RESUMO
A promising yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides, which are the major constituents of the outer membrane. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the response against five different LpxC inhibitors. All compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that FtsH-mediated proteolysis of the inactivated enzyme is impaired. LpxC inhibition sensitized E. coli to vancomycin and rifampin, which poorly cross the outer membrane of intact cells. Four of the five compounds led to an accumulation of lyso-phosphatidylethanolamine, a cleavage product of phosphatidylethanolamine, generated by the phospholipase PldA. The combined results suggested an imbalance in lipopolysaccharides and phospholipid biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids were consistently induced. Upregulated compound-specific proteins are involved in various functional categories, such as stress reactions, nucleotide, or amino acid metabolism and quorum sensing. Our work shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Moreover, we find that the response of E. coli to LpxC inhibition is distinct from the previously reported response in Pseudomonas aeruginosa.
Assuntos
Amidoidrolases , Inibidores Enzimáticos , Escherichia coli , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Lipopolissacarídeos/biossíntese , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Membrana Celular/efeitos dos fármacosRESUMO
Pseudopteroxazole (Ptx) and the pseudopterosins are marine natural products with promising antibacterial potential. While Ptx has attracted interest for its antimycobacterial activity, pseudopterosins are active against several clinically relevant pathogens. Both compound classes exhibit low cytotoxicity and accessibility to targeted synthesis, yet their antibacterial mechanisms remain elusive. In this study, we investigated the modes of action of Ptx and pseudopterosin G (PsG) in Bacillus subtilis employing an unbiased approach that combines gel-based proteomics with a mathematical similarity analysis of response profiles. Proteomic responses to sublethal concentrations of Ptx and PsG were compared to a library of antibiotic stress response profiles revealing that both induce a stress response characteristic for agents targeting the bacterial cell envelope by interfering with membrane-bound steps of cell wall biosynthesis. Microscopy-based assays confirmed that both compounds compromise the integrity of the bacterial cell wall without disrupting the membrane potential. Furthermore, LC-MSE analysis showed that the greater potency of PsG against B. subtilis, reflected in a lower MIC and a more pronounced proteomic response, may be rooted in a more effective association with and penetration of B. subtilis cells. We conclude that Ptx and PsG target the integrity of the gram-positive cell wall.
Assuntos
Antibacterianos , Bacillus subtilis , Diterpenos , Proteômica , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Diterpenos/farmacologia , Diterpenos/química , Antibacterianos/farmacologia , Antibacterianos/química , Proteômica/métodos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , GlicosídeosRESUMO
Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.
Assuntos
Bacillus subtilis , Lipossomas Unilamelares , Antibacterianos/farmacologia , Calcimicina/análogos & derivados , Calcimicina/farmacologia , Cálcio , Cobre/farmacologia , Ionóforos/farmacologia , Manganês/farmacologia , ProteômicaRESUMO
Pseudomonas aeruginosa is among the highest priority pathogens for drug development because of its resistance to antibiotics, extraordinary adaptability, and persistence. Antipseudomonal research is strongly encouraged to address the acute scarcity of innovative antimicrobial lead structures. In an effort to understand the physiological response of P. aeruginosa to clinically relevant antibiotics, we investigated the proteome after exposure to ciprofloxacin, levofloxacin, rifampicin, gentamicin, tobramycin, azithromycin, tigecycline, polymyxin B, colistin, ceftazidime, meropenem, and piperacillin-tazobactam. We further investigated the response to CHIR-090, which represents a promising class of lipopolysaccharide biosynthesis inhibitors currently under evaluation. Radioactive pulse-labeling of newly synthesized proteins followed by two-dimensional polyacrylamide gel electrophoresis was used to monitor the acute response of P. aeruginosa to antibiotic treatment. The proteomic profiles provide insights into the cellular defense strategies for each antibiotic. A mathematical comparison of these response profiles based on upregulated marker proteins revealed similarities of responses to antibiotics acting on the same target area. This study provides insights into the effects of commonly used antibiotics on P. aeruginosa and lays the foundation for the comparative analysis of the impact of novel compounds with precedented and unprecedented modes of action.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Proteômica , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
