RESUMO
Pseudomonas aeruginosa easily produces drug-resistant mutants. A large number of mutational resistome genes exist in the genome of P. aeruginosa. In this study, whole genome sequencing analysis of a multidrug-resistant P. aeruginosa strain isolated by in vitro antibiotic treatment showed a mutation in the cpxS gene. Random mutagenesis of cpxS was conducted and introduced into the PA14ΔcpxS strain. Numerous CpxS mutants, including 14 different single amino acid substitutions, were identified, which led to reduced antibiotic susceptibility. Moreover, some of them were also present in the published genomes of P. aeruginosa isolates. Around cpxS, a gene coding for a putative sensor kinase, the nearest gene coding for a response regulator is cpxR in the genome of P. aeruginosa. Deletion of cpxR restored antibiotic susceptibility in the above cpxS mutant strains. As an extension of our previous work, where the expression of the mexAB-oprM operon is directly activated by CpxR in P. aeruginosa, in this study, we showed that the expression of the mexA promoter was increased in the above cpxS mutant strains in a cpxR-dependent manner, and mexA is prerequisite for the reduced antibiotic susceptibility. Therefore, we propose that the putative sensor kinase CpxS, together with CpxR, comprises a two-component regulatory system regulating the expression of the mexAB-oprM operon in P. aeruginosa. Our work indicates that cpxS, as a novel member of mutational resistome, plays important roles on the development of multidrug resistance in P. aeruginosa.
Assuntos
Proteínas de Bactérias , Infecções por Pseudomonas , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Mutação/genética , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have been made to simplify CBS by refactoring a large number of genes with rearranged polycistrons and synthetic regulatory circuits. Here, a posttranslational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system, where the expression stoichiometry is particularly important. Operon-based nif genes from Klebsiella oxytoca were regrouped into giant genes either by fusing genes together or by expressing polyproteins that are subsequently cleaved with Tobacco Etch Virus protease. After several rounds of selection based on protein expression levels and tolerance toward a remnant C-terminal ENLYFQ-tail, a system with only five giant genes showed optimal nitrogenase activity and supported diazotrophic growth of Escherichia coli This study provides an approach for efficient translation from an operon-based system into a polyprotein-based assembly that has the potential for portable and stoichiometric expression of the complex nitrogenase system in eukaryotic organisms.
Assuntos
Proteínas de Bactérias , Escherichia coli , Klebsiella oxytoca/genética , Microrganismos Geneticamente Modificados , Fixação de Nitrogênio , Óperon , Poliproteínas , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Poliproteínas/biossíntese , Poliproteínas/genéticaRESUMO
Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. In this study, we demonstrate that CpxR, previously identified as a regulator of the cell envelope stress response in Escherichia coli, is directly involved in activation of expression of RND efflux pump MexAB-OprM in P. aeruginosa. A conserved CpxR binding site was identified upstream of the mexA promoter in all genome-sequenced P. aeruginosa strains. CpxR is required to enhance mexAB-oprM expression and drug resistance, in the absence of repressor MexR, in P. aeruginosa strains PA14. As defective mexR is a genetic trait associated with the clinical emergence of nalB-type multidrug resistance in P. aeruginosa during antibiotic treatment, we investigated the involvement of CpxR in regulating multidrug resistance among resistant isolates generated in the laboratory via antibiotic treatment and collected in clinical settings. CpxR is required to activate expression of mexAB-oprM and enhances drug resistance, in the absence or presence of MexR, in ofloxacin-cefsulodin-resistant isolates generated in the laboratory. Furthermore, CpxR was also important in the mexR-defective clinical isolates. The newly identified regulatory linkage between CpxR and the MexAB-OprM efflux pump highlights the presence of a complex regulatory network modulating multidrug resistance in P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos/fisiologia , Resistência a Múltiplos Medicamentos/fisiologia , Infecções por Pseudomonas/metabolismo , Proteínas da Membrana Bacteriana Externa/biossíntese , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana Transportadoras/biossíntese , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Uncoupling of biological nitrogen fixation from ammonia assimilation is a prerequisite step for engineering ammonia excretion and improvement of plant-associative nitrogen fixation. In this study, we have identified an amino acid substitution in glutamine synthetase, which provides temperature sensitive biosynthesis of glutamine, the intracellular metabolic signal of the nitrogen status. As a consequence, negative feedback regulation of genes and enzymes subject to nitrogen regulation, including nitrogenase is thermally controlled, enabling ammonia excretion in engineered Escherichia coli and the plant-associated diazotroph Klebsiella oxytoca at 23 °C, but not at 30 °C. We demonstrate that this temperature profile can be exploited to provide diurnal oscillation of ammonia excretion when variant bacteria are used to inoculate cereal crops. We provide evidence that diurnal temperature variation improves nitrogen donation to the plant because the inoculant bacteria have the ability to recover and proliferate at higher temperatures during the daytime.
