Abstract: In this study, we improved a method for rapid determination of viral RNA sequences (RDV) to overcome the limitations of previous versions. The RDV ver4.0 method can detect RNA sequences with at least 1,000 copies as starting material. A novel virus, which was isolated from field-collected Aedes aegypti larvae in the Phasi Charoen district of Thailand using C6/36 cells, was identified using the RDV ver4.0 protocol. The virus was named Phasi Charoen virus (PhaV). We used a high-throughput pyrosequencing approach to obtain more information about the genome sequence of PhaV. Analysis of a phylogenic tree based on amino acid sequences strongly suggested that PhaV belongs to the family Bunyaviridae.

Abstract: Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in female outpatients. We investigated the dissemination and antimicrobial susceptibilities of 101 S. saprophyticus isolates from the genitourinary tracts of patients in Japan. Eight of these isolates were mecA positive and showed beta-lactam resistance. Pulsed-field gel electrophoresis showed that only some isolates were isogenic, indicating that the mecA gene was apparently acquired independently by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). Type determination of SCCmec by multiplex PCR showed a nontypeable element in the eight mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. saprophyticus strain revealed that it was nontypeable with the current SCCmec classification due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are similar to those of type I SCCmec in S. aureus NCTC 10442. Furthermore, the genes around the mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a novel type.

Abstract: Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.

Abstract: Capsular polysaccharides (CPs) are important virulence factors of Staphylococcus aureus. The biosynthesis of type 5 and type 8 CPs (CP5 and CP8), which are produced by most clinical isolates of S. aureus, is catalyzed by 16 CP-assembling proteins. One of these proteins is the enzyme CapF, which catalyzes the synthesis of UDP-N-acetyl-L-fucosamine, a component of both CP5 and CP8. Here, the cloning, expression, purification, crystallization and diffraction analysis of CapF are reported. Optimization of the crystallization conditions by differential scanning calorimetry afforded a crystal of selenomethionine-substituted CapF that diffracted to a resolution of 2.80 A. The crystal belongs to space group P3(2)21, with unit-cell parameters a = b = 119.6, c = 129.5 A.

Abstract: The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.

Abstract: Ebh, a giant protein found in staphylococci, contains several domains, including a large central region with 52 imperfect repeats of a domain composed of 126 amino acids. We used electron microscopy to observe the rod-like structure of a partial Ebh protein containing 10 repeating units. This is the first report of the direct observation of an Ebh structure containing a large number of repeating units, although structures containing one, two, or four repeating units have been reported. The observed structure of the partial Ebh protein was distorted and had a length of ca. 520A and a width of ca. 21A. The observed structures were consistent with those deduced from crystal structure analysis, suggesting that the Ebh domains are connected to form a rod-like structure. The crystal structure data revealed distorted, string-like features in the simulated structure of the whole-length Ebh protein. Superposition of fragments of the simulated whole-length structure of the Ebh protein onto each electron micrograph showed a high level of correlation between the observed and calculated structures. These results suggest that Ebh is composed of highly flexible filate molecules. The highly repetitive structure and the associated unique structural flexibility of Ebh support the proposed function of this protein, i.e. binding to sugars in the cell wall. This binding might result in intra-cell-wall cross-linking that contributes to the rigidity of bacterial cells.

Abstract: To elucidate the heme acquisition system in pathogenic bacteria, we investigated the heme-binding properties of the third NEAT domain of IsdH (IsdH-NEAT3), a receptor for heme located on the surfaces of pathogenic bacterial cells, by using x-ray crystallography, isothermal titration calorimetry, examination of absorbance spectra, mutation analysis, size-exclusion chromatography, and analytical ultracentrifugation. We found the following: 1) IsdH-NEAT3 can bind with multiple heme molecules by two modes; 2) heme was bound at the surface of IsdH-NEAT3; 3) candidate residues proposed from the crystal structure were not essential for binding with heme; and 4) IsdH-NEAT3 was associated into a multimeric heme complex by the addition of excess heme. From these observations, we propose a heme-binding mechanism for IsdH-NEAT3 that involves multimerization and discuss the biological importance of this mechanism.