AIMS: Actinobacteria are known to produce extracellular enzymes including DyPs. We set out to identify and characterize novel peroxidases from Streptomyces chartreusis NRRL 3882, because S. chartreusis belongs to the small group of actinobacteria with three different DyPs. METHODS AND RESULTS: The genome of the actinomycete S. chartreusis NRRL 3882 was mined for novel DyP-type peroxidases. Three genes encoding for DyP-type peroxidases were cloned and overexpressed in Escherichia coli. Subsequent characterization of the recombinant proteins included examination of operating conditions such as pH, temperature and H2 O2 concentrations, as well as substrate spectrum. Despite their high sequence similarity, the enzymes named SCDYP1-SCDYP3 presented distinct preferences regarding their operating conditions. They showed great divergence in H2 O2 tolerance and stability, with SCDYP2 being most active at concentrations above 50 mmol l-1 . Moreover, SCDYP1 and SCDYP3 preferred acidic pH (typical for DyP-type peroxidases), whereas SCDYP2 was most active at pH 8. CONCLUSIONS: Regarding the function of DyPs in nature, these results suggest that availability of different DyP variants with complementary activity profiles in one organism might convey evolutionary benefits. SIGNIFICANCE AND IMPACT OF THE STUDY: DyP-type peroxidases are able to degrade xenobiotic compounds and thus can be applied in biocatalysis and bioremediation. However, the native function of DyPs and the benefits for their producers largely remain to be elucidated.
Assuntos
Actinobacteria , Peroxidases , Actinobacteria/genética , Actinobacteria/metabolismo , Corantes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Proteínas Recombinantes/metabolismo , Streptomyces , Xenobióticos/metabolismoRESUMO
Actinomycetes are known for producing diverse secondary metabolites. Combining genomics with untargeted data-dependent tandem MS and molecular networking, we characterized the secreted metabolome of the tunicamycin producer Streptomyces chartreusis NRRL 3882. The genome harbors 128 predicted biosynthetic gene clusters. We detected >1,000 distinct secreted metabolites in culture supernatants, only 22 of which were identified based on standards and public spectral libraries. S. chartreusis adapts the secreted metabolome to cultivation conditions. A number of metabolites are produced iron dependently, among them 17 desferrioxamine siderophores aiding in iron acquisition. Eight previously unknown members of this long-known compound class are described. A single desferrioxamine synthesis gene cluster was detected in the genome, yet different sets of desferrioxamines are produced in different media. Additionally, a polyether ionophore, differentially produced by the calcimycin biosynthesis cluster, was discovered. This illustrates that metabolite output of a single biosynthetic machine can be exquisitely regulated not only with regard to product quantity but also with regard to product range. Compared with chemically defined medium, in complex medium, total metabolite abundance was higher, structural diversity greater, and the average molecular weight almost doubled. Tunicamycins, for example, were only produced in complex medium. Extrapolating from this study, we anticipate that the larger part of bacterial chemistry, including chemical structures, ecological functions, and pharmacological potential, is yet to be uncovered.
Assuntos
Metaboloma/fisiologia , Sideróforos , Streptomyces/química , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Desferroxamina/química , Desferroxamina/metabolismo , Redes e Vias Metabólicas , Metabolômica , Modelos Moleculares , Sideróforos/química , Sideróforos/metabolismoRESUMO
A series of ruthenium(II) complexes with N-heterocyclic carbene (NHC) ligands of the general type (arene)(NHC)Ru(II)X2 (where X = halide) was prepared, characterized, and evaluated as antibacterial agents in comparison to the respective metal free benzimidazolium cations. The ruthenium(II) NHC complexes generally triggered stronger bacterial growth inhibition than the metal free benzimidazolium cations. The effects were much stronger against Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) than against Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa), and all complexes were inactive against the fungus Candida albicans. Moderate inhibition of bacterial thioredoxin reductase was confirmed for selected complexes, indicating that inhibition of this enzyme might be a contributing factor to the antibacterial effects.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/enzimologia , Rutênio/química , Rutênio/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/antagonistas & inibidores , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Metano/análogos & derivados , Metano/química , Metano/farmacologia , Modelos MolecularesRESUMO
New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.
