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1.
J Bacteriol ; 195(7): 1421-7, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23335411

ABSTRACT

Peptidoglycan (PG) composition in intact cells of methicillin-resistant Staphylococcus aureus (MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.


Subject(s)
Bacterial Proteins/genetics , Cell Wall/chemistry , Glycine/analysis , Methicillin-Resistant Staphylococcus aureus/chemistry , Oligopeptides/analysis , Peptidoglycan/chemistry , Gene Knockout Techniques , Magnetic Resonance Spectroscopy
2.
Antimicrob Agents Chemother ; 55(9): 4386-93, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21709093

ABSTRACT

Finafloxacin is a new fluoroquinolone antibiotic with the unique property of increasing antibacterial activity at pH values lower than neutral. Whereas its antibacterial activity at neutral pH matches that of other quinolones in clinical use, it is expected to surpass this activity in tissues and body fluids acidified by the infection or inflammation processes. Pharmacokinetic parameters of oral single and multiple doses of up to 800 mg of finafloxacin and safety/tolerability observations were assessed in a phase I study including 95 healthy volunteers. Finafloxacin is well absorbed after oral administration, generating maximum concentrations (C(max)s) in plasma at least comparable to those of other fluoroquinolones, with a half-life of around 10 h. About one-third of the dose is excreted unchanged in the urine. Renal elimination appears to be a saturable process leading to slight increases of the area under the concentration-time curve extrapolated to infinity and dose normalized (AUC(∞,norm)) at dosages of 400 mg and above. Safety and tolerability data characterize finafloxacin as a drug with a favorable safety profile. In particular, adverse reactions regarded as class-typical of fluoroquinolones, such as, e.g., electrocardiogram (ECG) changes, neurotoxic effects, or hypoglycemia, were not observed in the study population.


Subject(s)
Fluoroquinolones/adverse effects , Fluoroquinolones/pharmacokinetics , Administration, Oral , Adult , Double-Blind Method , Female , Fluoroquinolones/blood , Fluoroquinolones/urine , Humans , Male , Middle Aged
3.
Antimicrob Agents Chemother ; 55(9): 4394-7, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21709094

ABSTRACT

Finafloxacin is a novel fluoroquinolone that exhibits enhanced antibacterial activity under acidic conditions. The aim of this study was to define the in vitro pH-activity relationship. Finafloxacin exhibited optimal antibacterial activity between pH 5.0 and 6.0 at which MICs were 4- to 8-fold lower than those determined at neutral pH. These observations were then confirmed against a larger collection of bacteria. These data suggest that finafloxacin could potentially offer a therapeutic advantage within acidic foci of infection.


Subject(s)
Anti-Bacterial Agents/pharmacology , Fluoroquinolones/pharmacology , Ciprofloxacin/pharmacology , Escherichia coli/drug effects , Hydrogen-Ion Concentration , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Ofloxacin/pharmacology , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects
4.
J Antimicrob Chemother ; 65(12): 2530-3, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20940181

ABSTRACT

OBJECTIVES: Finafloxacin is an investigational fluoroquinolone exhibiting broad-spectrum activity that is enhanced under slightly acidic conditions (pH 5.0-6.5). The impact of individual and combinations of chromosomal mutations (gyrA, parC and marR) and the plasmid-mediated fluoroquinolone resistance mechanisms QepA1, QnrA1, QnrB1, QnrS1 and AAC(6')-Ib-cr were investigated. METHODS: The MICs of finafloxacin, compared with those of ciprofloxacin, levofloxacin and moxifloxacin, were determined at pH 5.8 and 7.2. RESULTS: MICs of finafloxacin compared with other fluoroquinolones at pH 5.8 were lower by a factor of 2-256. MICs of finafloxacin were unaffected by QepA1. Moreover, finafloxacin appeared not to be a substrate for AAC(6')-Ib-cr. CONCLUSIONS: Compared with ciprofloxacin, levofloxacin and moxifloxacin, finafloxacin shows higher activity especially at pH 5.8 against Escherichia coli mutants expressing known fluoroquinolone resistance determinants alone and in combinations.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Escherichia coli/drug effects , Fluoroquinolones/pharmacology , Plasmids/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chromosomes, Bacterial/genetics , DNA Gyrase/chemistry , DNA Gyrase/genetics , DNA Gyrase/metabolism , DNA Topoisomerase IV/chemistry , DNA Topoisomerase IV/genetics , DNA Topoisomerase IV/metabolism , DNA, Bacterial/genetics , Escherichia coli/genetics , Humans , Hydrogen-Ion Concentration , Microbial Sensitivity Tests
5.
Biochemistry ; 48(14): 3100-8, 2009 Apr 14.
Article in English | MEDLINE | ID: mdl-19309106