Abstract: Staphylococcus aureus is well known to colonize on human skin where the physiological condition is characterized by hypervariable water activity, i.e., repeated dehydration or rehydration. To determine the facilitating factors for the colonization under hypervariable water activity, we studied the giant protein Ebh (extracellular matrix (ECM)-binding protein homologue). The ebh mutant RAM8 showed invaginated vacuoles along the septum, similar to that found in partial plasmolysis, and the cells burst under osmotic upshift. RAM8 was also relatively susceptible to abrupt hyperosmotic upshift, teicoplanin, and Triton X-100. By using the green fluorescent protein (GFP) as a reporter, Ebh was localized over the entire cell surface. This suggests that Ebh might contribute to structural homeostasis by forming a bridge between the cell-wall and cytoplasmic membrane to avoid plasmolysis under hyperosmotic condition.

Abstract: BACKGROUND AND OBJECTIVES: The sesquiterpene farnesol, a natural plant metabolite, is known to intensify the effect of antimicrobial agents. However, the mode of action of its antimicrobial synergism has remained poorly understood. In this study, we investigated farnesol's synergistic effects on commonly used antimicrobials, beta-lactams in particular, to explore its potential inhibitory effect on cell wall synthesis. METHODS: We investigated farnesol's effects on: (i) antimicrobial susceptibilities of methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA) to ampicillin, oxacillin, cefoxitin, bacitracin, teicoplanin, amikacin, ciprofloxacin and clarithromycin by MIC determination using the Etest; (ii) penicillin-binding protein PBP2' (2a) expression by western-blot analysis; (iii) beta-lactamase secretion and activity by in vivo and in vitro farnesol inhibition assays; (iv) staphyloxanthin production by thin-layer chromatography (TLC); and (v) cell wall synthesis by [14C]GlcNAc (where GlcNAc stands for N-acetylglucosamine) and [14C]mevalonate incorporation assays, and TLC-based lipid extract profile analysis. RESULTS: Farnesol induced variable degrees of increased susceptibility to all antimicrobials except clarithromycin in both MSSA and MRSA. A remarkable increase in susceptibilities to ampicillin, oxacillin and cefoxitin was observed in both MRSA strains, N315 and COL, whereas a moderate increase in susceptibility to bacitracin was observed in all the strains. Although no apparent suppression of PBP2' expression was observed, beta-lactamase secretion and beta-lactamase activity were significantly reduced by farnesol. In addition, farnesol completely suppressed staphyloxanthin production. Farnesol reduced the incorporation of GlcNAc, but significantly increased that of mevalonate. Farnesol induced accumulation of C55-PP, lipid I and lipid II. CONCLUSIONS: Farnesol increased beta-lactam susceptibility of MRSA by inhibition of cell wall biosynthesis through reduction of free C55 lipid carrier with subsequent retardation of murein monomer precursor transport across the cell membrane.

Abstract: SaeRS is a two-component system that has been characterized as a positive regulatory system for the expression of several virulence factors, including coagulase, alpha-, beta- and gamma-haemolysins, nuclease, and fibronectin-binding proteins in Staphylococcus aureus. Previously, the SaeRS system was found to be induced at the transcriptional level by beta-lactam. Here, we found that subinhibitory concentrations of beta-lactam induce haemolytic activity in the S. aureus N315 strain but not in the saeRS null mutant KSA. Comparison of the transcriptional profile of the N315 and KSA strains by microarray analysis reveals that the SaeRS system modulates the regulation of coagulase (coa), alpha-, beta- and gamma-haemolysins (hla, hlb and hlg), nuclease (SA0746), fibrinogen-binding proteins (emp, efb, SA1000 and SA1004), fibronectin-binding protein B (fnbB), and 13 other genes. Further, the use of cefoxitin as a signal inducer reveals that the SaeRS system appears to modulate 22 additional genes as a secondary regulon, including the staphylococcal accessory regulators SarA and SarT and the Clp protease ATPase subunits ClpB and ClpL. These observations suggest that beta-lactam is able to induce the SaeRS system, which acts as a crucial signal transduction system for S. aureus pathogenicity rather than antimicrobial resistance.

