SummaryThe two-component regulatory system CiaRH of Streptococcus pneumoniae has been implicated in b-lactam resistance, maintenance of cell integrity, competence and virulence, but the genes that are regulated directly by the system have not been defined. Using transcriptional mapping, in vitro CiaR binding, and in vivo analysis of CiaR-mediated regulation, 15 promoters were identified to be directly controlled by the response regulator CiaR. A direct repeat, TTTAAG-N5-TTTAAG, was found to be essential for CiaR binding and regulation. It is present, either completely or with subtle changes, in all promoter regions. Fourteen promoters of the regulon are activated by CiaR, and one was found to be controlled negatively. The genes that are transcribed from these promoters included ciaRH, loci that are predicted to be involved in the modification of teichoic acids (lic), in sugar metabolism (mal, man), stress response (htrA), chromosome segregation (parB), protease maturation (ppmA) and unknown functions. Remarkably, the five strongest promoters of the CiaR regulon drive expression of small RNAs. These small RNAs, designated csRNAs for ciadependent small RNAs, are non-coding, between 87 and 151 nt in size, and show a high degree of similarity to each other. The analysis of deletion mutants in the csRNA genes revealed that csRNA4 and csRNA5 affect stationary-phase autolysis. The identification of five small non-coding regulatory RNAs opens new perspectives to approach the physiological role of the CiaRH two-component regulatory system.

Streptococcus pneumoniae is one of the few species within the group of low-G ؉C gram-positive bacteria reported to contain no D-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for D-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, D-alanine-D-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released D-alanine from dltAproficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G؉C gram-positive bacteria, teichoic acids of S. pneumoniae contain D-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.Teichoic acids (TAs) are polymers with a relatively wide structural diversity that are present at the surfaces of many gram-positive bacteria (18). The most common types of TAs are comprised of either polyglycerol phosphate or polyribitol phosphate chains of variable length that are substituted with glycosyl residues or D-alanyl esters, or both. TAs may be covalently linked to peptidoglycan (wall teichoic acids [WTAs]) or anchored in the cytoplasmic membrane by their glycolipid moiety (lipoteichoic acids [LTAs]). As major constituents of the surfaces of gram-positive bacteria, TAs have an impact on a number of important biological processes, such as autolysis (60), binding of cations (25) and surface-associated proteins (9, 29), adhesion (1, 61), biofilm formation (21), coaggregation (13), resistance to antimicrobial agents (50, 51), protein secretion (46), acid tolerance (7), stimulation of immune response (20, 41), and virulence (1, 14, 52). In most of these processes, the degree of D-alanylation of TAs has been shown to be of outstanding importance. Addition of D-alanine to TAs reduces the negative charge of the cell envelope, thereby influencing the binding and interaction of various compounds. Incorporation of D-alanine in LTAs is accomplished in a t...

A new promoter probe system for Streptococcus pneumoniae has been developed that allows stable genomic integration of promoters cloned in front of a promoterless hybrid beta-galactosidase gene consisting of translation initiation signals of the protease gene htrA of S. pneumoniae fused to a truncated Escherichia colibeta-galactosidase gene lacZ. Chromosomal insertions of promoter-lacZ fusions are directed to the endogenous beta-galactosidase gene bgaA, thereby abolishing background beta-galactosidase activity. The new system was tested by measuring beta-galactosidase activity directed by the two promoters of the early competence genes comA and comC. The new integrative plasmid offers several advantages compared with existing systems and is especially suited for stable integration of small promoter fragments to conduct mutagenesis or deletion studies.

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a variety of processes such as competence development, autolysis, bacteriocin production, host colonization, and virulence. While the targets of the regulator CiaR are known, the role of phosphorylation in CiaR regulation has not been defined. To address this issue, the presumed phosphorylation site of CiaR, aspartic acid at position 51, was replaced by alanine. The mutant CiaRD51A protein was no longer able to activate CiaR-dependent promoters, strongly suggesting that the phosphorylated form of CiaR is active in regulation. However, depending on the growth medium, inactivation of the kinase gene ciaH resulted in a subtle increase of CiaR-dependent promoter activities or in a strong reduction. Therefore, CiaH may act as a kinase or phosphatase and CiaR is apparently able to obtain its phosphate independently of CiaH. On the other hand, promoter measurements in cells with an intact CiaRH system demonstrated a high, nearly constitutive, expression level of the CiaR regulon independent from the growth medium. Thus, in contrast to many other two-component regulatory systems, CiaRH has apparently evolved to maintain high levels of gene expression under a variety of conditions rather than responding strongly to a signal.

BackgroundThe screening of hospital admission patients for methicillin resistant Staphylococcus aureus (MRSA) is of undisputed value in controlling and reducing the overall MRSA burden; yet, a concerted parallel universal screening intervention throughout all hospitals of an entire German Federal State has not yet been performed.Methodology/Principal FindingsDuring a four-week period, all 24 acute care hospitals of the State of Saarland participated in admission prevalence screening. Overall, 436/20,027 screened patients revealed MRSA carrier status (prevalence, 2.2/100 patients) with geriatrics and intensive care departments associated with highest prevalence (7.6/100 and 6.3/100, respectively). Risk factor analysis among 17,975 admission patients yielded MRSA history (OR, 4.3; CI95 2.7–6.8), a skin condition (OR, 3.2; CI95 2.1–5.0), and/or an indwelling catheter (OR, 2.2; CI95 1.4–3.5) among the leading risks. Hierarchical risk factor ascertainment of the six risk factors associated with highest odd’s ratios would require 31% of patients to be laboratory screened to allow for detection of 67% of all MRSA positive admission patients in the State.Conclusions/SignificanceState-wide admission prevalence screening in conjunction with risk factor ascertainment yields important information on the distribution of the MRSA burden for hospitals, and allows for data-based decisions on local or institutional MRSA screening policies considering risk factor prevalence and expected MRSA identification rates.