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PubMed1980

Binding and factor XIIIa-mediated cross-linking of a 27-kilodalton fragment of fibronectin to Staphylococcus aureus.

A 27-kilodalton tryptic fragment, derived from the amino terminus of the 200-kilodalton fibronectin subunit, inhibited binding of intact fibronectin to Staphylococcus aureus and could be cross-linked to Staphylococcus aureus by blood coagulation Factor XIIIa. Interactions of fibronectin with Staphylococcus aureus via this fragment may be important for bacterial opsonization and attachment.

Amino Acid Sequence↗

PubMed2006

Isolates with low-level vancomycin resistance associated with persistent methicillin-resistant Staphylococcus aureus bacteremia.

Low-level vancomycin-resistant Staphylococcus aureus (vancomycin-intermediate S. aureus [VISA] and heterogenous VISA [hVISA]) is increasingly reported and leads to glycopeptide treatment failure. Various phenotypic features have been reported for these isolates, but the genetic changes leading to hVISA and VISA have yet to be clearly determined. We assessed phenotypic, antibiotic resistance, and genomic changes by using genomic DNA microarray comparison and sequencing of selected loci in five pairs of clinical hVISA/VISA strains and the initial methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained prior to vancomycin therapy. The isolates were from adult patients in Australia and New Zealand who had persistent MRSA bacteremia (>7 days) while receiving vancomycin therapy. In all cases, the initial isolates were found to be fully vancomycin-susceptible Staphylococcus aureus (VSSA). The hVISA/VISA phenotype was associated with increased cell wall thickness, reduced autolytic activity in four of five hVISA/VISA strains, and a striking reduction in biofilm formation compared to the parent strains in all pairs. All five pairs appeared to be isogenic, and genomic DNA microarray comparison suggested that major genetic changes are not required for the development of the resistant phenotype in these strains. No sequence differences were found in the agr locus or the tcaRA genes for any pair, but a marked reduction in RNAIII expression was found in four pairs. In summary, hVISA/VISA arises from fully VSSA during persistent infection that fails to respond to glycopeptide therapy and is associated with significant phenotypic changes, including a marked reduction in biofilm-forming ability. These clinically derived pairs of isolates will be a useful resource to elucidate the genetic mechanism of resistance in hVISA/VISA strains.

Bacteremia↗

PubMed1987

Phenotypic expression and genetic heterogeneity of lincosamide inactivation in Staphylococcus spp.

We examined the resistance phenotype and the genetic basis of lincosamide modification in 25 clinical isolates of Staphylococcus spp. inactivating lincomycin and clindamycin. The strains were resistant to high levels of lincomycin but remained susceptible to clindamycin. However, MBCs and inoculum effects showed that the activity of clindamycin was impaired. The distribution in these strains of nucleotide sequences related to linA and linA', the genes encoding lincosamide nucleotidylation in Staphylococcus haemolyticus BM4610 and S. aureus BM4611, respectively, was studied by dot blot hybridization. The genes responsible for lincosamide inactivation in Staphylococcus spp. were found to constitute a family of related sequences which are not species specific.

Anti-Bacterial Agents↗

PubMed1992

Nucleotide sequence and phylogeny of the tet(L) tetracycline resistance determinant encoded by plasmid pSTE1 from Staphylococcus hyicus.

The nucleotide sequence of the tetracycline resistance (tet) gene and its regulatory region, encoded by the plasmid pSTE1 from Staphylococcus hyicus, was determined. The tet gene was inducible by tetracycline and encoded a hydrophobic protein of 458 amino acids. Comparisons between the predicted amino acid sequences of the pSTE1-encoded Tet from S. hyicus and the previously sequenced Tet K variants from Staphylococcus aureus, Tet L variants from Bacillus cereus, Bacillus stearothermophilus, and Bacillus subtilis, Tet M variants from Streptococcus faecalis and Staphylococcus aureus as well as Tet O from Streptococcus mutans were performed. An alignment of Tet amino acid sequences revealed the presence of 30 conserved amino acids among these Tet variants. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated large evolutionary distances between the Tet M and Tet O variants on one hand and the Tet K and Tet L variants on the other hand. The pSTE1-encoded Tet proved to be closely related to the Tet L proteins originally found on small Bacillus plasmids. The observed extensive similarities in the nucleotide sequences of the tet genes and in the deduced Tet amino acid sequences allowed the assignment of the pSTE1-encoded Tet to the Tet proteins of class L.

Amino Acid Sequence↗

PubMed1996

Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus.

We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.

