling, we investigated the specific role of PhpP phosphatase on bacterial growth and on the development of competence for genetic transformation in cultures, by using genetic analysis. Specific null mutation in ...