Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S. aureus) and RN450M (methicillin-resistant S. aureus). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S. aureus.     

Molecular characterisation of the haemolytic isolates of Bacillus anthracis originated in Poland

Bacillus anthracis is generally considered non-haemolytic, when cultured on the solid media. However, strains capable to lyse sheep erythrocytes have been reported. Anthrolysin O, an orthologue of cereolysin was proposed as a putative haemolysin of B. anthracis. AIM: to determine whether anthrolysin O, haemolytic enterotoxin HBL and the pleiotropic regulator PlcR that activates antrholysin O production are associated with a haemolytic activity of B. anthracis strains isolated in Poland. MATERIAL: in total 8 B. anthracis strains - the fully virulent BL1 and seven the pXO2 lacking strains including: a vaccine strain Sterne 34F2 together with three haemolytic and three non-haemolytic strains isolated from different samples of the same animal died from anthrax in Poland. METHODS: The haemolytic activity was detected using Columbia agar plates supplemented with 5% of sheep blood. Anthrolvsin O, cereolysin and gene hblA encoding the key subunit of the HBL were detected by PCR. In addition, the plcR gene fragment containing the B. anthracis specific non-sense mutation was analysed by the DNA sequencing. Ten marker loci based MLVA genotyping was performed to distinguish tested strains. RESULTS: The alo gene encoding anthrolysin O was detected in both the haemolytic and non-haemolytic strains while hblA was absent. The B. anthracis specific plcR non-sense mutation was detected in both the groups of tested strains, suggesting that the haemolysis in tested strains may rather be conferred by the PlcR-independent factors. Moreover, haemolytic and non-haemolytic strains were indistinguishable by the MLVA. Obtained results may argue the haemolytic and non-haemolytic strains are isogenic and most probably a single mutational event is responsible for the haemolytic phenotype induction.     

Two thermostable nucleases coexisted in Staphylococcus aureus: evidence from mutagenesis and in vitro expression

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same genetic origin. However, two ORFs in S. aureus genomes were predicted to encode nucleases. One encoded an unnamed nuclease A (SNase) (termed nuc1 ), and the other encoded a thermonuclease (TNase) named nuc (termed nuc2 ). In order to verify whether the two thermostable nuclease proteins are coexpressed in S. aureus , the nuc1 and nuc2 genes were cloned and expressed in Escherichia coli , and both of the recombinant proteins showed thermostable nuclease activity in a toluidine blue-DNA assay. Furthermore, a nuc1 -deleted mutant of S. aureus strain RN4220 (termed RNΔ nuc1 ) was successfully constructed by homologous recombination. Selection and characterization of this mutant strain revealed that it still exhibited thermostable nuclease activity, but at a relative lower level than that of the parent strain. The nucleases secreted by the parent strain and nuc1 -deleted strain still showed functional activity after 30 min at 121 °C. The findings indicated that two types of thermostable nucleases, encoded by two different genes, coexisted in S. aureus .     

Isolation and characterization of a Tn551-autolysis mutant of Staphylococcus aureus.

A Lyt- mutant with reduced autolytic activity was isolated after Tn551 mutagenesis of the methicillin-susceptible Staphylococcus aureus laboratory strain RN450. The Lyt- phenotype could be transferred back into the parent and into a variety of other S. aureus strains by transduction of the transposon marker. Southern analysis has located the Tn551 insert to a 3.2-kb HindIII DNA fragment on the SmaI B fragment of the staphylococcal chromosome. The Lyt- phenotype included reduced rates of cell wall turnover and autolysis induced by detergent or methicillin treatment; however, the rate of methicillin-induced killing was not affected. Peptidoglycans prepared from the parental and mutant cells showed identical muropeptide compositions, as resolved by a high-resolution high-pressure liquid chromatography technique. On the other hand, LiCl extracts of the mutant cells contained reduced amounts of total protein and lower specific cell wall-degrading activity compared with those of extracts of parental cells. The profile of bacteriolytic enzymes as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple band differences between mutant and parental cells; a major lytic band with properties characteristic of the staphylococcal endo-beta-N-acetylglucosaminidase was completely absent from the Lyt- cells. The Lyt- phenotype transduced into a series of methicillin-resistant strains of both homogeneous and heterogeneous phenotypes caused only a modest decrease in the level of methicillin resistance, as determined by population analysis.     