Abstract: Staphylococcus aureus lipase (SAL) is known to possess broad substrate specificity for triacylglycerides. We found that a sub-minimum inhibitory concentration of farnesol (1000 mg L(-1)) inhibits this lipase activity on a Mueller-Hinton agar containing 1% Tween substrates. A quantitative lipase assay using p-nitrophenyl palmitate (pNPP) revealed that the inhibitory action of farnesol appears to be the result of the inhibition of lipase activity rather than of its secretion into the culture medium. The inhibition was observed in all the tested 8 methicillin-susceptible S. aureus and 31 methicillin-resistant S. aureus clinical isolates. Using homogeneous lipase purified by hydrophobic interaction chromatography, it was revealed that farnesol could competitively inhibit the lipase activity against the substrate pNPP.

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Abstract: In order to validate the current Clinical and Laboratory Standards Institute (CLSI) criteria for the detection of mecA-mediated resistance in Staphylococcus saprophyticus, 101 clinical isolates, including 8 mecA-positive isolates, were investigated. All the isolates were in the range of the resistant category for coagulase-negative staphylococci with the 1 microg oxacillin disk diffusion method and agar dilution method, despite 93 isolates (92%) being mecA-negative. On the other hand, the 30 microg cefoxitin disk diffusion method showed clearly distinguishable zone diameters between the mecA-positive and -negative isolates. However, four of the mecA-negative isolates that would be considered resistant were false positive, and the current interpretive criteria of the CLSI may thus require reconsideration. This study suggests that the cefoxitin disk diffusion method could be more suitable than the oxacillin disk diffusion method for detecting mecA-mediated resistance in S. saprophyticus.

Abstract: Staphylococcus saprophyticus is a uropathogenic Staphylococcus frequently isolated from young female outpatients presenting with uncomplicated urinary tract infections. We sequenced the whole genome of S. saprophyticus type strain ATCC 15305, which harbors a circular chromosome of 2,516,575 bp with 2,446 ORFs and two plasmids. Comparative genomic analyses with the strains of two other species, Staphylococcus aureus and Staphylococcus epidermidis, as well as experimental data, revealed the following characteristics of the S. saprophyticus genome. S. saprophyticus does not possess any virulence factors found in S. aureus, such as coagulase, enterotoxins, exoenzymes, and extracellular matrix-binding proteins, although it does have a remarkable paralog expansion of transport systems related to highly variable ion contents in the urinary environment. A further unique feature is that only a single ORF is predictable as a cell wall-anchored protein, and it shows positive hemagglutination and adherence to human bladder cell associated with initial colonization in the urinary tract. It also shows significantly high urease activity in S. saprophyticus. The uropathogenicity of S. saprophyticus can be attributed to its genome that is needed for its survival in the human urinary tract by means of novel cell wall-anchored adhesin and redundant uro-adaptive transport systems, together with urease.

Abstract: Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S.haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S.haemolyticus, Staphylococcus aureus, and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the "oriC environ," likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S.haemolyticus, S.aureus, and S.epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S.haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.