ABSTRACT

Compositional analysis of the peptidoglycan (PG) of a wild-type methicillin-resistant Staphylococcus aureus and its fem-deletion mutants has been performed on whole cells and cell walls using stable-isotope labeling and rotational-echo double-resonance NMR. The labels included [1-(13)C,(15)N]glycine and l-[epsilon-(15)N]lysine (for a direct measure of the number of glycyl residues in the bridging segment), [1-(13)C]glycine and l-[epsilon-(15)N]lysine (concentration of bridge links), and d-[1-(13)C]alanine and [(15)N]glycine (concentrations of cross-links and wall teichoic acids). The bridging segment length changed from 5.0 glycyl residues (wild-type strain) to 2.5 +/- 0.1 (FemB) with modest changes in cross-link and bridge-link concentrations. This accurate in situ measurement for the FemB mutant indicates a heterogeneous PG structure with 25% monoglycyl and 75% triglycyl bridges. When the bridging segment was reduced to a single glycyl residue 1.0 +/- 0.1 (FemA), the level of cross-linking decreased by more than 20%, resulting in a high concentration of open N-terminal glycyl segments.


Subject(s)
Bacterial Proteins/chemistry , Magnetic Resonance Spectroscopy/methods , Methicillin-Resistant Staphylococcus aureus/chemistry , Mutation , Peptidoglycan/chemistry , Carbon Isotopes , Methicillin-Resistant Staphylococcus aureus/genetics , Nitrogen Isotopes
6.
Antimicrob Agents Chemother ; 53(4): 1619-23, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19164157

ABSTRACT

The related lipo(depsi)peptide antibiotics daptomycin and friulimicin B show great potential in the treatment of multiply resistant gram-positive pathogens. Applying genome-wide in-depth expression profiling, we compared the respective stress responses of Bacillus subtilis. Both antibiotics target envelope integrity, based on the strong induction of extracytoplasmic function sigma factor-dependent gene expression. The cell envelope stress-sensing two-component system LiaRS is exclusively and strongly induced by daptomycin, indicative of different mechanisms of action in the two compounds.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacillus subtilis/drug effects , Daptomycin/pharmacology , Gene Expression Profiling , Peptides/pharmacology , Bacillus subtilis/genetics , Cell Membrane/drug effects , Cell Membrane/metabolism
7.
F1000 Biol Rep ; 1: 40, 2009 May 28.
Article in English | MEDLINE | ID: mdl-20948644

ABSTRACT

The need for new antibiotics to effectively treat antibiotic-resistant infections remains unfulfilled. Despite the well-publicised concern over this issue, only two novel antibiotic classes have been introduced in the past 20 years alongside several new agents of existing classes. Accordingly, the current antibiotic armoury remains inadequate to meet the challenges posed by resistance today. More worryingly, there are very few new agents being developed that can be expected to replace existing antibiotics that succumb to the rising tide of resistance.

9.
Antimicrob Agents Chemother ; 52(12): 4470-4, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18809938

ABSTRACT

A conditional expression system has been developed using the isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter to validate essential genes of Staphylococcus aureus in vivo. The system has been applied to prove the essentiality of ligA and to evaluate the function of tarI, which was found to be essential in vitro but not in vivo.


Subject(s)
Bacterial Proteins/genetics , DNA Ligases/genetics , Genes, Essential , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/growth & development , Staphylococcus aureus/genetics , Abscess/microbiology , Animals , Culture Media , Disease Models, Animal , Female , Gene Expression Regulation, Bacterial , Humans , Isopropyl Thiogalactoside/pharmacology , Mice , Mutation , Staphylococcus aureus/drug effects
10.
Antimicrob Agents Chemother ; 51(9): 3445-8, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17620372

ABSTRACT

We analyzed the emergence of daptomycin nonsusceptibility in a patient with persistent vancomycin-intermediate Staphylococcus aureus (VISA) bacteremia. The daptomycin-nonsusceptible VISA's cell wall demonstrated a reduction in muramic acid O-acetylation, a phenotypic parameter not previously reported for VISA; some isolates also contained a single point mutation in the mprF gene.


