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Plants and microbes are closely associated with each other in their ecological niches. Much has been studied about plant-microbe interactions, but little is known about the effect of phytochemicals on microbes at the molecular level. To access the products of cryptic biosynthetic gene clusters in bacteria, we incorporated an organic extract of hibiscus flowers into the culture media of different Actinobacteria isolated from plant rhizospheres. This approach led to the production of broad-spectrum dithiolopyrrolone (DTP) antibiotics, thiolutin (1) and aureothricin (2), by Streptomyces sp. MBN2-2. The compounds from the hibiscus extract responsible for triggering the production of these two DTPs were found to be hibiscus acid dimethyl ester (3) and hydroxycitric acid 1,3-dimethyl ester (4). It was subsequently found that the addition of either Fe2+ or Fe3+ to culture media induced the production of 1 and 2. The Chrome Azurol S (CAS) assay revealed that 3 and 4 can chelate iron, and therefore, the mechanism leading to the production of thiolutin and aureothricin appears to be related to changes in iron concentration levels. This work supports the idea that phytochemicals can be used to activate the production of cryptic microbial biosynthetic gene clusters and further understand plant-microbe interactions.
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Covering: up to the end of 2022In recent years rare Actinobacteria have become increasingly recognised as a rich source of novel bioactive metabolites. Actinomadura are Gram-positive bacteria that occupy a wide range of ecological niches. This review highlights about 230 secondary metabolites produced by Actinomadura spp., reported until the end of 2022, including their bioactivities and selected biosynthetic pathways. Notably, the bioactive compounds produced by Actinomadura spp. demonstrate a wide range of activities, including antimicrobial, antitumor and anticoccidial effects, highlighting their potential in various fields.
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Actinobacteria , Anti-Infecciosos , Actinomadura , Actinobacteria/metabolismo , Anti-Infecciosos/química , Bactérias , BiologiaRESUMO
Inhibition of bacterial cell wall synthesis by antibiotics such as ß-lactams is thought to cause explosive lysis through loss of cell wall integrity. However, recent studies on a wide range of bacteria have suggested that these antibiotics also perturb central carbon metabolism, contributing to death via oxidative damage. Here, we genetically dissect this connection in Bacillus subtilis perturbed for cell wall synthesis, and identify key enzymatic steps in upstream and downstream pathways that stimulate the generation of reactive oxygen species through cellular respiration. Our results also reveal the critical role of iron homeostasis for the oxidative damage-mediated lethal effects. We show that protection of cells from oxygen radicals via a recently discovered siderophore-like compound uncouples changes in cell morphology normally associated with cell death, from lysis as usually judged by a phase pale microscopic appearance. Phase paling appears to be closely associated with lipid peroxidation.
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Antibacterianos , Bacillus subtilis , Morte Celular , Antibacterianos/farmacologia , Bacillus subtilis/genética , Carbono , Parede Celular , Espécies Reativas de OxigênioRESUMO
Bacterial cells are surrounded by a peptidoglycan (PG) wall, which is a crucial target for antibiotics. It is well known that treatment with cell wall-active antibiotics occasionally converts bacteria to a non-walled "L-form" state that requires the loss of cell wall integrity. L-forms may have an important role in antibiotic resistance and recurrent infection. Recent work has revealed that inhibition of de novo PG precursor synthesis efficiently induces the L-form conversion in a wide range of bacteria, but the molecular mechanisms remain poorly understood. Growth of walled bacteria requires the orderly expansion of the PG layer, which involves the concerted action not just of synthases but also degradative enzymes called autolysins. Most rod-shaped bacteria have two complementary systems for PG insertion, the Rod and aPBP systems. Bacillus subtilis has two major autolysins, called LytE and CwlO, which are thought to have partially redundant functions. We have dissected the functions of autolysins, relative to the Rod and aPBP systems, during the switch to L-form state. Our results suggest that when de novo PG precursor synthesis is inhibited, residual PG synthesis occurs specifically via the aPBP pathway, and that this is required for continued autolytic activity by LytE/CwlO, resulting in cell bulging and efficient L-form emergence. The failure of L-form generation in cells lacking aPBPs was rescued by enhancing the Rod system and in this case, emergence specifically required LytE but was not associated with cell bulging. Our results suggest that two distinct pathways of L-form emergence exist depending on whether PG synthesis is being supported by the aPBP or RodA PG synthases. This work provides new insights into mechanisms of L-form generation, and specialisation in the roles of essential autolysins in relation to the recently recognised dual PG synthetic systems of bacteria.