Assuntos
Antibacterianos , Proteômica , Antibacterianos/farmacologia , Bacillus subtilis , Proteínas de Bactérias/genética , TetraciclinasRESUMO
Allicin, a broad-spectrum antimicrobial agent from garlic, disrupts thiol and redox homeostasis, proteostasis, and cell membrane integrity. Since medicine demands antimicrobials with so far unexploited mechanisms, allicin is a promising lead structure. While progress is being made in unraveling its mode of action, little is known on bacterial adaptation strategies. Some isolates of Pseudomonas aeruginosa and Escherichia coli withstand exposure to high allicin concentrations due to as yet unknown mechanisms. To elucidate resistance and sensitivity-conferring cellular processes, the acute proteomic responses of a resistant P. aeruginosa strain and the sensitive species Bacillus subtilis are compared to the published proteomic response of E. coli to allicin treatment. The cellular defense strategies share functional features: proteins involved in translation and maintenance of protein quality, redox homeostasis, and cell envelope modification are upregulated. In both Gram-negative species, protein synthesis of the majority of proteins is downregulated while the Gram-positive B. subtilis responded by upregulation of multiple regulons. A comparison of the B. subtilis proteomic response to a library of responses to antibiotic treatment reveals 30 proteins specifically upregulated by allicin. Upregulated oxidative stress proteins are shared with nitrofurantoin and diamide. Microscopy-based assays further indicate that in B. subtilis cell wall integrity is impaired.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteoma/análise , Pseudomonas aeruginosa/metabolismo , Ácidos Sulfínicos/farmacologia , Adaptação Fisiológica , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Dissulfetos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteoma/efeitos dos fármacos , Proteômica/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Especificidade da EspécieRESUMO
Four new acyclic diaminocarbenes (ADACs), viz. [(cyclo-Cn H2n-1 )2 N]2 C (n=5-7) and iPr2 N-C-N(cyclo-C6 H11 )2 , were synthesised by reacting the corresponding formamidinium hexafluorophosphates with NaN(SiMe3 )2 . Their nucleophilicities and electrophilicities were respectively judged from the 1 JCH values determined for the N2 CH unit of the corresponding formamidinium cations and from the 77 Se NMR chemical shifts of the selenourea derivatives obtained from the reaction of elemental selenium with the corresponding ADACs. An ambiphilic profile essentially identical to that of the "Alder carbene" (iPr2 N)2 C was found in each case. Similar to the latter carbene, the new ADACs undergo a well-defined thermal decomposition by ß-fragmentation, affording an alkene and a formamidine. The stabilities of [(cyclo-Cn H2n-1 )2 N]2 C depend strongly on the value of n, following the order 6>5>7, with the latter congener being too unstable for isolation. [(cyclo-C6 H11 )2 N]2 C shows no thermal decomposition at room temperature in solution and is thus significantly more stable than (iPr2 N)2 C. The stability of iPr2 N-C-N(cyclo-C6 H11 )2 is intermediate between that of (iPr2 N)2 C and [(cyclo-C6 H11 )2 N]2 C, its ß-fragmentation selectively affording propene and iPrN=CH-N(cyclo-C6 H11 )2 . [(cyclo-Cn H2n-1 )2 N]2 C (n=5-7) react readily with CO under mild conditions, selectively affording trisubstituted spirocyclic ß-lactam derivatives with an antimicrobial activity spectrum similar to that of penicillin G.
RESUMO
A simple "click-chemistry" approach was employed in order to functionalize the known antibiotic fragment sulfanilamide with a bidentate pyridyl-triazole pocket, which allowed for the synthesis of ruthenium(II) and rhenium(I) carbonyl chloride complexes. Six new complexes were prepared and comprehensively characterized, including five single crystal X-ray structures, photophysical characterization, and testing for antimicrobial activity. Interestingly, functionalization of the pyridine ring with an ortho-hydroxymethyl group resulted in a greater than 100-fold increase in the rate of ligand release in a dimethylsulfoxide solution. Subsequent studies indicated this process could be further accelerated by irradiation with 265 nm light. Structural characterization of four of the complexes indicates that this is the result of a lengthening and weakening of the Re-NPyridine bond (average (Ltri) = 2.19 Å vs LtriOH = 2.25 Å) due to the steric influence of the hydroxymethyl group. The organometallic rhenium(I) pyridyl-triazole functionality maintains its characteristic fluorescent properties despite the presence of the sulfonamide moiety. Two of the compounds showed modest antimicrobial activity against methicillin-resistant Staphylococcus aureus, whereas the structurally similar sulfamethoxazole alone showed no activity under the same conditions.
