Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages

The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.     

Biological characteristics of Staphylococcus aureus isolated from nude mice

The characteristics of 50 strains of Staphylococcus aureus isolated from BALB/c nude mice (nu/nu, nu/+) with or without subcutaneous abscesses [13] were examined. All the 50 strains belonged to biotype B according to the classification by Hájek and Marsálek. All of them were phage typable, showing a single phage pattern of 52A/79/47/53/77/83A/85. The coagulase type was classified as VII. All of the 50 strains were highly sensitive to penicillin, methylphenylisoxazolyl penicillin, erythromycin, spiramycin, lincomycin, chloramphenicol, tetracycline, kanamycin, gentamicin and cephaloridine, but were resistant to sulfisoxazole. Two S. aureus strains isolated from the nostril and finger of one person working in the mouse colony were identified as the same biotype as the murine strains but different in phage type, coagulase type and drug resistance pattern.     

Nitrate reductase system in Staphylococcus aureus wild type and mutants.

Respiratory nitrate reductase with lactate as a hydrogen donor has been studied in cells and spheroplast preparations of wild type and heme-deficienct mutants of Staphylococcus aureus. The activity is rapidly induced when suspensions of aerobically grown cells are incubated without aeration in a complete medium with nitrate. In ruptured spheroplast preparations, the activity with lactate as the donor is located in the membrane fraction, whereas at least 50% of the activity assayed with reduced benzyl viologen is in the cytoplasm. The reductase is inhibited by azide and cyanide, and the lactate-linked system is also sensitive to oxamate, 2-heptyl-4-hydroxyquinoline-N-oxide, dicoumarol, and p-chloromercuribenzoate. An inactive form of the reductase is apparently made during induction with tungstate; this can be activated by subsequent incubation with molybdate in the presence of chloramphenicol. Nitrate reductase activity with reduced benzyl viologen as the donor is induced in suspensions of heme-deficient mutants in the presence or absence of heme. The proportion of cytoplasmic activity is increased in the absence of heme. The staphylococcal nitrate reductase has many of the characteristics commonly associated with the respiratory enzyme in other organisms, but the apparent predominance of cytoplasmic activity is unusual.     

Characterization of AloI, a restriction-modification system of a new type.

We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.     

万古霉素耐药金黄色葡萄球菌生物学特性

目的观察金黄色葡萄球菌对万古霉素耐药后生物学特性的变化,探讨有效方法,为临床正确诊断奠定基础。方法使用万古霉素体外诱导将1株h-VRSA逐步诱导为VISA;观察原代菌及其诱导菌的形态结构、培养特性和生化反应结果并与标准菌株ATCC 29213比较,了解金黄色葡萄球菌对万古霉素耐药后生物学特性变化规律;比较手工法、仪器法和PCR检测法的细菌鉴定结果。结果金黄色葡萄球菌对万古霉素耐药后主要生物学特性改变为:细胞壁增厚、细胞表面粗糙,有结节状凸起、菌落变小,溶血环和色素消失,血浆凝固酶、甘露醇、甘露糖等生化反应由阳性转为阴性。生物学特性改变可导致手工法和仪器法细菌鉴定结果错误,但对PCR检测鉴定结果没有影响。结论金葡菌对万古霉素耐药后生物学特性改变可导致常规方法细菌鉴定结果错误,应引起临床的重视。     

Discovery of substrate for type I signal peptidase SpsB from Staphylococcus aureus.

Based on the kinetic model of substrate phage proteolysis, we have formulated a strategy for best manipulating the conditions in screening phage display libraries for protease substrates (Sharkov, N. A., Davis, R. M., Reidhaar-Olson, J. F., Navre, M., and Cai, D. (2001) J. Biol. Chem. 276, 10788-10793). This strategy is exploited in the present study with signal peptidase SpsB from Staphylococcus aureus. We demonstrate that highly active substrate phage clones can be isolated from a phage display library by systematically tuning the selection stringency in screening. Several of the selected clones exhibit superior reactivity over a control, the best clone, SIIIRIII-8, showing >100-fold improvement. Because no conserved sequence features were readily revealed that could allow delineation of the active and unreactive clones, the sequences identified in five of the active clones were tested as synthetic dodecamers, Ac-AGX(8)GA-NH(2). Using electrospray ionization mass spectrometry, we show that four of these peptides can be cleaved by SpsB and that Ala is the P1 residue exclusively and Ala or Leu the P3 residue, in keeping with the (-3, -1) rule for substrate recognition by signal peptidase. Our successful screening with SpsB demonstrated the general applicability of the screening strategy and allowed us to isolate the first peptide substrates for the enzyme.     

