Role of the twin-arginine translocation pathway in Staphylococcus.

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (DeltatatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of DeltatatAC and Deltatat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.

Molecular basis of resistance to muramidase and cationic antimicrobial peptide activity of lysozyme in staphylococci.

It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R115 (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid-bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA or oatA/graRS double mutants are much more susceptible to lysozyme than each of the single mutants.

Genome analysis of the meat starter culture bacterium Staphylococcus carnosus TM300.

The Staphylococcus carnosus genome has the highest GC content of all sequenced staphylococcal genomes, with 34.6%, and therefore represents a species that is set apart from S. aureus, S. epidermidis, S. saprophyticus, and S. haemolyticus. With only 2.56 Mbp, the genome belongs to a family of smaller staphylococcal genomes, and the ori and ter regions are asymmetrically arranged with the replichores I (1.05 Mbp) and II (1.5 Mbp). The events leading up to this asymmetry probably occurred not that long ago in evolution, as there was not enough time to approach the natural tendency of a physical balance. Unlike the genomes of pathogenic species, the TM300 genome does not contain mobile elements such as plasmids, insertion sequences, transposons, or STAR elements; also, the number of repeat sequences is markedly decreased, suggesting a comparatively high stability of the genome. While most S. aureus genomes contain several prophages and genomic islands, the TM300 genome contains only one prophage, PhiTM300, and one genomic island, nuSCA1, which is characterized by a mosaic structure mainly composed of species-specific genes. Most of the metabolic core pathways are present in the genome. Some open reading frames are truncated, which reflects the nutrient-rich environment of the meat starter culture, making some functions dispensable. The genome is well equipped with all functions necessary for the starter culture, such as nitrate/nitrite reduction, various sugar degradation pathways, two catalases, and nine osmoprotection systems. The genome lacks most of the toxins typical of S. aureus as well as genes involved in biofilm formation, underscoring the nonpathogenic status.

Characterization of the oxygen-responsive NreABC regulon of Staphylococcus aureus.

Here, we investigate the functionality of the oxygen-responsive nitrogen regulation system NreABC in the human pathogen Staphylococcus aureus and evaluate its role in anaerobic gene regulation and virulence factor expression. Deletion of nreABC resulted in severe impairment of dissimilatory nitrate and nitrite reduction and led to a small-colony phenotype in the presence of nitrate during anaerobic growth. For characterization of the NreABC regulon, comparative DNA microarray and proteomic analyses between the wild type and nreABC mutant were performed under anoxic conditions in the absence and presence of nitrate. A reduced expression of virulence factors was not observed in the mutant. However, both the transcription of genes involved in nitrate and nitrite reduction and the accumulation of corresponding proteins were highly decreased in the nreABC mutant, which was unable to utilize nitrate as a respiratory oxidant and, hence, was forced to use fermentative pathways. These data were corroborated by the quantification of the extracellular metabolites lactate and acetate. Using an Escherichia coli-compatible two-plasmid system, the activation of the promoters of the nitrate and nitrite reductase operons and of the putative nitrate/nitrite transporter gene narK by NreBC was confirmed. Overall, our data indicate that NreABC is very likely a specific regulation system that is essential for the transcriptional activation of genes involved in dissimilatory reduction and transport of nitrate and nitrite. The study underscores the importance of NreABC as a fitness factor for S. aureus in anoxic environments.

DNA microarray based detection of genes involved in safety and technologically relevant properties of food associated coagulase-negative staphylococci.

Aim of the work was to design a polynucleotide based DNA microarray as screening tool to detect genes in food associated coagulase-negative staphylococci (CNS). A focus was laid on genes with potential health concern and technological relevance. The microarray contained 220 probes for genes encoding antibiotic resistances, hemolysins, toxins, amino acid decarboxylases (e.g. biogenic amine formation), binding proteins to extracellular matrix (ECM), lipases, proteases, stress response factors, or nitrate dissimilation. Hybridization of genomic DNA isolated from 32 phenotypically characterized CNS permitted to detect numerous genes, corresponding with the phenotype. However, numerous hybridization signals were obtained for genes without any detectable phenotype. The antibiotic resistance genes blaZ, lnuA, and tetK involved in ß-lactam, lincomycin and tetracycline resistance, respectively, were rarely identified in CNS, however, all species contained some strains with such resistance genes. Decarboxylase genes involved in biogenic amine formation were detected regularly in Staphylococcus carnosus, S. condimenti, S. piscifermentans and S. equorum, but was rarely correlated with the phenotype. The same applied for the fibrinogen (clf) and fibronectin (fbp) binding protein genes, whose phenotype (binding assay) was only correlated in S. equorum and Staphylococcus succinus. Although some CNS showed hemolytic activity and enterotoxin production (Immunoblot) the corresponding genes could not be verified. Technological relevant genes such as proteases or lipases revealed good hybridization signals. In addition, genes involved in nitrate dissimilation (nre, nar, nir), catalase (kat), or superoxide dismutase (sod) were well detected. Interestingly, genes involved in dissimilatory nitrate reduction were more prevalent in strains of S. carnosus, S. condimenti and S. piscifermentans than of S. equorum, S. succinus and S. xylosus.

