RESUMEN
Spontaneous mutations are important players in evolution. Nevertheless, there is a paucity of information about the mutagenic processes operating in most bacterial species. In this work, we implemented two forward mutational markers for studies in Caulobacter crescentus. We confirmed previous results in which A:T â G:C transitions are the most prevalent type of spontaneous base substitutions in this organism, although there is considerable deviation from this trend in one of the loci analyzed. We also investigated the role of dinB and imuC, encoding error-prone DNA polymerases, in spontaneous mutagenesis in this GC-rich organism. Both dinB and imuC mutant strains show comparable mutation rates to the parental strain. Nevertheless, both strains show differences in the base substitution patterns, and the dinB mutant strain shows a striking reduction in the number of spontaneous -1 deletions and an increase in C:G â T:A transitions in both assays.
RESUMEN
BACKGROUND: Heavy metal Resistance-Nodulation-Division (HME-RND) efflux systems help Gram-negative bacteria to keep the intracellular homeostasis under high metal concentrations. These proteins constitute the cytoplasmic membrane channel of the tripartite RND transport systems. Caulobacter crescentus NA1000 possess two HME-RND proteins, and the aim of this work was to determine their involvement in the response to cadmium, zinc, cobalt and nickel, and to analyze the phylogenetic distribution and characteristic signatures of orthologs of these two proteins. RESULTS: Expression assays of the czrCBA operon showed significant induction in the presence of cadmium and zinc, and moderate induction by cobalt and nickel. The nczCBA operon is highly induced in the presence of nickel and cobalt, moderately induced by zinc and not induced by cadmium. Analysis of the resistance phenotype of mutant strains showed that the ΔczrA strain is highly sensitive to cadmium, zinc and cobalt, but resistant to nickel. The ΔnczA strain and the double mutant strain showed reduced growth in the presence of all metals tested. Phylogenetic analysis of the C. crescentus HME-RND proteins showed that CzrA-like proteins, in contrast to those similar to NczA, are almost exclusively found in the Alphaproteobacteria group, and the characteristic protein signatures of each group were highlighted. CONCLUSIONS: The czrCBA efflux system is involved mainly in response to cadmium and zinc with a secondary role in response to cobalt. The nczCBA efflux system is involved mainly in response to nickel and cobalt, with a secondary role in response to cadmium and zinc. CzrA belongs to the HME2 subfamily, which is almost exclusively found in the Alphaproteobacteria group, as shown by phylogenetic analysis. NczA belongs to the HME1 subfamily which is more widespread among diverse Proteobacteria groups. Each of these subfamilies present distinctive amino acid signatures.
Asunto(s)
Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Metales Pesados/metabolismo , Familia de Multigenes , Transporte Biológico Activo , Análisis por Conglomerados , Evolución Molecular , Eliminación de Gen , Perfilación de la Expresión Génica , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
Most organisms that grow in the presence of oxygen possess catalases and/or peroxidases, which are necessary for scavenging the H(2)O(2) produced by aerobic metabolism. In this work we investigate the pathways that regulate the Caulobacter crescentus katG gene, encoding the only enzyme with catalase-peroxidase function in this bacterium. The transcriptional start site of the katG gene was determined, showing a short 5' untranslated region. The katG regulatory region was mapped by serial deletions, and the results indicate that there is a single promoter, which is responsible for induction at stationary phase. An oxyR mutant strain was constructed; it showed decreased katG expression, and no KatG protein or catalase-peroxidase activity was detected in stationary-phase cell extracts, implying that OxyR is the main positive regulator of the C. crescentus katG gene. Purified OxyR protein bound to the katG regulatory region between nucleotides -42 and -91 from the transcription start site, as determined by a DNase I footprinting assay, and a canonical OxyR binding site was found in this region. Moreover, OxyR binding was shown to be redox dependent, given that only oxidized proteins bound adjacent to the -35 sequence of the promoter and the katG P1 promoter was activated by OxyR in an H(2)O(2)-dependent manner. On the other hand, this work showed that the iron-responsive regulator Fur does not regulate C. crescentus katG, since a fur mutant strain presented wild-type levels of katG transcription and catalase-peroxidase production and activity, and the purified Fur protein was not able to bind to the katG regulatory region.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Proteínas de Unión al ADN/metabolismo , Peroxidasas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Caulobacter crescentus/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Peróxido de Hidrógeno , Datos de Secuencia Molecular , Peroxidasas/genética , Unión Proteica , Proteínas Represoras/genética , Factores de Transcripción/genética , Sitio de Iniciación de la TranscripciónRESUMEN
In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/genética , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Estrés Oxidativo , Proteínas Represoras/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Caulobacter crescentus/metabolismo , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Homeostasis , Mutación , Proteínas Represoras/genéticaRESUMEN
Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. ß-galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Estrés Oxidativo/fisiología , Zinc/metabolismo , Proteínas Bacterianas/genética , Homeostasis , Mutación , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Free-living bacteria must respond to a wide range of temperature changes, and have developed specific mechanisms to survive in extreme environments. In this work we describe a remarkable resistance of mesophilic bacterium Caulobacter crescentus to several cycles of freezing at -80 degrees C, which was able to grow at low temperatures. Exponentially growing cells and late stationary-phase cells presented higher freezing resistance at both -20 and -80 degrees C than early stationary-phase cells. Cryotolerance was observed when log-phase cultures grown at 30 degrees C were preincubated at 5, 15 or 20 degrees C before freezing at -20 degrees C. A transposon library was screened to identify mutants sensitive to freezing at -80 degrees C and three strains presenting <10% survival were isolated. Identification of genes disrupted in each mutant showed that they encoded an AddA family DNA helicase, a DEAD/DEAH box RNA helicase and a putative RND (resistance, nodulation, cell division) efflux system component. These strains showed longer generation times than wild-type cells when growing at 15 degrees C, with the RNA helicase mutant presenting a severe growth defect. These analyses suggest that the singular intrinsic resistance to freezing of C. crescentus is in fact a consequence of several independent traits, especially the maintenance of a proper degree of supercoiling of nucleic acids.