Effects of streptomycin and novobiocin on Staphylococcus aureus gene expression.

Streptomycin and novobiocin induced production of protein A and inhibited production of alpha- and beta-hemolysins in mutants of Staphylococcus aureus strains RN450 and RN1 resistant to these antibiotics. Streptomycin, but not novobiocin, also inhibited propagation of bacteriophages of serological group B, whereas phages of group A were unaffected. Streptomycin had to be present at adsorption of the phage, and 10 mM CACL2 reversed the inhibitory effect. Lysogenization and competence induction occurred in the presence of streptomycin, suggesting that some early phage genes were expressed.     

金黄色葡萄球菌核酸酶基因缺失突变株RN4220Δnuc1的构建

金黄色葡萄球菌存在两个核酸酶编码基因,一个是葡萄球菌核酸酶（Staphylococcal nuclease,SNase）,命名为nuc1,另一个是耐热核酸酶（Thermonuclease,TNase）,命名为nuc2,nuc2是一个新的候选基因,以往认为金黄色葡萄球菌中的核酸酶只源于一个编码基因nuc1,为了进一步研究nuc2基因的功能,首先要将金黄色葡萄球菌nuc1基因缺失。本研究目的就是通过构建同源重组质粒pBT2Δnuc1,将其电转入金黄色葡萄球菌菌株RN4220中,获得nuc1基因缺失突变株。经过了七轮培养和筛选,同源重组几率为2%（7/345）,筛选出的nuc1突变株用PCR方法和RT-PCR进行了验证,从而获得了nuc1基因缺失突变株RN4220Δnuc1。     

Inactivation of the SauI Type I Restriction-Modification System Is Not Sufficient To Generate Staphylococcus aureus Strains Capable of Efficiently Accepting Foreign DNA