Subject(s)
Anti-Bacterial Agents/pharmacology , Daptomycin/pharmacology , Drug Resistance, Bacterial/genetics , Endocarditis, Bacterial/microbiology , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Vancomycin Resistance/genetics , Acetylation , Aminoacyltransferases , Bacteremia/microbiology , Bacterial Proteins/genetics , Cell Wall/chemistry , Electrophoresis, Gel, Pulsed-Field , Humans , Muramic Acids/metabolism , Mutation/genetics , Phenotype
12.
Nat Med ; 11(10): 1082-7, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16200071

ABSTRACT

Here we show that a new class of antibiotics-acyldepsipeptides-has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.


Subject(s)
Anti-Bacterial Agents/classification , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacteria/metabolism , Depsipeptides/pharmacology , Endopeptidase Clp/metabolism , Escherichia coli Proteins/metabolism , Animals , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/toxicity , Bacillus subtilis/drug effects , Bacteria/enzymology , Depsipeptides/metabolism , Depsipeptides/pharmacokinetics , Depsipeptides/toxicity , Drug Resistance, Multiple, Bacterial , Escherichia coli/drug effects , Female , Mice , Molecular Structure , Mutation , Pneumococcal Infections/drug therapy , Pneumococcal Infections/microbiology , Protein Binding , Protein Processing, Post-Translational , Rats , Rats, Wistar , Sepsis/drug therapy , Sepsis/microbiology
13.
J Bacteriol ; 187(7): 2249-56, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15774866

ABSTRACT

The NfrA protein, a putative essential oxidoreductase in the soil bacterium Bacillus subtilis, is induced under heat shock and oxidative stress conditions. In order to characterize the function of an homologous NfrA protein in Staphylococcus aureus, an nfrA deletion strain was constructed, the protein was purified, the enzymatic activity was determined, and the transcriptional regulation was investigated. The experiments revealed that NfrA is not essential in S. aureus. The purified protein oxidized NADPH but not NADH, producing NADP in the presence of flavin mononucleotide, suggesting that NfrA is an NADPH oxidase in S. aureus. In addition, the NfrA enzyme showed nitroreductase activity and weak disulfide reductase activity. Transcription was strongly induced by ethanol, diamide, and nitrofurantoin. Hydrogen peroxide induced nfrA transcription only at high concentrations. The expression of nfrA was independent of the alternative sigma factor sigma(B). Furthermore, the transcriptional start site was determined, which allowed identification of a PerR box homologous sequence upstream of the nfrA promoter. The observations presented here suggest that NfrA is a nonessential NADPH oxidoreductase which may play a role in the oxidative stress response of S. aureus, especially in keeping thiol-disulfide stress in balance.


Subject(s)
Flavin Mononucleotide/physiology , NADPH Oxidases/metabolism , Staphylococcus aureus/enzymology , Chromosome Mapping , Diamide , Ethanol , Gene Expression Regulation, Bacterial , NADP/physiology , NADPH Oxidases/chemistry , NADPH Oxidases/genetics , Nitrofurantoin , Oxidative Stress , Staphylococcus aureus/genetics , Transcription, Genetic/drug effects
14.
Mass Spectrom Rev ; 24(4): 549-65, 2005.
Article in English | MEDLINE | ID: mdl-15389844

ABSTRACT

Gene-expression profiling technologies in general, and proteomic technologies in particular have proven extremely useful to study the physiological response of bacterial cells to various environmental stress conditions. Complex protein toolkits coordinated by sophisticated regulatory networks have evolved to accommodate bacterial survival under ever-present stress conditions such as varying temperatures, nutrient availability, or antibiotics produced by other microorganisms that compete for habitat. In the last decades, application of man-made antibacterial agents resulted in additional bacterial exposure to antibiotic stress. Whereas the targeted use of antibiotics has remarkably reduced human suffering from infectious diseases, the ever-increasing emergence of bacteria that are resistant to antibiotics has led to an urgent need for novel antibiotic strategies. The intent of this review is to present an overview of the major achievements of proteomic approaches to study adaptation networks that are crucial for bacterial survival with a special emphasis on the stress induced by antibiotic treatment. A further focus will be the review of the, so far few, published efforts to exploit the knowledge derived from bacterial proteomic studies directly for the antibacterial drug-discovery process.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Bacteria/drug effects , Bacteria/metabolism , Bacterial Proteins/metabolism , Drug Design , Electrophoresis, Gel, Two-Dimensional/methods , Proteomics/methods , Anti-Bacterial Agents/chemistry , Bacterial Proteins/analysis , Bacterial Proteins/chemistry , Electrophoresis, Gel, Two-Dimensional/trends , Gene Expression Profiling/methods , Gene Expression Profiling/trends , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/physiology , Mass Spectrometry/methods , Mass Spectrometry/trends , Proteomics/trends
15.
Antimicrob Agents Chemother ; 48(10): 3749-57, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15388430