Assuntos
Cobre/química , Metais/química , Sulfanilamida/análogos & derivados , Sulfanilamida/química , Catálise , Química Click , Cristalografia por Raios X , Modelos Moleculares , Estrutura MolecularRESUMO
Xanthoangelol is a geranylated chalcone isolated from fruits of Amorpha fructicosa that exhibits antibacterial effects at low micromolar concentration against Gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium and Enterococcus faecalis. We demonstrate that Xanthoangelol treatment of Gram-positive bacteria affects bacterial membrane integrity and leads to a leakage of intracellular metabolites. This correlates with a rapid collapse of the membrane potential and results in a fast and strong bactericidal effect. Proteomic profiling of Xanthoangelol-treated cells revealed signatures of cell wall and/or membrane damage and oxidative stress. Xanthoangelol specifically disturbs the membrane of Gram-positive bacteria potentially by forming pores resulting in cell lysis. In contrast, Xanthoangelol treatment of human cells showed only mildly hemolytic and cytotoxic effects at higher concentrations. Therefore, geranylated chalcones such as Xanthoangelol are promising lead structures for new antimicrobials against drug-resistant gram-positive pathogens.
Assuntos
Antibacterianos/farmacologia , Chalcona/análogos & derivados , Bactérias Gram-Positivas/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Antibacterianos/química , Linhagem Celular , Chalcona/química , Chalcona/farmacologia , Fabaceae/química , Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismoRESUMO
Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.
RESUMO
Classical tetracyclines targeting the protein biosynthesis machinery are commonly applied in human and veterinary medicine. The development and spread of resistance seriously compromise the successful treatment of bacterial infections. The atypical tetracycline chelocardin holds promise as it retains activity against tetracycline-resistant strains. It has been suggested that chelocardin targets the bacterial membrane, thus differing in mode of action from that of classical tetracyclines. We investigated the mechanism of action of chelocardin using global proteome analysis. The proteome profiles after sublethal chelocardin stress were compared to a reference compendium containing antibiotic response profiles of Bacillus subtilis. This approach revealed a concentration-dependent dual mechanism of action. At low concentrations, like classical tetracyclines, chelocardin induces the proteomic signature for peptidyl transferase inhibition demonstrating that protein biosynthesis inhibition is the dominant physiological challenge. At higher concentrations B. subtilis mainly responds to membrane stress indicating that at clinically relevant concentrations the membrane is the main antibiotic target of chelocardin. Studying the effects on the membrane in more detail, we found that chelocardin causes membrane depolarization but does not lead to formation of large pores. We conclude that at growth inhibiting doses chelocardin not only targets protein biosynthesis but also corrupts the integrity of the bacterial membrane. This dual mechanism of action might prove beneficial in slowing the development of new resistance mechanisms against this atypical tetracycline.