Genetic manipulation of Staphylococcus aureus is limited by the availability of only a single strain, RN4220, that is capable of easily accepting foreign DNA. Inactivation of the hsdR gene of the SauI type I restriction-modification system was shown previously to be responsible for the high transformation efficiency of RN4220 (D. E. Waldron and J. A. Lindsay, J Bacteriol. 188:5578-5585, 2006). However, deletion of this gene in three different S. aureus strains was not sufficient to make them readily transformable, which would be remarkably useful for genetic studies of this pathogenic organism. These results indicate that another unknown factor(s) is required for the transformable phenotype in S. aureus.Staphylococcus aureus is a major pathogen that causes both nosocomial and community-acquired infections, which range from superficial skin infections to severe systemic diseases. It is an extremely versatile pathogen which has developed resistance to virtually all known classes of antibiotics and which expresses various virulence factors that allow it to cause infection in different environments.Molecular genetic studies of S. aureus have resulted in a better understanding of both the virulence and antibiotic resistance mechanisms of this organism, which is crucial for discovery of new approaches to treat staphylococcal infections. One of the limitations in genetically manipulating S. aureus is the fact that there is only a single strain available which can easily accept plasmid DNA isolated from Escherichia coli. This strain, RN4220, is a chemical mutant obtained in the early 1980s by highly mutagenizing NCTC8325-4 (= RN450) with nitrosoguanidine and selecting for a mutant that was able to accept and maintain S. aureus plasmids (10; B. Kreiswirth, personal communication).One of the most important bacterial defenses against uptake of foreign DNA is restriction-modification (R-M) systems. These systems, comprising restriction endonucleases and methyltransferases, recognize and modify specific DNA sequences, protecting “self” DNA from restriction while eliminating potentially harmful foreign DNA which lacks appropriate modification (12). There are three distinct well-characterized types of classical R-M systems, including type II restriction enzymes, which cut DNA within specific recognition sequences and are therefore widely used as molecular biology tools (3). S. aureus strains may contain different R-M systems, including the Sau3AI and Sau96I type II systems (present in isolates of particular lytic groups) (19, 20) or the Sau42I BcgI-like R-M system expressed by S. aureus φ42 lysogens (5). However, the only chromosomal R-M system widely distributed in the sequenced S. aureus isolates is the SauI type I system (21). Type I R-M systems require the products of three genes, hsdR (restriction), hsdM (modification), and hsdS (sequence specificity), and cut DNA at sites remote from the recognition sequence (12). The staphylococcal SauI system includes a single hsdR gene and two copies of the hsdM and hsdS genes, and there is substantial variation between the hsdS genes from different isolates (21). It was recently shown that transformable RN4220 carries a stop mutation in the sauI hsdR gene and that complementation with a functional copy of this gene restores a nontransformable phenotype (21). An hsdR mutant does not cleave foreign unmodified DNA, but it does modify incoming DNA, which therefore is not cleaved when it is transferred to other S. aureus strains that contain an identical R-M system. For this reason, RN4220 has become an essential intermediate for laboratory manipulation of S. aureus, despite its limited clinical relevance. DNA first introduced into RN4220 by electroporation can then be transferred into other laboratory strains (for example, by phage transduction).The relevance of the SauI type I R-M system for horizontal transfer of foreign DNA in different S. aureus isolates in nature has been confirmed with bovine isolates that are hypersusceptible to gene transfer from enterococci and have stop mutations in each of the two SauI hsdS gene copies (18). These “hyperrecipient” strains may be ideal backgrounds for the acquisition of new antibiotic resistance markers, such as the vanA gene complex present in vancomycin-resistant S. aureus strains (18). However, the existence of a second pathway in S. aureus that blocks horizontal transfer of foreign DNA or of an unknown but necessary factor essential for SauI activity has also been proposed based on the fact that some strains that are hypersusceptible to gene transfer (such as B111 used by Noble et al. [13]) do not have a mutation in any of the five hsd genes (18).The availability of laboratory and clinical strains other than RN4220 that are capable of accepting foreign DNA would be remarkably useful for genetic studies of S. aureus, including studies of virulence and antibiotic resistance mechanisms present only in relevant clinical isolates. Therefore, we have tried to reproduce the hsdR mutation in RN4220 in different backgrounds of widely used laboratory strains in order to generate useful, easily transformable strains.     

Whole-genome sequencing of Staphylococcus aureus strain RN4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain

Staphylococcus aureus RN4220, a cloning intermediate, is sometimes used in virulence, resistance, and metabolic studies. Using whole-genome sequencing, we showed that RN4220 differs from NCTC8325 and contains a number of genetic polymorphisms that affect both virulence and general fitness, implying a need for caution in using this strain for such studies.     

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.     

The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus

The expression of many virulence determinants in Staphylococcus aureus is tightly coordinated generally by global regulatory elements such as accessory gene regulator (agr), staphylococcal accessory regulator and the alternative sigma factor sigmaB. We have compared the two-dimensional (2-D) protein pattern of extracellular protein extracts of wild-type cells with the 2-D patterns of the respective regulatory mutants in order to identify proteins whose amount is influenced by a mutation in agr or sigB. In order to quantify changes in the level of interesting proteins we used the Ettan-fluorescence difference gel electrophoresis technique (Amersham Biosciences). As in most bacteria, the amount of extracellular proteins was strongly regulated and increased mainly in the stationary phase of growth at high cell densities. By comparing the extracellular protein pattern of the RN6390 rsbU strain with that of an isogenic agr mutant RN6911 we show that the level of about 70 protein spots changed in the mutant. To analyze the role of sigmaB in virulence gene expression an RsbU+ (RN6390 RsbU+) derivative was included in this study. The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Deltaagr/DeltarsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes.     

