Chantal Granadel

Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases

The adcCBA putative operon of Streptococcus pneumoniae, an important human pathogen, was identified in a search for transformation‐deficient mutants. It was found to exhibit homology to ATP‐binding cassette (ABC) transport operons encoding streptococcal adhesins such as FimA of Streptococcus parasanguis and PsaA of S. pneumoniae. The latter was recently shown to be essential for virulence as judged by intranasal or intraperitoneal challenge of mice. We suggested previously that AdcA, together with a set of 14 proteins, including PsaA and homologous adhesins, defines a new family of external solute‐binding proteins specific for metals. In this work, Northern analysis revealed the existence of two adcB–adcA specific transcripts originating within adcC or further upstream, consistent with the hypothesis that adc is an operon. Investigation of growth of adc and psaA mutants in synthetic medium revealed that the addition of Zn improved the growth rate of the former, whereas the latter exhibited an absolute requirement for added Mn. A psaA–adc double mutant turned out to be essentially non‐viable unless both metals were added in the appropriate ratio. Taken together, these results suggest a previously undocumented requirement of S. pneumoniae for Zn and Mn. The addition of Zn also restored near‐normal spontaneous transformation of adc mutant cells in standard transformation medium. Zn was found to be specifically required soon after contact of cells with the competence‐stimulating peptide, revealing an unsuspected need for Zn in transformation of S. pneumoniae. The removal of Mn from standard transformation medium also resulted in transformation deficiency of psaA mutant cells. Taken together, these results lead us to propose that Adc is an ABC‐type Zn permease, the first such protein complex identified in any organism, and that Psa is an ABC‐type Mn permease complex.

Cross‐regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae

Two operons, comAB and comCDE, play a key role in the co‐ordination of spontaneous competence development in cultures of Streptococcus pneumoniae. ComAB is required for export of the comC‐encoded competence‐stimulating peptide (CSP). Upon CSP binding, the histidine kinase ComD activates ComE, its cognate response regulator, required for autoinduction of comCDE and for induction of the late competence genes. To understand better the early control of competence development, mutants upregulating comCDE (ComCDEUP) were isolated using a comC–lacZ transcriptional fusion. Mutants were generated by polymerase chain reaction mutagenesis of the comCDE region and by in vitro transposon mutagenesis of the chromosome. Both types of ComCDEUP mutants exhibited similar phenotypes. They differed from wild type in displaying trypsin‐resistant transformation, competence under acid growth conditions and expression of comCDE under microaerobiosis; increased production of CSP in the mutants could account for the various phenotypes. The ComCDEUP transposon mutations included four independent insertions in the ciaR gene, which encodes the response regulator of a two‐component system previously found to affect competence, and two immediately upstream of the comAB operon. The latter two resulted in comAB overexpression, indicating that CSP export is rate limiting. Among comDE point mutations, a single amino acid change in ComD (T233I) conferred constitutive, CSP‐independent competence and resulted in comAB overexpression, providing support for the hypothesis that ComE regulates comAB; a ComE mutant (R120S) exhibited altered kinetics of competence shut‐off. Collectively, these data indicate that pheromone autoinduction, cross‐regulation of the comAB and comCDE operons and, possibly, competence shut‐off contribute to the early control of competence development in S. pneumoniae. They argue for a metabolic control of competence, mediated directly or indirectly by CiaR, and they suggest that both comAB and comCDE are potential targets for regulation.

Development of competence in Streptococcus pneumoniae: pheromone autoinduction and control of quorum sensing by the oligopeptide permease

Competence for genetic transformation in the human pathogen Streptococcus pneumoniae is a transient physiological property. A competence‐stimulating peptide, CSP, was recently identified as the processed product of the comC gene. As conflicting results have been reported regarding CSP autoinduction, we monitored the CSP‐induced expression of comCDE in derivatives of strain R6 using comC::lacZ fusions. Autoinduction was demonstrated in this genetic background. The kinetics of CSP‐induced transcription of comCDE and of a late competence‐induced (cin) operon were compared. While the comCDE mRNA level was highest 5 min after CSP addition then decreased, maximal cin expression required 10 min exposure to CSP. Transformation frequencies paralleled cin expression. After 20 min exposure to CSP, both mRNAs disappeared almost completely, providing evidence for an intrinsic mechanism for shutting off CSP signal transduction. Investigation of spontaneous competence development in mixed cultures indicated that transformation of wild‐type cells was delayed in the presence of CSP non‐producers, consistent with a direct role of CSP in quorum sensing. The effect of varying inoculum size on the timing of competence development was investigated. While competence developed in wild‐type cultures at a similar critical density, about OD550 = 0.15, a mutant lacking the three oligopeptide‐binding lipoproteins transformed at a 50‐fold reduced cell density. The latter effect was mimicked in a strain harbouring a duplication of comC. Altogether, these results suggest that CSP does not accumulate passively in pneumoccal cultures, but that comCDE basal expression can be modulated.

New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor

In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non‐competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so‐called competence‐induced cell lysis. Competence‐induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence‐induced aggregation is abolished in a lytA–lytC double mutant, and absolutely requires CbpD and its N‐terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)‐induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP‐controlled mechanism that allows competent pneumococci to commit fratricide by killing non‐competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.

Programmed protection of foreign DNA from restriction allows pathogenicity island exchange during pneumococcal transformation.

In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of S. pneumoniae through programmed protection and incorporation of foreign DNA.