Assuntos
Tetraciclinas/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , ProteomaRESUMO
Antimicrobial peptides are a potent class of antibiotics. In the Gram-positive model organism Bacillus subtilis the synthetic peptide RWRWRW-NH2 integrates into the bacterial membrane and delocalizes essential peripheral membrane proteins involved in cell wall biosynthesis and respiration. A lysine residue has been added to the hexapeptide core structure, either C or N-terminally. Lipidation of the lysine residues by a C8-acyl chain significantly improved antibacterial activity against both Gram-positive and Gram-negative bacteria. Here, we report a comparative proteomic study in B. subtilis on the mechanism of action of the lipidated and non-lipidated peptides. All derivatives depolarized the bacterial membrane without forming pores and all affected cell wall integrity. Proteomic profiling of the bacterial stress responses to the small RW-rich antimicrobial peptides was reflective of non-disruptive membrane integration. Overall, our results indicate that antimicrobial peptides can be derivatized with lipid chains enhancing antibacterial activity without significantly altering the mechanism of action. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Arginina/química , Arginina/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Relação Estrutura-Atividade , Triptofano/química , Triptofano/metabolismoRESUMO
UNLABELLED: Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed. IMPORTANCE: Biological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, including Rhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome in R. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Molibdênio/metabolismo , Fixação de Nitrogênio , Nitrogenase/metabolismo , Rhodobacter capsulatus/enzimologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Nitrogênio/metabolismo , Nitrogenase/genética , Proteoma/genética , Proteoma/metabolismo , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismoRESUMO
DnaK-DnaJ-GrpE and GroES-GroEL are the major chaperone machineries in bacteria. In many species, dnaKJ and groESL are encoded in bicistronic operons. Quantitative proteomics revealed that DnaK and GroEL amounts in Salmonella dominate over DnaJ and GroES respectively. An imperfect transcriptional terminator in the intergenic region of dnaKJ is known to result in higher transcript levels of the first gene. Here, we examined the groESL operon and asked how the second gene in a heat shock operon can be preferentially expressed and found that an RNA structure in the 5'untranslated region of groES is responsible. The secondary structure masks the Shine-Dalgarno (SD) sequence and AUG start codon and thereby modulates translation of groES mRNA. Reporter gene assays combined with structure probing and toeprinting analysis revealed a dynamic temperature-sensitive RNA structure. Following an increase in temperature, only the second of two RNA hairpins melts and partially liberates the SD sequence, thus facilitating translation. Translation of groEL is not temperature-regulated leading to an excess of the chaperonin in the cell at low temperature. Discussion in a broader context shows how structured RNA segments can differentially control expression of temperature-affected operons in various ways.
Assuntos
Proteínas de Bactérias/biossíntese , Chaperoninas/biossíntese , Regulação Bacteriana da Expressão Gênica , Óperon , RNA Mensageiro/metabolismo , Salmonella typhimurium/efeitos da radiação , Regiões 5' não Traduzidas , Proteínas de Bactérias/genética , Sequência de Bases , Chaperoninas/genética , Genes Reporter , Temperatura Alta , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos da radiação , RNA Mensageiro/química , RNA Mensageiro/genética , Salmonella typhimurium/genéticaRESUMO
The cryptophyte phycocyanin Cr-PC577 from Hemiselmis pacifica is a close relative of Cr-PC612 found in Hemiselmis virescens and Hemiselmis tepida. The two biliproteins differ in that Cr-PC577 lacks the major peak at around 612 nm in the absorption spectrum. Cr-PC577 was thus purified and characterized with respect to its bilin chromophore composition. Like other cryptophyte phycobiliproteins, Cr-PC577 is an (αß)(α'ß) heterodimer with phycocyanobilin (PCB) bound to the α-subunits. While one chromophore of the ß-subunit is also PCB, mass spectrometry identified an additional chromophore with a mass of 585 Da at position ß-Cys-158. This mass can be attributed to either a dihydrobiliverdin (DHBV), mesobiliverdin (MBV), or bilin584 chromophore. The doubly linked bilin at position ß-Cys-50 and ß-Cys-61 could not be identified unequivocally but shares spectral features with DHBV. We found that Cr-PC577 possesses a novel chromophore composition with at least two different chromophores bound to the ß-subunit. Overall, our data contribute to a better understanding of cryptophyte phycobiliproteins and furthermore raise the question on the biosynthetic pathway of cryptophyte chromophores.
