Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most ß-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care ß-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by ß-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting ß-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific ß-lactam agents.

Comparative Chemical Profiling and Antimicrobial/Anticancer Evaluation of Extracts from Farmed versus Wild Agelas oroides and Sarcotragus foetidus Sponges.

Marine sponges are highly efficient in removing organic pollutants and their cultivation, adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality. Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus, compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture, sponge farming can serve as a valuable resource of biomolecules, with promising potential in pharmaceutical and biomedical applications.

Hydrophobic DES Based on Menthol and Natural Organic Acids for Use in Antifouling Marine Coatings.

Marine biofouling negatively impacts industries with off-shore infrastructures, such as naval, oil, and aquaculture. To date, there are no ideal sustainable, economic, and environmentally benign solutions to deal with this phenomenon. The advances achieved in green solvents, as well as its application in different industries, such as pharmaceutical and biotechnology, have promoted the emergence of deep eutectic systems (DES). These eutectic systems have applications in various fields and can be revolutionary in the marine-based industrial sector. In this study, the main objective was to investigate the potential use of hydrophobic DES (HDES) based on menthol and natural organic acids for their use as marine antifouling coatings. Our strategy encompassed the physicochemical characterization of different formulations, which allowed us to identify the most appropriate molar ratio and intermolecular interactions for HDES formations. The miscibility of the resulting HDES with the marine coating has been evaluated and proven to be successful. The Men/OL (1:1) system proved to be the most promising in terms of cost-production and thus was the one used in subsequent antifouling tests. The cytotoxicity of this HDES was evaluated using an in vitro cell model (HaCat cells) showing no significant toxicity. Furthermore, the application of this system incorporated into coatings that are used in marine structures was also studied using marine species (Mytilus edulis mussels and Patella vulgata limpets) to evaluate both their antifouling and ecotoxicity effects. HDES Men/OL (1:1) incorporated in marine coatings was promising in reducing marine macrofouling and also proved to be effective at the level of microfouling without viability impairment of the tested marine species. It was revealed to be more efficient than using copper oxide, metallic copper, or ivermectin as antifouling agents. Biochemical assays performed on marine species showed that this HDES does not induce oxidative stress in the tested species. These results are a strong indication of the potential of this HDES to be sustainable and efficiently used in marine fouling control technologies.

Exopolysaccharide Production from Marine-Derived Brevundimonas huaxiensis Obtained from Estremadura Spur Pockmarks Sediments Revealing Potential for Circular Economy.

Marine environments represent an enormous biodiversity reservoir due to their numerous different habitats, being abundant in microorganisms capable of producing biomolecules, namely exopolysaccharides (EPS), with unique physical characteristics and applications in a broad range of industrial sectors. From a total of 67 marine-derived bacteria obtained from marine sediments collected at depths of 200 to 350 m from the Estremadura Spur pockmarks field, off the coast of Continental Portugal, the Brevundimonas huaxiensis strain SPUR-41 was selected to be cultivated in a bioreactor with saline culture media and glucose as a carbon source. The bacterium exhibited the capacity to produce 1.83 g/L of EPS under saline conditions. SPUR-41 EPS was a heteropolysaccharide composed of mannose (62.55% mol), glucose (9.19% mol), rhamnose (19.41% mol), glucuronic acid (4.43% mol), galactose (2.53% mol), and galacturonic acid (1.89% mol). Moreover, SPUR-41 EPS also revealed acyl groups in its composition, namely acetyl, succinyl, and pyruvyl. This study revealed the importance of research on marine environments for the discovery of bacteria that produce new value-added biopolymers for pharmaceutical and other biotechnological applications, enabling us to potentially address saline effluent pollution via a sustainable circular economy.

Marine-Derived Actinomycetes: Biodegradation of Plastics and Formation of PHA Bioplastics-A Circular Bioeconomy Approach.