Amino Acid Sequence↗

PubMed1999

Staphylococcal surface display of immunoglobulin A (IgA)- and IgE-specific in vitro-selected binding proteins (affibodies) based on Staphylococcus aureus protein A.

An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from a Staphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinant S. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.

Antibodies, Bacterial↗

PubMed2000

The macrocyclic peptide antibiotic micrococcin P(1) is secreted by the food-borne bacterium Staphylococcus equorum WS 2733 and inhibits Listeria monocytogenes on soft cheese.

Staphylococcus equorum WS 2733 was found to produce a substance exhibiting a bacteriostatic effect on a variety of gram-positive bacteria. The metabolite was purified to homogeneity by ammonium sulfate precipitation and semipreparative reversed-phase high-performance liquid chromatography. Electrospray mass spectrometry confirmed the high purity of the compound and revealed a molecular mass of 1,143 Da. By two-dimensional nuclear magnetic resonance spectroscopy the substance was identified as micrococcin P(1) which is a macrocyclic peptide antibiotic that has not yet been reported for the genus Staphylococcus. A total of 95 out of 95 Listeria strains and 130 out of 135 other gram-positive bacteria were inhibited by this substance, while none of 37 gram-negative bacteria were affected. The antilisterial potential of this food-grade strain as a protective starter culture was evaluated by its in situ application in cheese-ripening experiments under laboratory conditions. A remarkable growth reduction of Listeria monocytogenes could be achieved compared to control cheese ripened with a nonbacteriocinogenic type strain of Staphylococcus equorum. In order to prove that inhibition was due to micrococcin P(1), a micrococcin-deficient mutant was constructed which did not inhibit L. monocytogenes in cheese-ripening experiments.

Anti-Bacterial Agents↗

PubMed2002

Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme.

Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the corresponding enzyme in Staphylococcus aureus and that the ilvE gene was constitutively expressed as a monocistronic transcript. To study the influence of ilvE on BCAA catabolism, we constructed an ilvE deletion mutant by gene replacement. The IlvE protein from S. carnosus was shown mainly to catalyze the transamination of isoleucine, valine, leucine, and, to some extent, methionine using pyridoxal 5'-phosphate as a coenzyme. The ilvE mutant degraded less than 5% of the BCAAs, while the wild-type strain degraded 75 to 95%. Furthermore, the mutant strain produced approximately 100-fold less of the methyl-branched carboxy acids, 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which derived from the BCAA catabolism, clearly emphasizing the role of IlvE in aroma formation. In contrast to previous reports, we found that IlvE was the only enzyme that catalyzed the deamination of BCAAs in S. carnosus. The ilvE mutant strain showed remarkably lower growth rate and biomass yield compared to those of the wild-type strain when grown in rich medium. Normal growth rate and biomass yield were restored by addition of the three BCAA-derived alpha-keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.

Amino Acids, Branched-Chain↗

PubMed2003

Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants.

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.

Bacterial Adhesion↗

PubMed2005

Survey of extreme solvent tolerance in gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene.

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.

Animals↗

PubMed1996

Diminished platelet binding in vitro by Staphylococcus aureus is associated with reduced virulence in a rabbit model of infective endocarditis.

The direct binding of platelets by bacteria is a postulated central mechanism in the pathogenesis of endocarditis. To address the role of binding more definitively, we employed Tn551 insertional mutagenesis of Staphylococcus aureus parental strain ISP479 to generate an isogenic variant (strain PS12) that bound platelets minimally. As compared with the binding of ISP479, the binding of PS12 to platelet monolayers was reduced by 67.2%. Similarly, the binding of PS12 to platelets in suspension was reduced by 71.3%, as measured by flow cytometry. The low-binding phenotype was transducible into both ISP479 and S. aureus Newman. Southern blotting indicated that a single copy of Tn551 was inserted within the chromosomes of PS12 and the transductants. When tested in a rabbit model, animals inoculated with PS12 were significantly less likely to develop endocarditis and had lower densities of organisms (CFU per gram) within vegetations and a decreased incidence of renal abscess formation, as compared with animals inoculated with the parental strain. The diminished virulence of PS12 was not attributable to a reduction in the initial attachment of organisms to the damaged endocardium, since 30 min after inoculation, PS12-infected animals had microbial densities on the valve surface comparable to those seen with the parental strain. These results indicate that the direct binding of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Staphylococcus-platelet binding appears to be critical for pathogenetic events occurring after the initial colonization of the valve surface, such as vegetation formation and septic embolization.

Animals↗

PubMed1997

Characterization of the canine type C enterotoxin produced by Staphylococcus intermedius pyoderma isolates.