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Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a protein machine called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening > 2,000 extracts from a diverse collection of actinobacteria, we identified a compound, mirubactin C, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed using synthetic mirubactin C. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin C rescuing activity is not due simply to iron sequestration. The results support an emerging view that the functions of bacterial siderophores extend well beyond simply iron binding and uptake.
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Metabolic profiling of the extracts from a library of actinobacteria led to the identification of a novel polyketide, demurilactone A, produced by Streptomyces strain DEM21308. The structure of the compound was assigned based on a detailed investigation of 1D/2D NMR spectra and HR-MS. Whole genome DNA sequencing, followed by bioinformatics analysis and insertional mutagenesis, identified type I polyketide synthases encoded by the dml gene cluster to direct the biosynthesis of this polyene macrolide. While the number of modules is consistent with the carbon backbone of the assigned structure, some discrepancies were identified in the domain organization of five modules. Close investigation of the amino acid sequences identified several mutations in the conserved motifs of nonfunctional domains. Furthermore, the absolute configuration of hydroxy-bearing stereocenters was proposed based on analyses of the ketoreductase domains. Remarkably, although demurilactone A has little detectable activity against normal-walled bacteria, it specifically inhibits the growth of cell wall-deficient "L-form" Bacillus subtilis at a minimum inhibitory concentration value of 16 µg/mL. Time-lapse microscopy analyses revealed that demurilactone affects membrane dynamics, probably by reducing membrane fluidity. This compound could be a powerful reagent for studying long-standing questions about the involvement of L-forms in recurrent infection.
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Bacillus subtilis , Streptomyces , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Inibidores do Crescimento/metabolismo , Policetídeo Sintases/genética , Streptomyces/genética , Streptomyces/química , MacrolídeosRESUMO
SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.
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Bacillus subtilis , Cromossomos Bacterianos , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos MultiproteicosRESUMO
Cell wall deficient bacterial L-forms are induced by exposure to cell wall-targeting antibiotics and immune effectors such as lysozyme. L-forms of different bacteria (including Escherichia coli) have been reported in human infections, but whether this is a normal adaptive strategy or simply an artifact of antibiotic treatment in certain bacterial species remains unclear. Here we show that members of a representative, diverse set of pathogenic E. coli readily proliferate as L-forms in supratherapeutic concentrations of the broad-spectrum antibiotic meropenem. We report that they are completely resistant to antibiotics targeting any penicillin-binding proteins in this state, including PBP1A/1B, PBP2, PBP3, PBP4, and PBP5/6. Importantly, we observed that reversion to the cell-walled state occurs efficiently, less than 20 h after antibiotic cessation, with few or no changes in DNA sequence. We defined for the first time a logarithmic L-form growth phase with a doubling time of 80 to 190 min, followed by a stationary phase in late cultures. We further demonstrated that L-forms are metabolically active and remain normally susceptible to antibiotics that affect DNA torsion and ribosomal function. Our findings provide insights into the biology of L-forms and help us understand the risk of ß-lactam failure in persistent infections in which L-forms may be common. IMPORTANCE Bacterial L-forms require specialized culture techniques and are neither widely reported nor well understood in human infections. To date, most of the studies have been conducted on Gram-positive and stable L-form bacteria, which usually require mutagenesis or long-term passages for their generation. Here, using an adapted osmoprotective growth media, we provide evidence that pathogenic E. coli can efficiently switch to L-forms and back to a cell-walled state, proliferating aerobically in supratherapeutic concentrations of antibiotics targeting cell walls with few or no changes in their DNA sequences. Our work demonstrates that L-form switching is an effective adaptive strategy in stressful environments and can be expected to limit the efficacy of ß-lactam for many important infections.
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Proteínas de Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Resistência beta-Lactâmica , beta-Lactamas/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Meropeném/farmacologia , Muramidase/farmacologia , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Ligação às Penicilinas/farmacologiaRESUMO
A genomic and bioactivity informed analysis of the metabolome of the extremophile Amycolatopsis sp. DEM30355 has allowed for the discovery and isolation of the polyketide antibiotic tatiomicin. Identification of the biosynthetic gene cluster was confirmed by heterologous expression in Streptomyces coelicolor M1152. Structural elucidation, including absolute stereochemical assignment, was performed using complementary crystallographic, spectroscopic and computational methods. Tatiomicin shows antibiotic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytological profiling experiments suggest a putative antibiotic mode-of-action, involving membrane depolarisation and chromosomal decondensation of the target bacteria.
