The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.

Streptococcus pneumoniae is the leading cause of death in children worldwide and forms highly organized biofilms in the nasopharynx, lungs, and middle ear mucosa. The luxS-controlled quorum-sensing (QS) system has recently been implicated in virulence and persistence in the nasopharynx, but its role in biofilms has not been studied. Here we show that this QS system plays a major role in the control of S. pneumoniae biofilm formation. Our results demonstrate that the luxS gene is contained by invasive isolates and normal-flora strains in a region that contains genes involved in division and cell wall biosynthesis. The luxS gene was maximally transcribed, as a monocistronic message, in the early mid-log phase of growth, and this coincides with the appearance of early biofilms. Demonstrating the role of the LuxS system in regulating S. pneumoniae biofilms, at 24 h postinoculation, two different D39⌬luxS mutants produced ϳ80% less biofilm biomass than wild-type (WT) strain D39 did. Complementation of these strains with luxS, either in a plasmid or integrated as a single copy in the genome, restored their biofilm level to that of the WT. Moreover, a soluble factor secreted by WT strain D39 or purified AI-2 restored the biofilm phenotype of D39⌬luxS. Our results also demonstrate that during the early mid-log phase of growth, LuxS regulates the transcript levels of lytA, which encodes an autolysin previously implicated in biofilms, and also the transcript levels of ply, which encodes the pneumococcal pneumolysin. In conclusion, the luxS-controlled QS system is a key regulator of early biofilm formation by S. pneumoniae strain D39.

SummaryBacteria attach to their appropriate environmental niche by using adhesins. To maximize their contact with the environment, adhesins are often present on the ends of long hairlike structures called pili. Recently, attention has focused on pili of Grampositive bacteria because they may be vaccine candidates in important human pathogens. These pili differ from the well-studied pili of Gram-negative bacteria because their subunits are covalently linked, they do not require specific chaperones for assembly, and the tip protein (likely to be the adhesin) is not required to initiate formation of the pilus structure. In Gram-positive bacteria, the genes for pili occur in clusters, which may constitute mobile genetic elements. These clusters include the transpeptidase(s) of the sortase family that is/are required for polymerization of the subunit proteins. However, efficient covalent attachment of the completed pilus structure to the cell wall is accomplished, in cases where this has been studied, by the 'housekeeping' sortase, which is responsible for attachment to the peptidoglycan of most surface proteins containing cell wall sorting signals. This enzyme is encoded elsewhere on the genome. Because pili of Grampositive bacteria have not been extensively investigated yet, we hope that this MicroReview will help to pinpoint the areas most in need of further study.

The β‐lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of β‐lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no β‐lactamase‐producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the β‐lactams, the penicillin‐binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non‐PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non‐PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non‐PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during β‐lactam treatment counteract the effects caused by the inhibition of penicillin‐binding proteins.

The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.

The ␤-galactosidase gene of Streptococcus pneumoniae, bgaA, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of grampositive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for lacZ reporter experiments, the bgaA gene was disrupted, resulting in a complete absence of endogenous ␤-galactosidase activity. The results are consistent with ␤-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.Streptococcus pneumoniae is an important human pathogen, causing invasive diseases such as pneumonia, bacteremia, and meningitis. In order to analyze gene expression in this organism, the availability of reporter constructs is highly desirable. The Escherichia coli ␤-galactosidase gene lacZ has been used in several studies. It has long been known that S. pneumoniae produces a ␤-galactosidase that can be purified from the growth medium (8, 11), necessitating the isolation of mutants devoid of this enzyme activity for gene expression studies. However, the ␤-galactosidase-negative S. pneumoniae strains described so far were spontaneously obtained and were not further characterized (6,20). A ␤-galactosidase activity of S. pneumoniae has been isolated from culture supernatants. The objectives of the present study were the identification of the gene encoding the ␤-galactosidase from S. pneumoniae and the construction of a genetically defined ␤-galactosidase-negative mutant suitable for work with lacZ reporter constructs in S. pneumoniae.Identification of the bgaA gene. For sequence database searches, the BLAST program was used (2). A BLAST homology search of the unfinished S. pneumoniae capsular type 4 strain genome, obtained from The Institute for Genomic Research at http://www.tigr.org, revealed a 365-residue peptide with 26% identical amino acids compared to the ␤-galactosidase of Streptococcus thermophilus. The peptide represented an internal region of a putative 2,235-amino acid protein, the product of a 6,704-bp open reading frame. The region covered the two motifs characteristic of glycosyl hydrolase family 2 (9), both of which showed some anomalies in the S. pneumoniae protein: the highly conserved residues Y in motif I and H in motif II were both replaced by an N (Fig. 1). The presence of these alterations was verified by direct sequencing between codons 285 and 716, using PCR products obtained from chromosomal DNA of S. pneumoniae strain R6, a nonencapsulated derivative of Rockefeller University strain R36A (3). For direct sequencing, a BigDye terminator cycle sequencing kit (Perkin-Elmer, Warrington, England) was used.The deduced 2,235-amino-acid sequence (247.3 kDa) revealed several features not typical o...

Pili are a major surface feature of the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). The T3 pilus is composed of a covalently linked polymer of protein T3 (formerly Orf100 or Fct3) with an ancillary protein, Cpa, attached. A putative signal peptidase, SipA (also called LepA), has been identified in several pilus gene clusters of GAS. We demonstrate that the SipA2 allele of a GAS serotype M3 strain is required for synthesis of T3 pili. Heterologous expression in Escherichia coli showed that SipA2, along with the pilus backbone protein T3 and the sortase SrtC2, is required for polymerization of the T3 protein. In addition, we found that SipA2 is also required for linkage of the ancillary pilin protein Cpa to polymerized T3. Despite partial conservation of motifs of the type I signal peptidase family proteins, SipA lacks the highly conserved and catalytically important serine and lysine residues of these enzymes. Substitution of alanine for either of the two serine residues closest to the expected location of an active site serine demonstrated that these serine residues are both dispensable for T3 polymerization. Therefore, it seems unlikely that SipA functions as a signal peptidase. However, a T3 protein mutated at the P-1 position of the signal peptide cleavage site (alanine to arginine) was unstable in the presence of SipA2, suggesting that there is an interaction between SipA and T3. A possible chaperone-like function of SipA2 in T3 pilus formation is discussed.

A major contributor to the emergence of antibiotic resistance in Gram-positive bacterial pathogens is the expansion of acquired, inducible genetic elements. Although acquired, inducible antibiotic resistance is not new, the interest in its molecular basis has been accelerated by the widening distribution and often ‘silent’ spread of the elements responsible, the diagnostic challenges of such resistance and the mounting limitations of available agents to treat Gram-positive infections. Acquired, inducible antibiotic resistance elements belong to the accessory genome of a species and are horizontally acquired by transformation/recombination or through the transfer of mobile DNA elements. The two key, but mechanistically very different, induction mechanisms are: ribosome-sensed induction, characteristic of the macrolide–lincosamide–streptogramin B antibiotics and tetracycline resistance, leading to ribosomal modifications or efflux pump activation; and resistance by cell surface-associated sensing of β-lactams (e.g., oxacillin), glycopeptides (e.g., vancomycin) and the polypeptide bacitracin, leading to drug inactivation or resistance due to cell wall alterations.