Assuntos
Amônia , Grão Comestível , Grão Comestível/metabolismo , Amônia/metabolismo , Nitrogênio/metabolismo , Fixação de Nitrogênio , Nitrogenase/genética , Nitrogenase/metabolismo , Bactérias/metabolismoRESUMO
The LysR-family regulator MexT modulates the expression of the MexEF-OprN efflux system in the human pathogen Pseudomonas aeruginosa. Recently, we demonstrated that MexT regulates certain virulence phenotypes, including the type-three secretion system and early attachment independent of its role in regulating MexEF-OprN. In this study, transcriptome profiling was utilized to investigate the global nature of MexT regulation in P. aeruginosa PAO1 and an isogenic mexEF mutant. Twelve genes of unknown function were highly induced by overexpressing MexT independent of MexEF-OprN. A well-conserved DNA motif was identified in the upstream regulatory region of nine of these genes and upstream of mexE. Reporter fusion analysis demonstrated that the expression of the genes was significantly induced by MexT in P. aeruginosa and a heterogenous Escherichia coli strain and that the conserved sequence was required for this induction. The conserved DNA motif was further characterized as the MexT binding site by site-directed mutagenesis and electrophoretic mobility shift assays. Genes containing this conserved regulatory sequence were identified across other Pseudomonas species, and their expression was activated by MexT. Thus, a novel regulon directly modulated by MexT, that includes but is independent of mexEF-oprN, has been identified.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Regulon , Fatores de Transcrição/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , Pseudomonas/genética , Pseudomonas aeruginosa/metabolismo , Ativação TranscricionalRESUMO
In the human pathogen Pseudomonas aeruginosa, the LysR-family regulator MexT modulates the induction of the tripartite MexEF-OprN resistance nodulation-division multi-drug efflux system resulting in increased resistance to diverse antibiotics. The MexEF-OprN system is normally quiescent in wild-type cells, but is highly induced in nfxC-type phenotypic mutants in a MexT dependent manner. In addition to antibiotic resistance, induction of mexEF-oprN in nfxC-type mutants has been linked to reduced levels of homoserine lactone-dependent virulence traits, including pyocyanin, elastase, rhamnolipids and PQS and to reduced expression of type three secretion effector proteins. In this study, MexT is overexpressed in wild-type PAO1 and an isogenic mexEF deletion mutant to determine if MexT regulates diverse virulence phenotypes dependent or independent of MexEF-OprN. It is shown that MexT regulates type three secretion, pyocyanin production and early surface attachment independent of MexEF-OprN. In contrast, MexT modulation of the expression of the virulence genes rhlA, lasB and hcnB is dependent on MexEF-OprN, which apparently mediates these effects via efflux of cell-signaling intermediates. The data presented demonstrates that MexT may play a more global role in modulating P. aeruginosa virulence than previously reported and suggests that MexT regulates diverse targets that mediate phenotypic alterations independent of MexEF-OprN.
Assuntos
Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/fisiologia , Fatores de Transcrição/fisiologia , Fatores de Virulência/biossíntese , Aderência Bacteriana , Deleção de Genes , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Piocinas/biossíntese , VirulênciaRESUMO
A binding site for the Escherichia coli nucleoid binding protein FIS (factor for inversion stimulation) was identified upstream of a sigma54-dependent promoter, glnAp2. The binding and bending center of FIS is positioned at -55 with respect to the transcription start site (+1). Binding of FIS at this site activates the transcription of glnAp2 both in vivo and in vitro. Furthermore, we substituted the FIS-mediated DNA bending with other protein (cAMP receptor protein or integration host factor)-mediated DNA bending, without changing the position of the bending center. In vitro transcription assays indicated that all DNA bends centered at -55 activate transcriptional initiation of glnAp2, especially when linear templates were used.
Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/metabolismo , Regulação Bacteriana da Expressão Gênica , Glutamato-Amônia Ligase/metabolismo , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Elementos Facilitadores Genéticos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Glutamato-Amônia Ligase/química , Glutamato-Amônia Ligase/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Although DBP (di-n-butyl phthalate) is commonly encountered as an artificially-synthesized plasticizer with potential to impair fertility, we confirm that it can also be biosynthesized as microbial secondary metabolites from naturally occurring filamentous fungi strains cultured either in an artificial medium or natural water. Using the excreted crude enzyme from the fungi for catalyzing a variety of substrates, we found that the fungal generation of DBP was largely through shikimic acid pathway, which was assembled by phthalic acid with butyl alcohol through esterification. The DBP production ability of the fungi was primarily influenced by fungal spore density and incubation temperature. This study indicates an important alternative natural waterborne source of DBP in addition to artificial synthesis, which implied fungal contribution must be highlighted for future source control and risk management of DBP.