Plastics are present in the majority of daily-use products worldwide. Due to society's production and consumption patterns, plastics are accumulating in the environment, causing global pollution issues and intergenerational impacts. Our work aims to contribute to the development of solutions and sustainable methods to mitigate this pressing problem, focusing on the ability of marine-derived actinomycetes to accelerate plastics biodegradation and produce polyhydroxyalkanoates (PHAs), which are biodegradable bioplastics. The thin plastic films' biodegradation was monitored by weight loss, changes in the surface chemical structure (Infra-Red spectroscopy FTIR-ATR), and by mechanical properties (tensile strength tests). Thirty-six marine-derived actinomycete strains were screened for their plastic biodegradability potential. Among these, Streptomyces gougerotti, Micromonospora matsumotoense, and Nocardiopsis prasina revealed ability to degrade plastic films-low-density polyethylene (LDPE), polystyrene (PS) and polylactic acid (PLA) in varying conditions, namely upon the addition of yeast extract to the culture media and the use of UV pre-treated thin plastic films. Enhanced biodegradation by these bacteria was observed in both cases. S. gougerotti degraded 0.56% of LDPE films treated with UV radiation and 0.67% of PS films when inoculated with yeast extract. Additionally, N. prasina degraded 1.27% of PLA films when these were treated with UV radiation, and yeast extract was added to the culture medium. The main and most frequent differences observed in FTIR-ATR spectra during biodegradation occurred at 1740 cm-1, indicating the formation of carbonyl groups and an increase in the intensity of the bands, which indicates oxidation. Young Modulus decreased by 30% on average. In addition, S. gougerotti and M. matsumotoense, besides biodegrading conventional plastics (LDPE and PS), were also able to use these as a carbon source to produce degradable PHA bioplastics in a circular economy concept.

Influence of Sodium Bicarbonate on Wall Teichoic Acid Synthesis and ß-Lactam Sensitization in NaHCO3-Responsive and Nonresponsive Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) strains pose major treatment challenges due to their innate resistance to most ß-lactams under standard in vitro antimicrobial susceptibility testing conditions. A novel phenotype among MRSA, termed "NaHCO3 responsiveness," where certain strains display increased susceptibility to ß-lactams in the presence of NaHCO3, has been identified among a relatively large proportion of MRSA isolates. One underlying mechanism of NaHCO3 responsiveness appears to be related to decreased expression and altered functionality of several genes and proteins involved in cell wall synthesis and maturation. Here, we studied the impact of NaHCO3 on wall teichoic acid (WTA) synthesis, a process intimately linked to peptidoglycan (PG) synthesis and functionality, in NaHCO3-responsive versus -nonresponsive MRSA isolates. NaHCO3 sensitized responsive MRSA strains to cefuroxime, a specific penicillin-binding protein 2 (PBP2)-inhibitory ß-lactam known to synergize with early WTA synthesis inhibitors (e.g., ticlopidine). Combining cefuroxime with ticlopidine with or without NaHCO3 suggested that these latter two agents target the same step in WTA synthesis. Further, NaHCO3 decreased the abundance and molecular weight of WTA only in responsive strains. Additionally, NaHCO3 stimulated increased autolysis and aberrant cell division in responsive strains, two phenotypes associated with disruption of WTA synthesis. Of note, studies of key genes involved in the WTA biosynthetic pathway (e.g., tarO, tarG, dltA, and fmtA) indicated that the inhibitory impact of NaHCO3 on WTA biosynthesis in responsive strains likely occurred posttranslationally. IMPORTANCE MRSA is generally viewed as resistant to standard ß-lactam antibiotics. However, a NaHCO3-responsive phenotype is observed in a substantial proportion of clinical MRSA strains in vitro, i.e., isolates which demonstrate enhanced susceptibility to standard ß-lactam antibiotics (e.g., oxacillin) in the presence of NaHCO3. This phenotype correlates with increased MRSA clearance in vivo by standard ß-lactam antibiotics, suggesting that patients with infections caused by such MRSA strains might be amenable to treatment with ß-lactams. The mechanism(s) behind this phenotype is not fully understood but appears to involve mecA-PBP2a production and maturation axes. Our study adds significantly to this body of knowledge in terms of additional mechanistic targets of NaHCO3 in selected MRSA strains. This investigation demonstrates that NaHCO3 has direct impacts on S. aureus wall teichoic acid biosynthesis in NaHCO3-responsive MRSA. These findings provide an additional target for new agents being designed to synergistically kill MRSA using ß-lactam antibiotics.