The type C staphylococcal enterotoxins (SECs) are a group of highly conserved proteins with substantial antigenic cross-reactivity. Although Staphylococcus intermedius and coagulase-positive species of staphylococci are reported to produce SEC and other SEs, toxins produced by species other than Staphylococcus aureus have not been previously characterized. In this study we report the molecular, biological, and immunological properties of the canine SEC (SECcanine) expressed by pathogenic isolates of S. intermedius. The mature form of SECcanine has 239 amino acid residues and a pI of 7.0. Typical of the SEs, purified SECcanine induces an emetic response in monkeys and the proliferation of T cells in a Vbeta-dependent manner. Although SECcanine has >95% sequence identity to previously described SEC variants, its sequence is most related to SEC2 and SEC3. In contrast to the sequence similarity, the Vbeta profile induced by SECcanine is typical of that induced by SEC1. This result is likely explained by the conservation of a cysteine residue at position 26 in SECcanine; residues at this position have been previously shown to determine subtype-dependent differences in T-cell receptor interactions of other SEs. Overall, these results show that superantigen toxins produced by the multiple members of the genus Staphylococcus are highly conserved in respect to biological and structural properties. Further, the frequent association of SECcanine with pyoderma in dogs supports the notion that the toxins are important for staphylococcal survival and pathogenesis.

Alleles↗

PubMed2006

Accessory gene regulator locus of Staphylococcus intermedius.

The accessory gene regulator (agr) locus, a candidate system for the regulation of the production of virulence factors in Staphylococcus intermedius, has been characterized. Using PCR-based genome walking, we have obtained the first complete sequence (3,436 bp) of the accessory gene regulator (agr) gene in this organism. Sequence analysis of the agr gene has identified five open reading frames (ORFs), agrB, agrD, agrC, agrA, and hld. The translated ORF contained amino acid motifs characteristic of the response regulator and histidine protein kinase signal transducer of the classic two-component regulatory system. Sequencing of the agrD PCR products amplified from DNA from 20 different isolates has facilitated detection of genetic variation in the putative autoinducing peptide (AIP) within the agr gene of S. intermedius, revealing the presence of at least three agr specificity groups within this species. Classification of the agr gene from S. intermedius was supported by phylogenetic analysis. Real-time PCR also revealed that the effector molecule of the agr system, RNAIII, was regulated in an autocrine manner in S. intermedius and demonstrated positive correlation with the temporal gene expression patterns of luk and entC. Transcription of RNAIII was also dependent on self secreted cues. Cyclic self and nonself peptides were synthesized on the basis of the novel AIPs produced by S. intermedius, which lack the cysteine necessary to form the thiolactone ring in analogous peptides from Staphylococcus aureus and Staphylococcus epidermidis. Experiments with these synthetic cyclic peptides indicated that self peptides led to up-regulation of RNAIII--findings in support of the assumption that activation of the agr gene is initiated by growth- and species-specific factors generated during bacterial growth.

Amino Acid Sequence↗

PubMed1985

Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn918).

Streptococcus faecalis RC73 was found to harbor a conjugative plasmid (pAM373) which confers a mating response to a sex pheromone (cAM373) excreted by plasmid-free members of the same species. The pheromone was also detected in culture filtrates of all of 23 Staphylococcus aureus strains but in only 2 of 22 coagulase negative staphylococcus strains. Streptococcus sanguis Challis and G9B also produced the activity, but 10 other Streptococcus sanguis strains did not. The activity was also produced by Streptococcus faecium 9790. A tetracycline resistance (Tc) determinant present in S. faecalis RC73 was not associated with pAM373 but served as a useful marker in efforts to identify pAM373 among other plasmids present in the strain. Analyses of the Tc determinant showed that it was located on a conjugative transposon very similar to Tn916. Designated Tn918, the transposon could insert into pAM373 as well as into two other hemolysin plasmids. Whereas pAM373 derivatives transferred very well between strains of Streptococcus faecalis, the plasmid would not establish in Staphylococcus aureus or Streptococcus sanguis. However, a derivative of pAM373 carrying Tn918 proved to be a useful delivery vehicle for generating transposon insertions into multiple sites on the staphylococcal chromosome.

Animals↗

PubMed1992

Regulation of Staphylococcus xylosus xylose utilization genes at the molecular level.