Abstract: A specific phenotype of Staphylococcus aureus strains Mu50 and Mu3 is characterized by thickened cell wall and moderate resistance to vancomycin. The N315 strain is a prototype of methicillin-resistant S. aureus (MRSA), but it is methicillin susceptible, despite carrying the mecA resistance gene. Here, we revised differences in the sequences of Mu50 and N315, referencing that of Mu3 which were assumed to be of one lineage. The 362 ORFs diverse between Mu50 and N315 were picked up, and the corresponding ones in three strains were re-sequenced. This defined 213 ORFs diverse between Mu50 and N315, and 9 between Mu50 and Mu3. The fixed diversities of 174 ORFs (except for 39 silent ORFs from 213), including nucleotide substitution (NSs), frame shift, and truncation were grouped into three major functional categories, which were transport (14.9% in the 174 diverse ORFs), metabolism of carbohydrates (5.7%), and RNA synthesis (9.6%). The other gene categories had small diversities. These gene categories seemed to be functionally decisive for the Mu50-specific characters, the thickened cell wall and moderate vancomycin resistance. All of the diverse genes and the high quality sequence of Mu50 can be viewed at the web site (http://133.5.48.239/VRSA/).

Abstract: DNA microarray covering the whole genome of Staphylococcus aureus strain N315 was prepared to investigate transcription profiles. The microarray analyses revealed that vancomycin induces transcription of 139 genes. Forty-six genes among them failed to be induced in the vraSR null mutant KVR. Part of the genes regulated by VraSR system is associated with cell-wall biosynthesis, such as PBP2, SgtB and MurZ. Other cell-wall synthesis inhibitors also induced VraSR, suggesting that the sensor kinase VraS responds to the damage of cell-wall structure or inhibition of cell-wall biosynthesis. Additionally, the vraSR null mutants derived from hetero- and homo-methicillin-resistant S. aureus showed significant decrease of resistance against teicoplanin, beta-lactam, bacitracin and fosfomycin but not of D-cycloserine and levofloxacin. The observation strongly indicates that VraSR constitutes a positive regulator of cell-wall peptidoglycan synthesis, and that is deeply involved in the expression of beta-lactam and glycopeptide resistance in S. aureus.

Abstract: BACKGROUND: A new type of meticillin-resistant Staphylococcus aureus (MRSA), designated community-acquired MRSA, is becoming increasingly noticeable in the community, some strains of which cause fatal infections in otherwise healthy individuals. By contrast with hospital-acquired MRSA, community-acquired MRSA is more susceptible to non b-lactam antibiotics. We investigated the high virulence potential of certain strains of this bacterium. METHODS: We ascertained the whole genome sequence of MW2, a strain of community-acquired MRSA, by shotgun cloning and sequencing. MW2 caused fatal septicaemia and septic arthritis in a 16-month-old girl in North Dakota, USA, in 1998. The genome of this strain was compared with those of hospital-acquired MRSA strains, including N315 and Mu50. FINDINGS: Meticillin resistance gene (mecA) in MW2 was carried by a novel allelic form (type IVa) of staphylococcal cassette chromosome mec (SCCmec), by contrast with type II in N315 and Mu50. Type IVa SCCmec did not carry any of the multiple antibiotic resistance genes reported in type II SCCmec. By contrast, 19 additional virulence genes were recorded in the MW2 genome. All but two of these virulence genes were noted in four of the seven genomic islands of MW2. INTERPRETATION: MW2 carried a range of virulence and resistance genes that was distinct from those displayed on the chromosomes of extant S aureus strains. Most genes were carried by specific allelic forms of genomic islands in the MW2 chromosome. The combination of allelic forms of genomic islands is the genetic basis that determines the pathogenicity of medically important phenotypes of S aureus, including those of community-acquired MRSA strains.

Abstract: Significant advances have been made in recent years in our understanding of how methicillin resistance is acquired by Staphylococcus aureus. Integration of a staphylococcal cassette chromosome mec (SCCmec) element into the chromosome converts drug-sensitive S. aureus into the notorious hospital pathogen methicilin-resistant S. aureus (MRSA), which is resistant to practically all beta-lactam antibiotics. SCCmec is a novel class of mobile genetic element that is composed of the mec gene complex encoding methicillin resistance and the ccr gene complex that encodes recombinases responsible for its mobility. These elements also carry various resistance genes for non-beta-lactam antibiotics. After acquiring an SCCmec element, MRSA undergoes several mutational events and evolves into the most difficult-to-treat pathogen in hospitals, against which all extant antibiotics including vancomycin are ineffective. Recent epidemiological data imply that MRSA has embarked on another evolutionary path as a community pathogen, as at least one novel SCCmec element seems to have been successful in converting S. aureus strains from the normal human flora into MRSA.