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Staphylococcus aureus Resistente à Meticilina , Policetídeos , Streptomyces coelicolor , Amycolatopsis , Antibacterianos/química , Staphylococcus aureus Resistente à Meticilina/genética , Streptomyces coelicolor/genéticaRESUMO
Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the ß-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis. Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.
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Micetoma , Amoxicilina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ácido Clavulânico/uso terapêutico , Humanos , Micetoma/microbiologia , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
Almost all major classes of bacteria are surrounded by a peptidoglycan cell wall, which is a crucial target for antibiotics. It is now understood that many bacteria can tolerate loss of the cell wall provided that they are in an isotonic environment. Furthermore, in some cases the cells can continue to proliferate in a state known as the L-form. L-form proliferation occurs by an unusual blebbing or tubulation mechanism that is completely independent of the normally essential division machine or cell wall synthetic enzymes, and is resistant to cell wall-active antibiotics. However, the growth is limited by reactive oxygen species generated by the respiratory chain pathway. In this work, we examined the walled to L-form transition in a pathogenic Gram-negative bacterium, Streptobacillus moniliformis, which naturally lacks the respiratory chain pathway, under aerobic conditions. L-form-like cells often emerged spontaneously, but proliferation was not observed unless the cells were treated with cell wall-active antibiotics. Time-lapse imaging revealed that cell division of S. moniliformis L-forms involves unusual membrane dynamics with an apparent imbalance between outer membrane and cytoplasmic volume growth. The results suggest that outer membrane expansion may be an important general factor for L-form proliferation of diderm bacteria.
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Formas L , Antibacterianos/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/metabolismo , Proliferação de Células/fisiologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Citoplasma/metabolismo , Formas L/fisiologia , Streptobacillus/efeitos dos fármacos , Streptobacillus/crescimento & desenvolvimentoRESUMO
ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.
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Bacillus subtilis/citologia , Citidina Trifosfato/metabolismo , Pirofosfatases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Cromossomos Bacterianos/genética , Citidina Trifosfato/fisiologia , Proteínas do Citoesqueleto/genética , Pirofosfatases/fisiologiaRESUMO
Transcriptional regulation allows adaptive and coordinated gene expression, and is essential for life. Processive antitermination systems alter the transcription elongation complex to allow the RNA polymerase to read through multiple terminators in an operon. Here, we describe the discovery of a novel bipartite antitermination system that is widespread among conjugative elements from Gram-positive bacteria, which we named conAn. This system is composed of a large RNA element that exerts antitermination, and a protein that functions as a processivity factor. Besides allowing coordinated expression of very long operons, we show that these systems allow differential expression of genes within an operon, and probably contribute to strict regulation of the conjugation genes by minimizing the effects of spurious transcription. Mechanistic features of the conAn system are likely to decisively influence its host range, with important implications for the spread of antibiotic resistance and virulence genes.
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Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Bacteriano/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genéticaRESUMO
This study was designed to identify and investigate bioactive natural product compounds that alter the cellular shape of the fission yeast Schizosaccharomyces pombe and induce a "rounded" or "small" cellular morphological phenotype. Bioassays using a range of antifungal agents against a multidrug-sensitive fission yeast strain, SAK950 showed that many induced a "rounded" phenotype. We then investigated whether 46 of the actinomycete strains identified in our previous study as inducing a similar phenotype produced antifungal agents of similar classes. We show that five of the strains produced streptothricin and that 26 strains produced polyenes, including fungichromin, filipin and candicidin, the last of which was produced by 24 strains. A taxonomic study of the strains indicated that the majority of the candicidin only producers were Streptomyces hydrogenans and S. albidoflavus whilst those that additionally produced streptothricin were related to S. enissocaesilis. A follow-up study to investigate the natural products made by related strains indicated that they followed a similar pattern. The identification of several compounds from the actinomycete strains similar to the antifungal agents initially tested confirm the validity of an approach using the S. pombe morphological phenotype and actinomycete taxonomy as a predictive tool for natural product identification.