The Diversity, Metabolomics Profiling, and the Pharmacological Potential of Actinomycetes Isolated from the Estremadura Spur Pockmarks (Portugal).

The Estremadura Spur pockmarks are a unique and unexplored ecosystem located in the North Atlantic, off the coast of Portugal. A total of 85 marine-derived actinomycetes were isolated and cultured from sediments collected from this ecosystem at a depth of 200 to 350 m. Nine genera, Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Actinopolymorpha, Nocardiopsis, Saccharomonospora, Stackebrandtia, and Verrucosispora were identified by 16S rRNA gene sequencing analyses, from which the first two were the most predominant. Non-targeted LC-MS/MS, in combination with molecular networking, revealed high metabolite diversity, including several known metabolites, such as surugamide, antimycin, etamycin, physostigmine, desferrioxamine, ikarugamycin, piericidine, and rakicidin derivatives, as well as numerous unidentified metabolites. Taxonomy was the strongest parameter influencing the metabolite production, highlighting the different biosynthetic potentials of phylogenetically related actinomycetes; the majority of the chemical classes can be used as chemotaxonomic markers, as the metabolite distribution was mostly genera-specific. The EtOAc extracts of the actinomycete isolates demonstrated antimicrobial and antioxidant activity. Altogether, this study demonstrates that the Estremadura Spur is a source of actinomycetes with potential applications for biotechnology. It highlights the importance of investigating actinomycetes from unique ecosystems, such as pockmarks, as the metabolite production reflects their adaptation to this habitat.

Unveiling the Mechanism of Action of 7α-acetoxy-6ß-hydroxyroyleanone on an MRSA/VISA Strain: Membrane and Cell Wall Interactions.

The number of cases of failure in the treatment of infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, 7α-Acetoxy-6ß-hydroxyroyleanone (AHR), a diterpene isolated from different Plectranthus species, showed antibacterial activity, namely against Methicillin-resistant Staphylococcus aureus (MRSA) strains. The high antibacterial activity and low cytotoxicity render this natural compound an interesting alternative against resistant bacteria. The aim of this study is to understand the mechanism of action of AHR on MRSA, using the MRSA/Vancomycin-intermediate S. aureus (VISA) strain CIP 106760, and to study the AHR effect on lipid bilayers and on the cell wall. Although AHR interacted with lipid bilayers, it did not have a significant effect on membrane passive permeability. Alternatively, bacteria treated with this royleanone displayed cell wall disruption, without revealing cell lysis. In conclusion, the results gathered so far point to a yet undescribed mode of action that needs further investigation.

Antifouling Napyradiomycins from Marine-Derived Actinomycetes Streptomyces aculeolatus.

The undesired attachment of micro and macroorganisms on water-immersed surfaces, known as marine biofouling, results in severe prevention and maintenance costs (billions €/year) for aquaculture, shipping and other industries that rely on coastal and off-shore infrastructures. To date, there are no sustainable, cost-effective and environmentally safe solutions to address this challenging phenomenon. Therefore, we investigated the antifouling activity of napyradiomycin derivatives that were isolated from actinomycetes from ocean sediments collected off the Madeira Archipelago. Our results revealed that napyradiomycins inhibited ≥80% of the marine biofilm-forming bacteria assayed, as well as the settlement of Mytilus galloprovincialis larvae (EC50 < 5 µg/ml and LC50/EC50 >15), without viability impairment. In silico prediction of toxicity end points are of the same order of magnitude of standard approved drugs and biocides. Altogether, napyradiomycins disclosed bioactivity against marine micro and macrofouling organisms, and non-toxic effects towards the studied species, displaying potential to be used in the development of antifouling products.

Distinct Phenotypic and Genomic Signatures Underlie Contrasting Pathogenic Potential of Staphylococcus epidermidis Clonal Lineages.