Assuntos
Criptófitas/metabolismo , Ficobiliproteínas/química , Biliverdina/análogos & derivados , Biliverdina/química , Cromatografia Líquida de Alta Pressão , Criptófitas/fisiologia , Complexos de Proteínas Captadores de Luz/química , Espectrometria de Massas , Peso Molecular , Ficobilinas/química , Ficobiliproteínas/metabolismo , Ficobiliproteínas/fisiologia , Ficocianina/química , Subunidades Proteicas/química , Análise de Sequência de ProteínaRESUMO
Clorobiocin is a well-known, highly effective inhibitor of DNA gyrase belonging to the aminocoumarin antibiotics. To identify potentially novel derivatives of this natural product, we conducted an untargeted investigation of clorobiocin biosynthesis in the known producer Streptomyces roseochromogenes DS 12.976 using LC-MSE, molecular networking, and analysis of fragmentation spectra. Previously undescribed clorobiocin derivatives uncovered in this study include bromobiocin, a variant halogenated with bromine instead of chlorine, hydroxylated clorobiocin, carrying an additional hydroxyl group on its 5-methyl-pyrrole 2-carboxyl moiety, and two other derivatives with modifications on their 3-dimethylallyl 4-hydroxybenzoate moieties. Furthermore, we identified several compounds not previously considered clorobiocin pathway products, which provide new insights into the clorobiocin biosynthetic pathway. By supplementing the medium with different concentrations of potassium bromide, we confirmed that the clorobiocin halogenase can utilize bromine instead of chlorine. The reaction, however, is impeded such that non-halogenated clorobiocin derivatives accumulate. Preliminary assays indicate that the antibacterial activity of bromobioin against Bacillus subtilis and efflux-impaired Escherichia coli matches that of clorobiocin. Our findings emphasize that yet unexplored compounds can be discovered from established strains and biosynthetic gene clusters by means of metabolomics analysis and highlight the utility of LC-MSE-based methods to contribute to unraveling natural product biosynthetic pathways. IMPORTANCE: The aminocoumarin clorobiocin is a well-known gyrase inhibitor produced by the gram-positive bacterium Streptomyces roseochromogenes DS 12.976. To gain a deeper understanding of the biosynthetic pathway of this complex composite of three chemically distinct entities and the product spectrum, we chose a metabolite-centric approach. Employing high-resolution LC-MSE analysis, we investigated the pathway products in extracted culture supernatants of the natural producer. Novel pathway products were identified that expand our understanding of three aspects of the biosynthetic pathway, namely the modification of the noviose, transfer and methylation of the pyrrole 2-carboxyl moiety, and halogenation. For the first time, brominated products were detected. Their levels and the levels of non-halogenated products increased in medium supplemented with KBr. Based on the presented data, we propose that the enzyme promiscuity contributes to a broad product spectrum.
Assuntos
Antibacterianos , Vias Biossintéticas , Metabolômica , Novobiocina , Streptomyces , Streptomyces/metabolismo , Streptomyces/genética , Antibacterianos/farmacologia , Antibacterianos/biossíntese , Antibacterianos/metabolismo , Antibacterianos/química , Novobiocina/análogos & derivados , Novobiocina/biossíntese , Novobiocina/farmacologia , Novobiocina/metabolismo , Cromatografia LíquidaRESUMO
Cold atmospheric pressure plasmas are used for surface decontamination or disinfection, e.g. in clinical settings. Protein aggregation has been shown to significantly contribute to the antibacterial mechanisms of plasma. To investigate the potential role of the redox-activated zinc-binding chaperone Hsp33 in preventing protein aggregation and thus mediating plasma resistance, we compared the plasma sensitivity of wild-type E. coli to that of an hslO deletion mutant lacking Hsp33 as well as an over-producing strain. Over-production of Hsp33 increased plasma survival rates above wild-type levels. Hsp33 was previously shown to be activated by plasma in vitro. For the PlasmaDerm source applied in dermatology, reversible activation of Hsp33 was confirmed. Thiol oxidation and Hsp33 unfolding, both crucial for Hsp33 activation, occurred during plasma treatment. After prolonged plasma exposure, however, unspecific protein oxidation was detected, the ability of Hsp33 to bind zinc ions was decreased without direct modifications of the zinc-binding motif, and the protein was inactivated. To identify chemical species of potential relevance for plasma-induced Hsp33 activation, reactive oxygen species were tested for their ability to activate Hsp33 in vitro. Superoxide, singlet oxygen and potentially atomic oxygen activate Hsp33, while no evidence was found for activation by ozone, peroxynitrite or hydroxyl radicals.