RESUMEN
The aims of this study were to functionally characterize and analyze the transcriptional regulation and transcriptome of the Rhizobium etli rpoE4 gene. An R. etli rpoE4 mutant was sensitive to oxidative, saline, and osmotic stresses. Using transcriptional fusions, we determined that RpoE4 controls its own transcription and that it is negatively regulated by rseF (regulator of sigma rpoE4; CH03274), which is cotranscribed with rpoE4. rpoE4 expression was induced not only after oxidative, saline, and osmotic shocks, but also under microaerobic and stationary-phase growth conditions. The transcriptome analyses of an rpoE4 mutant and an rpoE4-overexpressing strain revealed that the RpoE4 extracytoplasmic function sigma factor regulates about 98 genes; 50 of them have the rpoE4 promoter motifs in the upstream regulatory regions. Interestingly, 16 of 38 genes upregulated in the rpoE4-overexpressing strain encode unknown putative cell envelope proteins. Other genes controlled by RpoE4 include rpoH2, CH00462, CH02434, CH03474, and xthA1, which encode proteins involved in the stress response (a heat shock sigma factor, a putative Mn-catalase, an alkylation DNA repair protein, pyridoxine phosphate oxidase, and exonuclease III, respectively), as well as several genes, such as CH01253, CH03555, and PF00247, encoding putative proteins involved in cell envelope biogenesis (a putative peptidoglycan binding protein, a cell wall degradation protein, and phospholipase D, respectively). These results suggest that rpoE4 has a relevant function in cell envelope biogenesis and that it plays a role as a general regulator in the responses to several kinds of stress.
Asunto(s)
Proteínas Bacterianas/fisiología , Presión Osmótica/fisiología , Estrés Oxidativo/genética , Rhizobium etli/fisiología , Factor sigma/fisiología , Proteínas Bacterianas/genética , Secuencia de Bases , Fabaceae/microbiología , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/fisiología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhizobium etli/genética , Rhizobium etli/crecimiento & desarrollo , Rhizobium etli/metabolismo , Homología de Secuencia de Ácido Nucleico , Factor sigma/genéticaRESUMEN
The physiological role and transcriptional expression of Rhizobium etli sigma factors rpoH1 and rpoH2 are reported in this work. Both rpoH1 and rpoH2 were able to complement the temperature-sensitive phenotype of an Escherichia coli rpoH mutant. The R. etli rpoH1 mutant was sensitive to heat shock, sodium hypochlorite and hydrogen peroxide, whereas the rpoH2 mutant was sensitive to NaCl and sucrose. The rpoH2 rpoH1 double mutant had increased sensitivity to heat shock and oxidative stress when compared with the rpoH1 single mutant. This suggests that in R. etli, RpoH1 is the main heat-shock sigma factor, but a more complete protective response could be achieved with the participation of RpoH2. Conversely, RpoH2 is involved in osmotic tolerance. In symbiosis with bean plants, the R. etli rpoH1 and rpoH2 rpoH1 mutants still elicited nodule formation, but exhibited reduced nitrogenase activity and bacterial viability in early and late symbiosis compared with nodules produced by rpoH2 mutants and wild-type strains. In addition, nodules formed by R. etli rpoH1 and rpoH2 rpoH1 mutants showed premature senescence. It was also determined that fixNf and fixKf expression was affected in rpoH1 mutants. Both rpoH genes were induced under microaerobic conditions and in the stationary growth phase, but not in response to heat shock. Analysis of the upstream region of rpoH1 revealed a sigma70 and a probable sigmaE promoter, whereas in rpoH2, one probable sigmaE-dependent promoter was detected. In conclusion, the two RpoH proteins operate under different stress conditions, RpoH1 in heat-shock and oxidative responses, and RpoH2 in osmotic tolerance.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Presión Osmótica , Rhizobium etli/fisiología , Factor sigma/metabolismo , Estrés Fisiológico , Secuencia de Bases , Fabaceae/microbiología , Fabaceae/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Mutación , Estrés Oxidativo , Regiones Promotoras Genéticas , Rhizobium etli/genética , Nódulos de las Raíces de las Plantas/microbiología , Nódulos de las Raíces de las Plantas/fisiología , Factor sigma/genética , SimbiosisRESUMEN