ABSTRACT

The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriolysis/drug effects , Cell Wall/chemistry , Glycopeptides/pharmacology , Lysostaphin/pharmacology , Staphylococcus aureus/drug effects , Blotting, Northern , Chromatography, High Pressure Liquid , Cytoplasm/chemistry , Cytoplasm/metabolism , Peptidoglycan/pharmacology , Phosphorus/chemistry , Teichoic Acids/metabolism , Vancomycin Resistance
16.
Curr Opin Microbiol ; 7(5): 445-50, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15451498

ABSTRACT

The continued evolution of resistance to antibiotics has led to wide ranging consultation at National and International levels as to how to address this issue. In addition to attempting to limit the spread of resistance there is growing consensus that a cornerstone requirement is the development of new antibiotics to help redress the balance of resistance versus available antibiotics. The availability of new technologies such as genomics has opened up new approaches for antibacterial research. It would appear that from an industry perspective, the research and development of antibiotics should be an attractive option. However, this is not the current perception at the majority of large pharmaceutical companies. In addition, the perceived failure of new technologies to create another golden age of new antibacterial classes has led many companies to prioritise other areas of research and, in some cases, to exit antibacterial research. In response, a plethora of small biotech companies have emerged with an interest in antibacterial discovery and large pharmaceutical companies may look to these as a source of development candidates although, to date, these have contributed a very low number of truly novel antibiotic lead compounds. As a reaction to these changes several initiatives are ongoing to examine ways to incentivise antibacterial research and development and ensure a healthy pipeline of compounds in the 21st Century.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Infections/drug therapy , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/trends , Drug Industry , Research , Drug Evaluation, Preclinical/economics , Drug Industry/economics , Drug Industry/trends , Drug Resistance, Bacterial , Humans , Research/trends
17.
Curr Opin Microbiol ; 7(5): 451-9, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15451499

ABSTRACT

Recent scientific publications demonstrate the increasing interest in measurement of genome-wide gene expression on transcript and protein level in response to treatment with antibacterial agents. Nevertheless, the number of large bacterial transcriptome and proteome datasets available so far is limited, although a high number and diversity of antibiotic-triggered expression profiles aid to optimally exploit these technologies. The first published examples substantiate the need to establish these so-called reference compendia of bacterial expression profiles, to discover the molecular mechanism-of-action of uncharacterized bioactive substances. In addition, such compendia open up ways for novel cell-based drug screening approaches.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Drug Evaluation, Preclinical/methods , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Proteome/analysis , Bacteria/genetics , Genes, Bacterial , Mutation , Transcription, Genetic
18.
J Biol Chem ; 278(41): 39435-42, 2003 Oct 10.
Article in English | MEDLINE | ID: mdl-12867414

ABSTRACT

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.


Subject(s)
Chromans/pharmacology , DNA Ligases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Adenosine Monophosphate/metabolism , Base Sequence , Chromans/chemistry , DNA Ligase ATP , DNA Ligases/chemistry , DNA Ligases/genetics , DNA Ligases/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Enzyme Inhibitors/chemistry , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , In Vitro Techniques , Kinetics , Models, Molecular , Molecular Structure , Mutation , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
19.
Antimicrob Agents Chemother ; 47(3): 948-55, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12604526

ABSTRACT

We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacillus subtilis/drug effects , Proteomics , Anti-Infective Agents, Urinary/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/drug effects , DNA Fingerprinting , Electrophoresis, Polyacrylamide Gel , Image Processing, Computer-Assisted , Isotope Labeling , Methionine/metabolism , Nitrofurantoin/pharmacology , Pyrimidinones/pharmacology , Sulfur Radioisotopes
20.
Microbiol Immunol ; 46(9): 601-12, 2002.
Article in English | MEDLINE | ID: mdl-12437027

ABSTRACT

atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non-covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non-growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin-induced lysis of the cells.


Subject(s)
Mutation , N-Acetylmuramoyl-L-alanine Amidase/genetics , N-Acetylmuramoyl-L-alanine Amidase/physiology , Staphylococcus aureus/enzymology , Animals , Cell Wall , DNA Primers , Endopeptidases/metabolism , Gene Deletion , Mice , Models, Genetic , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Plasmids/genetics , Staphylococcal Infections/genetics , Staphylococcus aureus/cytology , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism
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