Assuntos
Dibutilftalato/metabolismo , Água Doce/análise , Fungos/metabolismo , Ácido Chiquímico/química , Esporos Fúngicos/metabolismo , Dibutilftalato/análise , Água Doce/microbiologia , Fungos/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimento , TemperaturaRESUMO
Based on the complete Sinorhizobium meliloti genome sequence we established DNA microarrays as a comprehensive tool for systematic genome-wide gene expression analysis in S. meliloti 1021. For these PCR fragment-based microarrays, called Sm6kPCR, a collection of probes for the 6207 predicted protein-coding genes consisting of 6046 gene-specific PCR fragments and 161 70 mer oligonucleotides was arrayed in high density on glass slides. To obtain these PCR fragments primer pairs were designed to amplify internal gene-specific DNA fragments of 80-350 bp. Additionally, these primers were characterized by a 5' extension that allowed for reamplification using standard primers after the first amplification employing the specific primers. In order to ascertain the quality of the Sm6kPCR microarrays and to validate gene expression studies in S. meliloti parallel hybridizations based on RNA samples obtained from cells cultured under identical conditions were performed. In addition, gene expression in S. meliloti in response to an osmotic upshift imposed by the addition of 0.38 M NaCl was monitored. 137 genes were identified showing significant changes in gene expression resulting from the osmotic upshift. From these genes 52 were induced and 85 genes were repressed. Among the genes displaying different RNA levels some functional groups could be identified that are particularly remarkable. Repression was observed for 8 genes related to motility and chemotaxis, 7 genes encoding amino acid biosynthesis enzymes and 15 genes involved in iron uptake whereas 14 genes involved in transport of small molecules and 4 genes related to polysaccharide biosynthesis were induced.
Assuntos
Mapeamento Cromossômico/instrumentação , Perfilação da Expressão Gênica/instrumentação , Regulação Bacteriana da Expressão Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Sinorhizobium meliloti/genética , Equilíbrio Hidroeletrolítico/genética , Mapeamento Cromossômico/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Perfilação da Expressão Gênica/métodos , Genoma Bacteriano , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Sinorhizobium meliloti/classificação , Especificidade da EspécieRESUMO
The expression of σ(54)-dependent Pseudomonas putida Pu promoter is activated by XylR activator when cells are exposed to a variety of aromatic inducers. In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP), a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1) of CRP, which interacts directly with the αCTD (C-terminal domain of α-subunit) of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed.
Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Polimerase Sigma 54/genética , Xilenos/farmacologia , Pegada de DNA , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Pseudomonas putida/enzimologia , Transcrição GênicaRESUMO
Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1(optimized)) at the 5' end. The PparoA1(optimized) gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T(1) progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1(optimized) gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Glicina/análogos & derivados , Nicotiana/efeitos dos fármacos , Nicotiana/fisiologia , Pseudomonas putida/metabolismo , Proteínas de Bactérias/genética , Segregação de Cromossomos/genética , Cruzamentos Genéticos , Glicina/farmacologia , Padrões de Herança/genética , Dados de Sequência Molecular , Fenótipo , Filogenia , Plantas Geneticamente Modificadas , Plasmídeos/genética , Pseudomonas putida/efeitos dos fármacos , Nicotiana/genética , Transformação Genética/efeitos dos fármacos , GlifosatoRESUMO
Spontaneous nfxC-type phenotypic mutants of Pseudomonas aeruginosa are characterized by chloramphenicol resistance and increased expression of the multidrug efflux pump, MexEF-OprN. These mutants frequently arise during the propagation of bacteria in laboratory conditions. This study shows that some of the discrepancies in transcriptomic/phenotypic profiles of two rsmA mutants, PAZH13 (rsmA mutant in strain PAO1) and PAKΔrsmA, are due to the nfxC-type nature of PAZH13. This indicates that awareness of the possible occurrence of these spontaneous nfxC-type phenotypes is necessary. Screening of mexE levels could provide clarity when an nfxC-type phenotype is suspected.
RESUMO
Sigma54-RNA polymerase (Esigma54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of Esigma54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Esigma54, DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Esigma54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Esigma54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-Esigma54 complex.
Assuntos
DNA Bacteriano/química , Proteínas de Escherichia coli , Glutamato-Amônia Ligase , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/metabolismo , Sequência de Bases , Proteína Receptora de AMP Cíclico , Pegada de DNA , Desoxirribonuclease I/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Conformação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Glyphosate has been used globally as a safe herbicide for weed control. It inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA), which is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. A Pseudomonas putida strain, 4G-1, was isolated from a soil heavily contaminated by glyphosate in China. Its AroA-encoding gene (aroA) has been cloned, sequenced, and expressed in Escherichia coli. Phylogenetic analysis revealed that this AroA belongs neither to class I nor to class II AroA enzymes. When compared with E. coli AroA, 4G-1 AroA shows similar values for K(m)[PEP], K(m)[S3P], and specific enzyme activity. Moreover, 4G-1 AroA exhibits high tolerance to glyphosate, which indicates a protein with a high potential for structural and functional studies of AroA in general and its potential usage for the generation of transgenic crops resistant to the herbicide.