Regulation of gene expression in a type II restriction-modification system

Type II restriction-modification systems are comprised of a restriction endonuclease and methyltransferase. The enzymes are coded by individual genes and recognize the same DNA sequence. Endonuclease makes a double-stranded break in the recognition site, and methyltransferase covalently modifies DNA bases within the recognition site, thereby preventing cleavage by the endonuclease. The concerted action of these enzymes plays the role of a primitive immune system and protects the bacterial host cell from invasion by foreign (for example, viral) DNA. However, uncontrolled expression of restriction-modification system genes can result in the death of a bacterial host cell because of endonuclease cleavage of the host DNA. In the present review, data on the regulation of expression of the type II restriction-modification enzymes genes are discussed.     

Identification of a high-affinity glycine betaine transport system in Staphylococcus aureus.

Staphylococcus aureus accumulates proline and glycine betaine when cells are grown at low water activity. In the present study, we have identified a high-affinity glycine betaine transport system in this bacterium. Optimal activity for this transport system was measured in the presence of high NaCl concentrations, but transport activity was not stimulated by high concentrations of other solutes.     

KpnBI is the prototype of a new family (IE) of bacterial type I restriction-modification system

KpnBI is a restriction-modification (R-M) system recognized in the GM236 strain of Klebsiella pneumoniae. Here, the KpnBI modification genes were cloned into a plasmid using a modification expression screening method. The modification genes that consist of both hsdM (2631 bp) and hsdS (1344 bp) genes were identified on an 8.2 kb EcoRI chromosomal fragment. These two genes overlap by one base and share the same promoter located upstream of the hsdM gene. Using recently developed plasmid R-M tests and a computer program RM Search, the DNA recognition sequence for the KpnBI enzymes was identified as a new 8 nt sequence containing one degenerate base with a 6 nt spacer, CAAANNNNNNRTCA. From Dam methylation and HindIII sensitivity tests, the methylation loci were predicted to be the italicized third adenine in the 5′ specific region and the adenine opposite the italicized thymine in the 3′ specific region. Combined with previous sequence data for hsdR, we concluded that the KpnBI system is a typical type I R-M system. The deduced amino acid sequences of the three subunits of the KpnBI system show only limited homologies (25 to 33% identity) at best, to the four previously categorized type I families (IA, IB, IC, and ID). Furthermore, their identity scores to other uncharacterized putative genome type I sequences were 53% at maximum. Therefore, we propose that KpnBI is the prototype of a new ‘type IE’ family.     

Regulation of gene expression in type II restriction-modification system

Type II restriction-modification systems are comprised of a restriction endonuclease and methyltransferase. The enzymes are coded by individual genes and recognize the same DNA sequence. Endonuclease makes a double-stranded break in the recognition site, and methyltransferase covalently modifies the DNA bases within the recognition site, thereby down-regulating endonuclease activity. Coordinated action of these enzymes plays a role of primitive immune system and protects bacterial host cell from the invasion of foreign (for example, viral) DNA. However, uncontrolled expression of the restriction-modification system genes can result in the death of bacterial host cell because of the endonuclease cleavage of host DNA. In the present review, the data on the expression regulation of the type II restriction-modification enzymes are discussed.     

Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.     

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.     

2-Oxoglutarate transport system in Staphylococcus aureus

2-[(14)C]oxoglutarate uptake in resting cells of Staphylococcus aureus 17810S occurs via two kinetically different systems: (1) a secondary, electrogenic 2-oxoglutarate:H(+) symporter (K(m)=0.105 mM), energized by an electrochemical proton potential (Delta mu H(+)) that is generated by the oxidation of endogenous amino acids and sensitive to ionophores, and (2) a Delta mu H(+)-independent facilitated diffusion system (K(m)=1.31 mM). The 2-oxoglutarate transport system of S. aureus 17810S can be classified as a new member of the MHS (metabolite:H(+) symporter) family. This transporter takes up various dicarboxylic acids in the order of affinity: succinate = malate > fumarate > 2-oxoglutarate > glutamate. Energy conservation with 2-oxoglutarate was studied in starved cells of strain 17810S. Initial transport of 2-oxoglutarate in these cells is energized by Delta mu H(+) generated via hydrolysis of residual ATP. Subsequent oxidation of the accumulated 2-oxoglutarate generates Delta mu H(+) for further, autoenergized transport of this 2-oxoacid and also for Delta mu H(+)-linked resynthesis of ATP. In the cadmium-sensitive S. aureus 17810S, Cd(2+) accumulation strongly inhibits energy conservation with 2-oxoglutarate at the level of Delta mu H(+) generation, without direct blocking of the 2-oxoglutarate transport system or ATP synthase complex. In the cadmium-resistant S. aureus 17810R, Cd(2+) does not affect energy conservation due to its extrusion by the Cd(2+) efflux system (Cd(2+)-ATPase of P-type), which prevents Cd(2+) accumulation.