The recombination genes involved in Holliday junction migration (ruvB, recG, radA) and heteroduplex editing (mutS) were studied in the alpha-proteobacterium Rhizobium etli. The genes were interrupted with a loxPSp interposon and R. etli mutants, either single or in combination, were constructed by marker exchange. Our results show that these systems play a differential role in sensitivity to DNA damaging agents and recombination in R. etli. RuvB appears to be the main system for tolerance toward agents instigating single- or double-strand breaks (such as UV light, methyl methanesulphonate and nalidixic acid) while the RecG and RadA systems play minor roles in tolerance to these agents. Using five different recombination assays, we have found that a ruvB null mutant showed a notable reduction in recombination proficiency, while a radA mutant was only weakly affected. A null mutation in recG had the opposite effect, enhancing recombination in most of our assays. This effect was more clearly seen in an assay that measured recombination between divergent sequences (i.e. homeologous), but is unaffected by inactivation of mutS. These data indicate that RecG in R. etli limits intra- and intergenomic plasticity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Daño del ADN , ADN Cruciforme/metabolismo , Mutágenos/toxicidad , Recombinación Genética/efectos de los fármacos , Rhizobium etli/efectos de los fármacos , Rhizobium etli/genética , Proteínas Bacterianas/genética , Transporte Biológico/efectos de los fármacos , Clonación Molecular , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Genes Bacterianos , Mutación/genéticaRESUMEN
Gene conversion has been defined as the nonreciprocal transfer of information between homologous sequences. Despite its broad interest for genome evolution, the occurrence of this mechanism in bacteria has been difficult to ascertain due to the possible occurrence of multiple crossover events that would mimic gene conversion. In this work, we employ a novel system, based on cointegrate formation, to isolate gene conversion events associated with crossovers in the nitrogen-fixing bacterium Rhizobium etli. In this system, selection is applied only for cointegrate formation, with gene conversions being detected as unselected events. This minimizes the likelihood of multiple crossovers. To track the extent and architecture of gene conversions, evenly spaced nucleotide changes were made in one of the nitrogenase structural genes (nifH), introducing unique sites for different restriction endonucleases. Our results show that (i) crossover events were almost invariably accompanied by a gene conversion event occurring nearby; (ii) gene conversion events ranged in size from 150 bp to 800 bp; (iii) gene conversion events displayed a strong bias, favoring the preservation of incoming sequences; (iv) even small amounts of sequence divergence had a strong effect on recombination frequency; and (v) the MutS mismatch repair system plays an important role in determining the length of gene conversion segments. A detailed analysis of the architecture of the conversion events suggests that multiple crossovers are an unlikely alternative for their generation. Our results are better explained as the product of true gene conversions occurring under the double-strand break repair model for recombination.
Asunto(s)
Intercambio Genético , Conversión Génica , Rhizobium etli/genética , Proteínas Bacterianas/genética , Mapeo Cromosómico , Cromosomas Bacterianos , Reparación del ADN , Modelos Genéticos , Oxidorreductasas/genética , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Single-strand gaps (SSGs) and double-strand breaks (DSBs) are the major initiation sites for recombination. In bacteria, the SSGs are repaired by RecFOR, while the DSBs are processed by RecBCD in gram-negative bacteria and AddAB in gram-positive bacteria. Unexpectedly, instead of recBCD genes, the addAB genes were found in members of the alpha-proteobacteria group (gram negative). Taking Rhizobium etli as a model, the role of recF and addAB genes in homologous recombination and repair of damaged DNA was evaluated. Inactivation of either recF or addA provoked strong sensitivity to UV radiation and mitomycin C, while an additive effect was observed in the recF-addA mutant. The DSBs generated by nalidixic acid caused low viability only in the addA mutant. The recombination frequency of large and small plasmids was reduced in the recF mutant (24- and 36-fold, respectively), whereas a slight decrease (threefold) in the addA mutant was observed. Moreover, an additive effect (47- and 90-fold, respectively) was observed in the double mutant, but it was not as dramatic as that in a recA mutant. Interestingly, the frequency of deletion and Campbell-type recombination was slightly affected in either single or double mutants. These results suggest that another pathway exists that allows plasmid and Campbell-type recombination in the absence of recF and addA genes.