We have investigated the regulation of the operon encoding xylose utilization in Staphylococcus xylosus C2a and Staphylococcus carnosus TM300. For in vivo studies, transcriptional fusions of the xylAB regulatory region to the lipase gene from Staphylococcus hyicus were constructed. Repression of lipase activity depended on a functional xylR gene and an xyl operator palindrome downstream of the promoter, while induction was obtained in the presence of xylose. Inactivation of either xylR or the xyl operator led to constitutive expression in the absence of xylose. Crude protein extracts from xylR+ staphylococci led to gel mobility shifts of the xyl regulatory DNA in the absence but not in the presence of xylose. A copper-phenanthroline footprint of the shifted band revealed protection of 28 phosphodiesters from cleavage in each strand of the xyl operator. Thus, the Xyl repressor covers the DNA over more than 2.5 helical turns. Glucose repression of the xyl operon occurs at the level of transcription and is independent of a functional xylR gene. A potential cis-active sequence element for glucose repression is discussed on the basis of sequence similarities to respective elements from bacilli.

Base Sequence↗

PubMed1995

Cell surface display of recombinant proteins on Staphylococcus carnosus.

A novel expression system for surface display of heterologous proteins on Staphylococcus carnosus cells has been developed. Taking advantage of the promoter and secretion signals, including a propeptide region, from the lipase gene of Staphylococcus hyicus and the cell wall-spanning and membrane-binding region of protein A from Staphylococcus aureus, efficient surface display of an 80-amino-acid peptide from a malaria blood stage antigen could be achieved. A serum albumin binding protein from streptococcal protein G was used both as a general reporter molecule and to increase the accessibility of the surface-displayed proteins. Immunoblotting, immunogold staining, and immunofluorescence on intact recombinant S. carnosus cells verified the presence of the propeptide, the malaria antigen, and the albumin-binding reporter protein on the bacterial surface. For the first time, fluorescence-activated cell sorting was used to analyze the presence of surface-displayed hybrid receptors on gram-positive bacteria.

Antigens, Protozoan↗

PubMed1997

Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus.

A gene encoding an autolytic activity was identified in an autolysis-deficient mutant (Lyt-) of Staphylococcus aureus which produces only a single band in autolytic-activity gels (N. Mani, P. Tobin, and R. K. Jayaswal, J. Bacteriol. 175:1493-1499, 1993). An open reading frame, designated lytM, of 948 bp that could encode a polypeptide of 316 amino acid residues was identified. The calculated molecular mass of the lytM gene product (34.4 kDa) corresponded to that of the autolytic activity detected (approximately 36 kDa) in the Lyt- mutant. Results deduced from amino acid sequence analysis and N-terminal amino acid sequencing data suggest that LytM is a secreted protein. The C-terminal region of the putative protein encoded by lytM showed 51% identity with the N-terminal region of the mature lysostaphin from Staphylococcus simulans and 50% identity with the N-terminal region of ALE-1 from Staphylococcus capitis EPK1. Northern blot analysis showed that lytM expresses a transcript of approximately 955 bp, as predicted from the DNA sequence. Escherichia coli clones carrying the lytM gene exhibited autolytic-activity bands of approximately 36 kDa as well as of 19 and 22 kDa in activity gels. The lytM gene was mapped to the SmaI-D fragment on the S. aureus chromosome. Mapping data and results of hybridization experiments with primers generated from gene sequences of known autolytic genes of S. aureus clearly indicate that the lytM gene is distinct from other staphylococcal autolytic genes reported to date.

Amino Acid Sequence↗

PubMed2003

Jumping the barrier to beta-lactam resistance in Staphylococcus aureus.

Although the staphylococcal methicillin resistance determinant, mecA, resides on a mobile genetic element, staphylococcus cassette chromosome mec (SCCmec), its distribution in nature is limited to as few as five clusters of related methicillin-resistant Staphylococcus aureus (MRSA) clones. To investigate the potential role of the host chromosome in clonal restriction of the methicillin resistance determinant, we constructed plasmid pYK20, carrying intact mecA, and introduced it into several methicillin-susceptible Staphylococcus aureus strains, five of which were naive hosts (i.e., mecA not previously resident on the host chromosome) and five of which were experienced hosts (i.e., methicillin-susceptible variants of MRSA strains from which SCCmec was excised). We next assessed the effect of the recipient background on the methicillin resistance phenotype by population analysis, by assaying the mecA expression of PBP2a by Western blot analysis, and by screening for mutations affecting mecA. Each experienced host transformed with pYK20 had a resistance phenotype and expressed PBP2a similar to that of the parent with chromosomal SCCmec, but naive hosts transformed with pYK20 selected against its expression, indicative of a host barrier. Either inducible beta-lactamase regulatory genes blaR1-blaI or homologous regulatory genes mecR1-mecI, which control mecA expression, acted as compensatory elements, permitting the maintenance and expression of plasmid-carried mecA.

Bacterial Proteins↗