Asunto(s)
Proteínas Bacterianas/genética , Caulobacter crescentus/crecimiento & desarrollo , Frío , Congelación , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico , Proteínas Bacterianas/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiología , Medios de Cultivo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Elementos Transponibles de ADN , Biblioteca de Genes , Mutación , ARN Helicasas/genética , ARN Helicasas/metabolismoRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen with high resistance to a wide variety of antimicrobials. The multidrug resistance pump MexAB-OprM promotes the efflux of various antibiotics, mostly when mutations accumulate in the transcriptional regulators MexR, NalC and NalD, thereby causing MexAB-OprM overexpression. In this work, a characterization of 50 P. aeruginosa isolates obtained from Brazilian agricultural soils to determine the reasons of their resistance to aztreonam was done. The majority of the isolates showed higher aztreonam resistance than wild-type strain by MIC method. DNA sequence analysis of mexR, nalC and nalD genes from 13 of these isolates showed the amino acid substitution in NalC for all tested isolates, just one mutation was detected in MexR and none in NalD. Furthermore, an increase in the level of mexA expression by real-time RT-PCR analysis in eight isolates harboring mutations in NalC was found. Although there was not a relationship between MIC of aztreonam and the level of mexA expression, on the other hand, the results presented here suggest that novel mutations in NalC, including Arg97-Gly and Ala186-Thr, are related to MexAB-OprM overexpression causing aztreonam resistance in P. aeruginosa environmental isolates.
Asunto(s)
Antibacterianos/farmacología , Aztreonam/farmacología , Farmacorresistencia Bacteriana Múltiple , Proteínas de Transporte de Membrana/genética , Pseudomonas aeruginosa/genética , Proteínas Represoras/genética , Microbiología del Suelo , Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/genética , Brasil , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Operón , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Proteínas Represoras/metabolismoRESUMEN
A transposon library of 5700 mutants was constructed in Caulobacter crescentus strain NA1000. Thirteen mutant strains were isolated that presented growth deficiency in the presence of 20 microM cadmium chloride, and the disrupted genes were identified by DNA sequencing. Among the predicted products were found two putative transcriptional regulators, one histidine kinase/response regulator hybrid protein, two 2Fe-2S family proteins, and also members of a putative metal efflux system. Certain of these mutants also showed an increased sensitivity to zinc and copper, and the genes encoding the efflux system were induced by cadmium and zinc, indicating that this system may be important for homeostasis of both metal ions.
Asunto(s)
Cadmio/farmacología , Caulobacter crescentus/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Genes Bacterianos , Homeostasis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crecimiento & desarrollo , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Mutagénesis InsercionalRESUMEN
A Caulobacter crescentus rho::Tn5 mutant strain presenting a partially functional transcription termination factor Rho is highly sensitive to hydrogen peroxide in both exponential and stationary phases. The mutant was shown to be permanently under oxidative stress, based on fluorophore oxidation, and also to be sensitive to tert-butyl hydroperoxide and paraquat. However, the results showed that the activities of superoxide dismutases CuZnSOD and FeSOD and the alkylhydroperoxide reductase ahpC mRNA levels in the rho mutant were comparable to the wild-type control in the exponential and stationary phases. In contrast, the KatG catalase activity of the rho mutant strain was drastically decreased and did not show the expected increase in the stationary phase compared with the exponential phase. Transcription of the katG gene was increased in the rho mutant and the levels of the immunoreactive KatG protein do not differ considerably compared with the wild type in the stationary phase, suggesting that KatG activity is affected in a translational or a post-translational step.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Caulobacter crescentus/enzimología , Caulobacter crescentus/genética , Peroxidasas/biosíntesis , Factor Rho/deficiencia , Caulobacter crescentus/efectos de los fármacos , Elementos Transponibles de ADN , Perfilación de la Expresión Génica , Peróxido de Hidrógeno/toxicidad , Viabilidad Microbiana/efectos de los fármacos , Mutagénesis Insercional , Oxidantes/toxicidad , Estrés Oxidativo , Paraquat/toxicidad , Peroxirredoxinas/biosíntesis , Superóxido Dismutasa/biosíntesis , terc-Butilhidroperóxido/toxicidadRESUMEN
The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1) encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3), which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30(per cente) of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function. (au)