Background: Staphylococcus epidermidis is a common skin commensal that has emerged as a pathogen in hospitals, mainly related to medical devices-associated infections. Noteworthy, infection rates by S. epidermidis have the tendency to rise steeply in next decades together with medical devices use and immunocompromized population growth. Staphylococcus epidermidis population structure includes two major clonal lineages (A/C and B) that present contrasting pathogenic potentials. To address this distinction and explore the basis of increased pathogenicity of A/C lineage, we performed a detailed comparative analysis using phylogenetic and integrated pangenome-wide-association study (panGWAS) approaches and compared the lineages's phenotypes in in vitro conditions mimicking carriage and infection. Results: Each S. epidermidis lineage had distinct phenotypic signatures in skin and infection conditions and differed in genomic content. Combination of phenotypic and genotypic data revealed that both lineages were well adapted to skin environmental cues. However, they appear to occupy different skin niches, perform distinct biological functions in the skin and use different mechanisms to complete the same function: lineage B strains showed evidence of specialization to survival in microaerobic and lipid rich environment, characteristic of hair follicle and sebaceous glands; lineage A/C strains showed evidence for adaption to diverse osmotic and pH conditions, potentially allowing them to occupy a broader and more superficial skin niche. In infection conditions, A/C strains had an advantage, having the potential to bind blood-associated host matrix proteins, form biofilms at blood pH, resist antibiotics and macrophage acidity and to produce proteases. These features were observed to be rare in the lineage B strains. PanGWAS analysis produced a catalog of putative S. epidermidis virulence factors and identified an epidemiological molecular marker for the more pathogenic lineage. Conclusion: The prevalence of A/C lineage in infection is probably related to a higher metabolic and genomic versatility that allows rapid adaptation during transition from a commensal to a pathogenic lifestyle. The putative virulence and phenotypic factors associated to A/C lineage constitute a reliable framework for future studies on S. epidermidis pathogenesis and the finding of an epidemiological marker for the more pathogenic lineage is an asset for the management of S. epidermidis infections.

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan.

Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to ß-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus.

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure that is essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely, through the localization of late-stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in elongation and division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 probably constitute cognate pairs of interacting proteins. A lack of RodA-PBP3 resulted in more spherical cells due to deficient sidewall PGN synthesis, whereas depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilizing the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings or arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate sidewall and septal PGN incorporation, respectively, and that their activity must be balanced to maintain coccoid morphology.

Bacterial cellulose: a versatile biopolymer for wound dressing applications.

Although several therapeutic approaches are available for wound and burn treatment and much progress has been made in this area, room for improvement still exists, driven by the urgent need of better strategies to accelerate wound healing and recovery, mostly for cases of severe burned patients. Bacterial cellulose (BC) is a biopolymer produced by bacteria with several advantages over vegetal cellulose, such as purity, high porosity, permeability to liquid and gases, elevated water uptake capacity and mechanical robustness. Besides its biocompatibility, BC can be modified in order to acquire antibacterial response and possible local drug delivery features. Due to its intrinsic versatility, BC is the perfect example of a biotechnological response to a clinical problem. In this review, we assess the BC main features and emphasis is given to a specific biomedical application: wound dressings. The production process and the physical-chemical properties that entitle this material to be used as wound dressing namely for burn healing are highlighted. An overview of the most common BC composites and their enhanced properties, in particular physical and biological, is provided, including the different production processes. A particular focus is given to the biochemistry and genetic manipulation of BC. A summary of the current marketed BC-based wound dressing products is presented, and finally, future perspectives for the usage of BC as wound dressing are foreseen.

Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites.

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC-MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 (1) against S. aureus was confirmed.

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD.

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

Contribution of peptidoglycan amidation to beta-lactam and lysozyme resistance in different genetic lineages of Staphylococcus aureus.