Penicillin tolerance in Streptococcus pneumoniae, autolysis and the Psa ATP-binding cassette (ABC) manganese permease

Recently, Novak et al. (1998, Mol Microbiol 29: 1285±1296) reported their investigation on the phenomenon of penicillin tolerance in Streptococcus pneumoniae. A library of mutants in pneumococcal surface proteins was screened for the ability to survive in the presence of 10 ́ the minimum inhibitory concentration of antibiotic. A mutant harbouring an insertion in the known gene psaA was isolated among 10 candidate tolerance mutants. Inactivation of psaA was previously shown to result in reduced virulence of S. pneumoniae (as judged by intranasal or intraperitoneal challenge of mice) and in reduced adherence to A549 cells (type II pneumocytes), leading to the suggestion that PsaA was an adhesin (Berry and Paton, 1996, Infect Immun 64: 5255±5262). This gene is part of the psa locus (Fig. 1) that encodes an ATP-binding cassette (ABC) permease belonging to cluster 9, a family of ABC metal permeases (Dintilhac et al., 1997, Mol Microbiol 25: 727±740). Novak et al. (1998, Mol Microbiol 29: 1285±1296) reported that psa mutants displayed pleiotropic phenotypes: (i) reduced sensitivity to the lytic and killing effects of penicillin; (ii) growth in chains of 40±50 (psaC ) to 200±300 (psaD ) cells; (iii) autolysis defect and loss of sensitivity to low concentrations of deoxycholate (DOC), a species characteristic trait; (iv) absence of LytA, the major autolytic amidase; (v) almost complete loss of choline-binding proteins (ChBPs) (psaC and psaD ) and absence of CbpA; (vi) loss of transformability (except psaA); and (vii) manganese (Mn) requirement for growth in a chemically de®ned medium. Because penicillin tolerance was ®rst associated with an autolysis defect (Tomasz et al., 1970, Nature 227: 138± 140), the absence of LytA (phenotype iv) could itself explain phenotypes i and iii. Dysregulation of lytA could not be investigated because, according to Novak et al. (1998, Mol Microbiol 29: 1285±1296), the dif®culty in lysing psa mutant cells prohibited Northern analysis, although lysates of the psa mutants could be obtained for immunoblot analysis of LytA and of RecA and for Southern con®rmation of the psa mutations. Nevertheless, because expression of the lytA gene has been shown to be driven by three different promoters, including Pb which is the recA basal promoter (Mortier-BarrieÁre et al., 1998, Mol Microbiol 27: 159±170), and because wild-type levels of RecA were detected in the psa mutants (Novak et al., 1998, Mol Microbiol 29: 1285±1296), it seems dif®cult to account for the complete absence of LytA on the basis of altered expression. On the other hand, phenotypes i±iv are reminiscent of alterations observed after the replacement of choline (Ch) by ethanolamine (EA) in the cell wall of pneumococcus (Tomasz, 1968, Proc Natl Acad Sci USA 59: 86±93). Similar phenotypes were also displayed by Ch-independent mutants of S. pneumoniae (Severin et al., 1997, Microb Drug Res 3: 391±400; Yother et al., 1998, J Bacteriol 180: 2093±2101). S. pneumoniae has a nutritional requirement for Ch that is incorporated by covalent bonds into the cell wall teichoic acids (TA) and in the membrane-bound lipoteichoic acid (LTA). Ch residues bound to TA (ChTA) were shown to be absolutely required for LytA activity (Holtje and Tomasz, 1975; J Biol Chem 250: 6072±6076). The action of LytA has long been thought to be restricted to pneumococcal cell walls because of this requirement. However, recent reports suggest that ChTA is required Molecular Microbiology (1999) 32(4), 881±891

The puzzle of zmpB and extensive chain formation, autolysis defect and non-translocation of choline- binding proteins in Streptococcus pneumoniae

Choline‐binding proteins (CBPs) from Streptococcus pneumoniae are involved in several important processes. Inactivation of zmpB, a gene that encodes a surface‐located putative zinc metalloprotease, in a S. pneumoniae serotype 4 strain was recently reported to reveal a composite phenotype, including extensive chain formation, lysis defect and transformation deficiency. This phenotype was associated with the lack of surface expression of several CBPs, including the major autolysin LytA. LytA, normally 36 kDa in size, was reported to form an SDS‐resistant 80 kDa complex with CinA. ZmpB was therefore proposed to control translocation of CBPs to the surface, possibly through the proteolytic release of CBPs (and RecA) from CinA. Based on the use of 12 independent mariner insertions in the zmpB gene of the well‐characterized R6 laboratory strain, we could not confirm several of these observations. Our zmpB mutants: (i) did not form chains; (ii) lysed normally in the presence of deoxycholate, which indicates the presence of a functional autolysin; (iii) transformed at normal frequency; and (iv) contained bona fide CinA and LytA species. Polymorphism of ZmpB between R6 and the serotype 4 isolate could not account for the discrepancy, as inactivation of zmpB (through replacement by transposon‐inactivated zmpB R6 alleles) in the latter strain did not affect separation of daughter cells and autolysis. The conflicting observations could be explained by our finding that the reportedly serotype 4 zmpB‘mutant’ differed from its S. pneumoniae parent in lacking capsule and in exhibiting characteristic traits of the Streptococcus viridans group, including resistance to optochin.

RadC, a Misleading Name?

The pfam04002 annotation describes RadC as a bacterial DNA repair protein. Although the radC gene is expressed specifically during competence for genetic transformation in Streptococcus pneumoniae, we report that radC mutants exhibit normal uptake and processing of transforming DNA. They also display normal sensitivity to DNA-damaging agents, providing no support for the rad epithet.