Abstract: BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Abstract: We previously reported the first vancomycin-resistant Staphylococcus aureus (VRSA) clinical strain, Mu50, whose cell wall is remarkably thickened resulting from the activation of cell-wall synthesis. To explore the genetic basis for the vancomycin resistance, cDNA differential hybridization was performed using RNAs extracted from a set of closely related S. aureus strains with various levels of vancomycin susceptibilities. The strains were Mu3 (MIC = 2 microg/ml), Mu50 (MIC = 8 microg/ml), and a susceptible revertant of Mu50, Mu50omega (MIC = 0.5 microg/ml). In this study, we report identification of a novel response regulator, designated vraR (standing for vancomycin-resistance associated gene R) whose transcription was remarkably up-regulated in Mu3 and Mu50 as compared to Mu50omega. Experimental over-expression of VraR in vancomycin-susceptible strain N315P raised vancomycin resistance of the strain. Also, the genes coding for fructose utilization, fatty acid metabolism, and two putative ATP-binding cassette (ABC) transporter genes were found to be up-regulated in Mu3 and Mu50. On the other hand, Protein A expression was suppressed in Mu50, as compared with Mu3 and Mu50omega. We consider that the response regulator vraR is one of the key regulators modulating the level of vancomycin-resistance in S. aureus. Presumed increased uptake of fructose and altered fatty acid metabolism may also contribute to vancomycin resistance by supplying more precursor metabolites for cell-wall synthesis.

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Abstract: A novel operon, czrAB (zinc-responsible genes), was identified in the chromosome of Staphylococcus aureus. The operon consists of two genes, czrA and czrB. The czrA gene, coding for an 11.5 kDa protein, was homologous to cadC, arsR of S. aureus plasmid pI258 and smtB of Synechococcus PCC7942. The czrB, coding for a 36 kDa membrane spanning protein, was homologous to the czcD gene, cobalt, zinc and the cadmium-resistant factor of Bacillus subtilis and Alcaligenes eutrophus. In the presence of zinc (0.1-10 mM), the transcription of czrAB was enhanced in a concentration-dependent manner. Other heavy metals, such as cobalt, copper, manganese and nickel showed no effect on czrAB expression. The disruptant of the czrB gene became sensitive to zinc ion (MIC, 2 mM; MBC, 10 mM), and the complementation with the plasmid recovered the resistance to zinc at the same concentration as a parental strain (MIC, 5 mM; MBC, 20 mM). The disruptant accumulated intracellular zinc up to 0.4 mg per g dry weight of the organism, while that of the parental strain was 0.25 mg per g dry weight. The findings indicated that the novel operon czrAB should play a role in the transportation of zinc across the cell membrane to maintain the proper intracellular concentration.

Abstract: The heat-shock proteins are coded for the polycistronic operons hsp70 and hsp60 in Staphylococcus aureus. The hsp70 operon is comprised of five genes, hrc37, hsp20, hsp70, hsp40 and orf35, and the hsp60 is comprised of two genes, hsp10 and hsp60. The hsp70 operon transcribed five different sizes of mRNA from three promoters: P1, the most active promoter, transcribed 6.0 and 3.6 kb mRNAs; P2 transcribed a single 1.8 kb mRNA; and P3 transcribed 4.2 and 2.4 kb mRNAs. The hsp60 operon transcribed a single 2.1 kb transcript from only one promoter, P1. Both operons had a common structure of inverted repeat element (CIRCE, Controlling Inverted Repeat of Chaperon Expression) at the promoter region. All of the transcripts were heat (46 C) inducible. One of the unidentified genes, hrc37, was characterized. The disruptant of the hrc37 in the hsp70 operon enhanced the transcription of both operons at 37 C (derepression). Complementation of the disruptant with the cloned hrc37 plasmid recovered the repression of the transcription of both operons at 37 C. The product of hrc37, Hrc37, was found to bind to the CIRCE element. These findings indicated that Hrc37 from the hsp70 operon repressed the transcription of both the hsp70 and hsp60 operons by binding to the CIRCE element located at the promoter region.