Inhibition of staphylococcal biofilm formation by nitrite.

Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesin (PIA) synthesis and biofilm formation by the human pathogens Staphylococcus aureus and Staphylococcus epidermidis. In this study we characterized an adaptive response of S. aureus SA113 to nitrite-induced stress and show that it involves concomitant impairment of PIA synthesis and biofilm formation. Transcriptional analysis provided evidence that nitrite, either as the endogenous product of respiratory nitrate reduction or after external addition, causes repression of the icaADBC gene cluster, mediated likely by IcaR. Comparative microarray analysis revealed a global change in gene expression during growth in the presence of 5 mM sodium nitrite and indicated a response to oxidative and nitrosative stress. Many nitrite-induced genes are involved in DNA repair, detoxification of reactive oxygen and nitrogen species, and iron homeostasis. Moreover, preformed biofilms could be eradicated by the addition of nitrite, likely the result of the formation of toxic acidified nitrite derivatives. Nitrite-mediated inhibition of S. aureus biofilm formation was abrogated by the addition of nitric oxide (NO) scavengers, suggesting that NO is directly or indirectly involved. Nitrite also repressed biofilm formation of S. epidermidis RP62A.

Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation.

A lipoprotein diacylglyceryl transferase (lgt) deletion mutant of Staphylococcus aureus SA113 was constructed. The lipoprotein and prelipoprotein expression, the growth behavior, and the ability of the mutant to elicit an immune response in various host cells were studied. In the wild type, the majority of [14C]palmitate-labeled lipoproteins were located in the membrane fraction, although some lipoproteins were also present on the cell surface and in the culture supernatant. The lgt mutant completely lacked palmitate-labeled lipoproteins and released high amounts of some unmodified prelipoproteins, e.g., the oligopeptide-binding protein OppA, the peptidyl-prolyl cis-trans isomerase PrsA, and the staphylococcal iron transporter SitC, into the culture supernatant. The growth of the lgt mutant was hardly affected in rich medium but was retarded under nutrient limitation. The lgt mutant and its crude lysate induced much fewer proinflammatory cytokines and chemokines in human monocytic (MonoMac6), epithelial (pulmonary A549), and endothelial (human umbilical vein endothelial) cells than the wild type. However, in whole blood samples, the culture supernatant of the lgt mutant was equal or even superior to the wild-type supernatant in tumor necrosis factor alpha induction. Lipoprotein fractionation experiments provided evidence that a small proportion of the mature lipoproteins are released by the S. aureus wild type despite the lipid anchor and are trapped in part by the cell wall, thereby exposing the immune-activating lipid structure on the cell surface. Bacterial lipoproteins appear to be essential for a complete immune stimulation by gram-positive bacteria.

Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions.

It is well known that biofilm formation by pathogenic staphylococci on implanted medical devices leads to "chronic polymer-associated infections." Bacteria in these biofilms are more resistant to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which genes are up-regulated in Staphylococcus aureus biofilm cells, we carried out a comparative transcriptome analysis. Biofilm growth was simulated on dialysis membranes laid on agar plates. Staphylococci were cultivated planktonically in Erlenmeyer flasks with shaking. mRNA was isolated at five time points from cells grown under both conditions and used for hybridization with DNA microarrays. The gene expression patterns of several gene groups differed under the two growth conditions. In biofilm cells, the cell envelope appeared to be a very active compartment since genes encoding binding proteins, proteins involved in the synthesis of murein and glucosaminoglycan polysaccharide intercellular adhesin, and other enzymes involved in cell envelope synthesis and function were significantly up-regulated. In addition, evidence was obtained that formate fermentation, urease activity, the response to oxidative stress, and, as a consequence thereof, acid and ammonium production are up-regulated in a biofilm. These factors might contribute to survival, persistence, and growth in a biofilm environment. Interestingly, toxins and proteases were up-regulated under planktonic growth conditions. Physiological and biochemical tests for the up-regulation of urease, formate dehydrogenase, proteases, and the synthesis of staphyloxanthin confirmed the microarray data.