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In the course of screening for compounds with differential growth inhibition activity on a mutant of Bacillus subtilis lacking all four class A penicillin-binding proteins (Δ4), we came across an isoquinoline derivative, IQ-1 carboxylic acid (IQC) with relatively high activity on the mutant compared to the wild type strain. Treated cells were slightly elongated and had altered chromosome morphology. Mutants of Δ4 resistant to IQC were isolated and subjected to whole genome sequencing. Most of the mutants were affected in the gene, pyrG, encoding CTP synthetase (CTPS). Purified wild type CTPS was inhibited in vitro by IQC. Two of the three mutant proteins purified showed decreased sensitivity to IQC in vitro. Finally, inhibition by IQC was rescued by addition of cytidine but not uridine to the growth medium, consistent with the notion that IQC acts by reducing the synthesis of CTP or a related compound. IQC provides a promising new starting point for antibiotic inhibitors of CTPS.
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Many bacteria can form wall-deficient variants, or L-forms, that divide by a simple mechanism that does not require the FtsZ-based cell division machinery. Here, we use microfluidic systems to probe the growth, chromosome cycle and division mechanism of Bacillus subtilis L-forms. We find that forcing cells into a narrow linear configuration greatly improves the efficiency of cell growth and chromosome segregation. This reinforces the view that L-form division is driven by an excess accumulation of surface area over volume. Cell geometry also plays a dominant role in controlling the relative positions and movement of segregating chromosomes. Furthermore, the presence of the nucleoid appears to influence division both via a cell volume effect and by nucleoid occlusion, even in the absence of FtsZ. Our results emphasise the importance of geometric effects for a range of crucial cell functions, and are of relevance for efforts to develop artificial or minimal cell systems.
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Bacillus subtilis/crescimento & desenvolvimento , Divisão Celular/fisiologia , Segregação de Cromossomos/fisiologia , Formas L/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip/microbiologia , Bacillus subtilis/citologia , Bacillus subtilis/fisiologia , Parede Celular/fisiologia , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/fisiologia , Formas L/citologia , Formas L/fisiologia , Modelos BiológicosRESUMO
During sporulation of Bacillus subtilis, the cell cycle is reorganized to generate separated prespore and mother cell compartments, each containing a single fully replicated chromosome. The process begins with reorganization of the nucleoid to form an elongated structure, the axial filament, in which the two chromosome origins are attached to opposite cell poles, with the remainder of the DNA stretched between these sites. When the cell then divides asymmetrically, the division septum closes around the chromosome destined for the smaller prespore, trapping the origin-proximal third of the chromosome in the prespore. A translocation pore is assembled through which a DNA transporter, SpoIIIE/FtsK, transfers the bulk of the chromosome to complete the segregation process. Although the mechanisms involved in attaching origin regions to the cell poles are quite well understood, little is known about other aspects of axial filament morphology. We have studied the behavior of the terminus region of the chromosome during sporulation using time-lapse imaging of wild-type and mutant cells. The results suggest that the elongated structure involves cohesion of the terminus regions of the sister chromosomes and that this cohesion is resolved when the termini reach the asymmetric septum or translocation pore. Possible mechanisms and roles of cohesion and resolution are discussed.IMPORTANCE Endospore formation in Firmicutes bacteria provides one of the most highly resistant life forms on earth. During the early stages of endospore formation, the cell cycle is reorganized so that exactly two fully replicated chromosomes are generated, before the cell divides asymmetrically to generate the prespore and mother cell compartments that are critical for the developmental process. Decades ago, it was discovered that just prior to asymmetrical division the two chromosomes enter an unusual elongated configuration called the axial filament. This paper provides new insights into the nature of the axial filament structure and suggests that cohesion of the normally separated sister chromosome termini plays an important role in axial filament formation.
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Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Esporos Bacterianos/fisiologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Microscopia de Fluorescência , MorfogêneseRESUMO
Bacteria surround their cell membrane with a net-like peptidoglycan layer, called sacculus, to protect the cell from bursting and maintain its cell shape. Sacculus growth during elongation and cell division is mediated by dynamic and transient multiprotein complexes, the elongasome and divisome, respectively. In this Review we present our current understanding of how peptidoglycan synthases are regulated by multiple and specific interactions with cell morphogenesis proteins that are linked to a dynamic cytoskeletal protein, either the actin-like MreB or the tubulin-like FtsZ. Several peptidoglycan synthases and hydrolases require activation by outer-membrane-anchored lipoproteins. We also discuss how bacteria achieve robust cell wall growth under different conditions and stresses by maintaining multiple peptidoglycan enzymes and regulators as well as different peptidoglycan growth mechanisms, and we present the emerging role of LD-transpeptidases in peptidoglycan remodelling.
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Proteínas de Bactérias/metabolismo , Peptidoglicano/metabolismo , Bactérias/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.