The patterns of extracellular protein formation by spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus

Spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus were isolated on 4% (w/v) Tryptone Soya Agar containing 4 and 40 times the m.i.c. for rifampicin. A number of colonies were selected at each rifampicin concentration and were grown aerobically in 3% (w/v) Tryptone Soya Broth for 24 h at 37 degrees C. In the case of S. aureus RN4220 all the mutants grew to bacterial densities up to approximately 1.7 times more than the parent organism. The corresponding levels of extracellular protein secretion varied over a 5-fold range, all the mutants being less productive than the parent. By contrast, mutants of the wild-type Wood 46 strain achieved bacterial densities of only 45-83% that of the parent whilst exoprotein secretion showed a smaller 1.7-fold variation. However, widely-differing patterns of exoproteins were revealed by SDS-polyacrylamide gel electrophoresis of the parent and mutant organisms of both strains.     

Cloning and sequencing of sarA of Staphylococcus aureus, a gene required for the expression of agr.

To evaluate the effect of a sar mutation on the agr locus, Northern (RNA) blotting was performed to determine the levels of RNAIII, the agr regulatory molecule, in two isogenic pairs of Staphylococcus aureus strains. Our results demonstrated that RNAIII was either significantly diminished or absent in both sar mutants compared with the parents. The RNAIII level was partially restored in sar mutants complemented with an intact sar gene (designated sarA). Additionally, we were able to complement selected sar phenotypes with a plasmid carrying RNAIII (pRN6735). These studies suggest that the sarA gene is necessary for the optimal expression of agr. The sarA gene of strain RN450 was subsequently cloned and sequenced. Sequence analysis revealed an open reading frame of 372 bp with a predicted molecular size of 14,718 Da and a deduced pI of 8.52. The deduced protein sequence has a predominance of charged residues (33%) and shares sequence similarity with the virF gene of Shigella flexneri.     

Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus

A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.     

Interaction of UV and N-methyl-N'-nitro-N-nitrosoguanidine: cytotoxicity and mutagenicity in V79 cells

Killing and mutation by UV in the MNNG-exposed population of V79 cells, as well as by MNNG in the UV-irradiated population of these cells have been studied. It was observed that pretreatment with MNNG increased the killing and mutation by UV, whereas, pretreatment with UV had no effect upon killing and mutation by MNNG. The increase in sensitivity to UV due to pretreatment with MNNG was lost if UV exposure was delayed for 24 h after MNNG treatment.     

Sister-chromatid exchanges (SCEs), cell survival and mutation in HeLa s3 cells with different sensitivity to alkylating agents; evidence that SCE induction and cell survival or mutation induction are dissociable

We previously isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR from HeLa S3 Mer- cells. In the present study, we have isolated 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant cells, ACr. The MR cells had only a little O6-methylguanine-DNA methyltransferase (MT) activity, while the ACr cells had increased MT activity and also became resistant to the cytotoxic effect of MNNG. We compared the induction of sister-chromatid exchanges (SCEs), cell survival and mutation in these HeLa S3 cells with different sensitivity to MNNG. The ACr cells were much more resistant than the parental HeLa S3 Mer- cells to cytotoxicity, mutagenicity and SCE induction by MNNG, showing a positive correlation between SCE induction and cell killing or mutation. In contrast, this positive relationship was not observed between HeLa S3 Mer- and MR cells. These results suggest that O6-methylguanine (O6-MeG) is involved in the induction of the biological effects of MNNG such as cytotoxicity, mutagenicity and SCEs, and also indicate that SCE induction does not always correlate with cell killing and mutation.     