The enzymes responsible for peptidoglycan amidation in Staphylococcus aureus, MurT and GatD, were recently identified and shown to be required for optimal expression of resistance to beta-lactams, bacterial growth, and resistance to lysozyme. In this study, we analyzed the impact of peptidoglycan amidation in representative strains of the most widespread clones of methicillin resistant S. aureus (MRSA). The inhibition of the expression of murT-gatD operon resulted in different phenotypes of resistance to beta-lactams and lysozyme according to the different genetic backgrounds. Further, clonal lineages CC1 and CC398 (community-acquired MRSA [CA-MRSA]) showed a stronger dependency on MurT-GatD for resistance to beta-lactams, when compared to the impact of the impairment of the cell wall step catalyzed by MurF. In the remaining backgrounds similar phenotypes of beta-lactam resistance were observed upon the impairment of both cell-wall-related genes. Therefore, for CA-related backgrounds, the predominant beta-lactam resistance mechanism seems to involve genes associated with secondary modifications of peptidoglycan. On the other hand, the lack of glutamic acid amidation had a more substantial impact on lysozyme resistance for cells of CA-MRSA backgrounds, than for hospital-acquired MRSA (HA-MRSA). However, no significant differences were found in the resistance level of the respective peptidoglycan structure, suggesting that the lysozyme resistance mechanism involves other factors. Taken together, these results suggested that the different genetic lineages of MRSA were able to develop different molecular strategies to overcome the selective pressures experienced during evolution.

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan.

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to ß-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

The glucosaminidase domain of Atl - the major Staphylococcus aureus autolysin - has DNA-binding activity.

In this communication, we describe evidence demonstrating the capacity of Atl, the major Staphylococcus aureus autolytic enzyme to bind DNA. Electrophoretic mobility shift assays (EMSA) show that both the Atl protein and the endo-ß-N-acetylglucosaminidase (GL) domain were able to bind DNA of nonspecific sequence. The implications of this unexpected observation for the physiology of S. aureus remain to be explored.

Identification of genetic determinants and enzymes involved with the amidation of glutamic acid residues in the peptidoglycan of Staphylococcus aureus.

The glutamic acid residues of the peptidoglycan of Staphylococcus aureus and many other bacteria become amidated by an as yet unknown mechanism. In this communication we describe the identification, in the genome of S. aureus strain COL, of two co-transcribed genes, murT and gatD, which are responsible for peptidoglycan amidation. MurT and GatD have sequence similarity to substrate-binding domains in Mur ligases (MurT) and to the catalytic domain in CobB/CobQ-like glutamine amidotransferases (GatD). The amidation of glutamate residues in the stem peptide of S. aureus peptidoglycan takes place in a later step than the cytoplasmic phase--presumably the lipid phase--of the biosynthesis of the S. aureus cell wall precursor. Inhibition of amidation caused reduced growth rate, reduced resistance to beta-lactam antibiotics and increased sensitivity to lysozyme which inhibited culture growth and caused degradation of the peptidoglycan.

Extensive and genome-wide changes in the transcription profile of Staphylococcus aureus induced by modulating the transcription of the cell wall synthesis gene murF.

A murF conditional mutant was used to evaluate the effect of suboptimal transcription of this gene on the transcriptome of the methicillin-resistant Staphylococcus aureus strain COL. The mutant was grown in the presence of optimal and suboptimal concentrations of the inducer, and the relative levels of transcription of genes were evaluated genome wide with an Affymetrix DNA microarray that included all open reading frames (ORFs) as well as intergenic sequences derived from four sequenced S. aureus strains. Using a sensitivity threshold value of 1.5, suboptimal expression of murF altered the transcription of a surprisingly large number of genes, i.e., 668 out of the 2,740 ORFs (close to one-fourth of all ORFs), of the genome of S. aureus strain COL. The genes with altered transcription were distributed evenly around the S. aureus chromosome, and groups of genes involved with distinct metabolic functions responded in unique and operon-specific manners to modulation in murF transcription. For instance, all genes belonging to the isd operon and all but 2 of the 35 genes of prophage L54a were down-regulated, whereas all but one of the 21 members of the vraSR regulon and most of the 79 virulence-related genes (those for fibronectin binding proteins A and B, clumping factor A, gamma hemolysin, enterotoxin B, etc.) were up-regulated in cells with suboptimal expression of murF. Most importantly, the majority of these altered gene expression profiles were reversible by resupplying the optimal concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) to the culture. The observations suggest the coordinate regulation of a large sector of the S. aureus transcriptome in response to a disturbance in cell wall synthesis.