Ralf Rosenstein

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

ABSTRACT 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.

Staphylococcal lipases: biochemical and molecular characterization.

To date, the nucleotide sequences of nine different lipase genes from six Staphylococcus species, three from S. epidermidis, two from S. aureus, and one each from S. haemolyticus, S. hyicus, S. warneri, and S. xylosus, have been determined. All deduced lipase proteins are similarly organized as pre-pro-proteins, with pre-regions corresponding to a signal peptide of 35 to 38 amino acids, a pro-peptide of 207 to 321 amino acids with an overall hydrophilic character, and a mature peptide comprising 383 to 396 amino acids. The lipases are secreted in the pro-form and are afterwards processed to the mature form by specific proteases. The pro-peptide of the S. hyicus lipase is necessary for efficient translocation and for protection against proteolytic degradation. Despite being very similar in their primary structures the staphylococcal lipases show significant differences in their biochemical and catalytic properties, such as substrate selectivity, pH optimum and interfacial activation. The lipase from S. hyicus is unique among the staphylococcal and bacterial lipases in that it has not only lipase activity, but also a high phospho-lipase activity. All staphylococcal lipases are dependent on Ca(2+), which is thought to have a function in stabilizing the tertiary structure of the lipases. Evidence exists that staphylococcal lipases like other bacterial lipases, possess a lid-like domain that might be involved in the interfacial activation of these enzymes.

Genome Analysis of the Meat Starter Culture Bacterium Staphylococcus carnosus TM300

ABSTRACT 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, ΦTM300, and one genomic island, νSCA1, 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 toxin production of coagulase-negative staphylococci isolated from food and starter cultures

In this study a comprehensive analysis of toxin production of food associated coagulase-negative staphylococci (CNS) was investigated. The strains belong to the following staphylococcal species, Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus equorum, Staphylococcus piscifermentans, Staphylococcus succinus, and Staphylococcus xylosus, which were isolated from fermented food and starter cultures. A collection of 330 strains were analyzed with respect to their hemolytic activity. 59% of the strains exhibited weak to moderate hemolytic activity with human blood and 34% with sheep blood after 48 h incubation. A selection of 35 strains were tested by immunoblot analysis for their ability to produce toxins, such as the most common staphylococcal enterotoxins (SEs), the toxic shock syndrome toxin 1 (TSST-1), and the exfoliative toxin A (ETA). 18 of the 35 strains produced at least one of the toxins with the SED and SEH being the most common. These indicate that the use of CNS in food production demands a safety evaluation.

Microevolution of cytochrome bd oxidase in Staphylococci and its implication in resistance to respiratory toxins released by Pseudomonas.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and frequently coinfect the lungs of cystic fibrosis patients. P. aeruginosa secretes an arsenal of small respiratory inhibitors, like pyocyanin, hydrogen cyanide, or quinoline N-oxides, that may act against the commensal flora as well as host cells. Here, we show that with respect to their susceptibility to these respiratory inhibitors, staphylococcal species can be divided into two groups: the sensitive group, comprised of pathogenic species such as S. aureus and S. epidermidis, and the resistant group, represented by nonpathogenic species such as S. carnosus, S. piscifermentans, and S. gallinarum. The resistance in the latter group of species was due to cydAB genes that encode a pyocyanin- and cyanide-insensitive cytochrome bd quinol oxidase. By exchanging cydB in S. aureus with the S. carnosus-specific cydB, we could demonstrate that CydB determines resistance. The resistant or sensitive phenotype was based on structural alterations in CydB, which is part of CydAB, the cytochrome bd quinol oxidase. CydB represents a prime example of both microevolution and the asymmetric pattern of evolutionary change.

The molecular organization of the lysostaphin gene and its sequences repeated in tandem

SummaryThe gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51669). It was shown by NH2-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.

Staphylococcal lipases: molecular characterisation, secretion, and processing

Up to date five different staphylococcal lipase genes, two of Staphylococcus aureus (sal-1 and sal-2), two of Staphylococcus epidermidis (sel-1 and sel-2) and one of Staphylococcus hyicus (sh1) have been cloned and sequenced. All corresponding proteins are organised as pre-pro-enzymes: the pre-region represents the signal peptide, the pro-region has a length between 207 and 267 amino acids, and the mature part comprises 380 to 400 amino acids. We found that the lipases are secreted in the pro-lipase form. The processing of the pro-form to the mature enzyme occurs extracellular by a specific protease. Interestingly the pro-lipase reveals not much less activity compared to the mature lipase. There are evidences that the pro-region acts as an intramolecular chaperone which facilitates translocation not only of the native lipase but also of a number of completely unrelated proteins fused to the pro-peptide. It was also observed that the pro-region protects the proteins from proteolytic degradation. While the Staphylococcus aureus and Staphylococcus epidermidis lipases have only lipase (esterase) activity, the related Staphylococcus hyicus enzyme (SHL) is distinguished by both lipase and phospho-lipase activity. The biochemical and catalytic properties of these lipases are described in the accompanying article (Simons, J.W., Götz, F., Egmont, M.R. and Verheij, H.M., 1998. Staphylococcal lipases: Biochemical properties. Accompanying article).

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.

Binding of ArsR, the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter.

AbstractarsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the −35 and −10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the −35 and −10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.

What Distinguishes Highly Pathogenic Staphylococci from Medium- and Non-pathogenic?

Members of the genus Staphylococcus are widespread as commensals of humans and animals where they colonize the skin or mucous membranes. While this coexistence remains mostly untroubled, especially for the healthy host, the bacteria may pose a serious threat for the human or animal host when they get access to inner layers of the body through breaches in skin or membranes. Among the members of the genus a wide span exists in the ability to cope with the hostile conditions encountered in the bloodstream of the living host as a scarce supply of certain nutrients, attacks of the immune system, or anti-infective measures undertaken in the clinical field. In this respect, Staphylococcus aureus is by far the most versatile species of the genus. Its equipment with a huge repertoire of different virulence factors and additional supportive gene products that increase the capability to survive within the living host makes S. aureus the leading pathogen not only within the genus but also one of the most threatening microorganisms regarding hospitally and community-acquired infections. Compared with S. aureus, the other virulent species of the genus like S. epidermidis, S. lugdunensis, S. saprophyticus, and S. haemolyticus have a more limited arsenal of virulence factors resulting in a specialized spectrum of diseases and a generally lower degree of pathogenicity. Besides the highly and medium-pathogenic staphylococci, the genus comprises also species like S. carnosus, S. xylosus, and S. equorum that are generally inconspicuous regarding clinical occurrences. Some strains of this group are used in the food industry and can be graded as non-pathogenic. This review aims to work out the differences between the pathogenic properties of highly and medium-pathogenic staphylococcal species and to draw a comparison between the pathogenic species and the food-grade S. carnosus TM300.