Involvement of the drug efflux protein TolC in mutagenicity induced by MNNG or Trp-P-2

In the development of mutation assay systems, a number of approaches have been performed with a particular view to improve sensitivity. The inhibition of mutagen-efflux from tester bacteria might lead to increased mutagenic activity as the concentration of mutagen increases inside the cell. In this study, we constructed a series of Escherichia coli CC strains lacking the TolC protein to determine if mutation is actually enhanced by the inhibition of mutagen reflux. TolC is an outer-membrane protein that forms part of an excretion system in E. coli. The frequency of induction of mutations by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and ethyl methanesulfonate (EMS) were significantly higher in TolC-deficient strain KA796-1/CC102 than in TolC-proficient strains, especially that of MNNG was seven times higher and detected at lower doses than in the parent strain. In a KA796-1/CC108 TolC-deficient strain, mutation induced by Trp-P-2 was detected at significant levels, even at low doses that did not induce detectable levels of mutation in the parent strain KA796/CC108. When the wild-type E. coli tolC gene was introduced into a strain lacking the gene, TolC function was restored and the frequency of induction by MNNG became similar to that of the wild-type. In contrast, introduction of a mutant tolC gene did not complement the TolC deficiency and the frequency of MNNG-induced mutations remained high. These results suggest that some mutagens are excreted at least in part via the TolC system, and that the lack of functional TolC increases the susceptibility of bacteria to many mutagens.     

Genetic and biochemical properties of thialysine-resistant mutants of Saccharomyces cerevisiae

Three groups of lysine-excreting, thialysine-resistant mutants of Saccharomyces cerevisiae were derived from the wild-type strain (X2180) by mutagenic treatment and selected on the basis of a cross-feeding assay. Mutants MNNG2-9, MNNG2-27, MNNG2-39 and MNNG2-62 (group 1) exhibited a 2:2 segregation for thialysine resistance following mating with a wild-type strain and a lower than wild-type lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was dominant in specific hybrids. Mutant MNNG2-2 (group II) was similar to group I mutants except that the thialysine-resistant phenotype was recessive in the hybrid. Mutant MNNG3-142 (group III) exhibited an irregular ratio of segregation of thialysine resistance and a significantly lower lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was recessive in the hybrid. The growth of both group I and group III mutants was temperature-sensitive. The thialysine-resistant mutants exhibited pleiotropic properties including the increased production and excretion of lysine, thermosensitive growth and an impairment of lysyl-tRNA synthetase activity.     

Aphidicolin allows a rapid and simple evaluation of DNA-repair synthesis in damaged human cells

The decrease in microbial mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) was compared in an animal mediation with rats and in direct incubation with human as well as rat blood and blood components. The mutagenic activity was assayed by reverse mutation from streptomycin (SM) dependence to non-dependence in Escherichia coli, strain Sd-B (TC). The mutagenic response curves of both MNNG and MNU were approximately linear and parallel at non-cytotoxic concentrations. However, the mutagenic capabilities of MNNG were estimated to be 10-fold more potent than those of MNU. The mutagenic activity in blood and liver preparations from rats killed immediately after intravenous injection of MNNG, 50 mg/kg, was negative. Results with MNU, 100 mg/kg, were positive in both cases.For the detection of mutagenicity, blood was diluted 50 times for the final testing mixture (1 ml) to avoid bactericidal effects of the blood itself. When a larger amount of liver preparation was used in the tests, and diluted 8 times, mutagenic activity was still detected 15 min after injection of MNU, 80 mg/kg. Comparisons of the diminished rate of mutagenicity between MNNG and MNU during certain periods of incubation with blood indicated that MNNG was inactivated much more rapidly than MNU with both human and rat blood. Plasma showed a moderate inactivating effect on both MNNG and MNU. Red blood cells inactivated MNNG at a remarkably rapid rate similar to that of whole blood, but was less effective on MNU. In further experiments with red- cell components, the cell contents inactivated both MNNG and MNU at rates similar to those with red cells, but cell membrane had absolutely no effect in decreasing the mutagenicity in either MNNG or MNU.