Abstract: The Staphylococcus aureus HSP70 operon produces a polycistronic RNA in response to heat shock, and ORF37 is the first protein to be translated. The promoter of this operon contains a palindromic nucleotide sequence that may form a stem-loop structure. Structural analysis of the promoter regions by atomic force microscopy (AFM) revealed a quadruplet that consists of a pair of stem-loops. A novel "SL2S' (Stem-Loop-Loop-Stem) model was proposed for this structure. AFM also revealed the binding of ORF37 to the quadruplet, establishing a molecular mechanism for this heat shock gene expression; ORF37 acts as a regulator by binding to the SL2S structure in the promoter.

Abstract: The growth of Staphylococcus aureus occurs at a wide range of pH(5-10), while the optimal is pH 7.0-7.5. The molecular mechanism of such pH tolerant properties should be elucidated because the production of the virulence factors was greatly affected by environmental pH. The effect of pH shift on the composition of cytosolic proteins in S. aureus was examined. A protein with a molecular mass of 23 kDa was remarkably enhanced by a pH upshift from 7 to 10. This alkaline shock protein (ASP23) was isolated and purified by ion-exchange chromatography. The N-terminal sequences of the purified protein and the protease-digested peptides were analyzed. The 320-bp DNA fragment that was designed from the peptide analysis was amplified. Using the amplified fragment as a probe, the ASP gene, asp23, was cloned. The deduced primary sequence of ASP23 comprised 169 amino acids with a calculated molecular weight of 19,191. Northern analysis revealed that asp23 was positively regulated at the transcription level by alkaline shock. Homology search revealed that asp23 is a novel gene. Although the physiological role of ASP23 has yet to be further analysed, we suggest that ASP23 plays a key role in alkaline pH tolerance of S. aureus.

Abstract: We have identified two new heat shock protein genes, orf37 and orf35, in Staphylococcus aureus, located upstream and downstream of grpE(hsp20), dnaK(hsp70), and dnaJ(hsp40) homologous genes in the order orf37-hsp20-hsp70-hsp40-orf35. The transcripts of both orf37 and orf35 were increased by thermal upshift of the culture from 37 to 46 degrees C. The heat shock promoters were located upstream of orf37 and upstream of hsp40. The deduced peptide of orf37 showed similarity with those of orfA in Clostridium acetobutylicum and orf39 in Bacillus subtilis. orf35 was unique in S. aureus and has not yet been described in other bacteria.

Abstract: Methicillin-resistance of S.aureus (MRSA) was diminished or depressed at 44 degrees C. In order to investigate whether bacterial heat shock response is correlated with methicillin resistance, we examined the inducibility of the heat shock proteins (HSPs) in MRSA, and cloned and sequenced of their genes. A temperature shift from 37 degrees C to 46 degrees C enhanced the production of at least 8 kinds of cytoplasmic proteins as measured with two-dimensional PAGE. The induced protein profile was almost the same as methicillin sensitive S.aureus, and stress conditions due to ethanol, cadmium or low pH. Two of these proteins were HSP60 and HSP10. Their N-terminal amino acid sequences were 79%, and 83%, homologous with thermobacterium PS3, respectively. A positively hybridized 4.2 kbp DNA fragment encoding both proteins was isolated from the chromosomal DNA of MRSA. The resulting sequence revealed two reading frames and showed high homology to those of hsp60 (groEL) and hsp10 (groES) of bacteria (E.coli) and several other species. The genes of HSP60 and HSP10 in S.aureus comprised an operon as in E.coli. The relationship between those HSPs and PBP2' is currently under investigation.