Functional Analysis of Regulatory Modules Involved in the General Stress Response and Symbiosis in Rhizobium etli.

Rhizobium etli is a soil-dwelling member of the Alpha-proteobacteria, capable of infecting the roots of its leguminous host Phaseolus vulgaris, the common bean plant, in order to establish a nitrogen-fixing symbiosis. Both in free-living state and during symbiosis, R. etli frequently has to cope with stressful and often harmful conditions. Thanks to a combination of strategies, the bacterium is able to react to these possibly life‑threatening assaults. Mechanisms include both highly stress‑specific responses and a complex, more general stress response, responsible for the development of multiple stress resistance in stationary phase cells. In this dissertation, a functional analysis of several regulatory modules involved in stress response and symbiosis in R. etli was performed. In a first part, we focused on the R. etli counterpart of the Alpha‑proteobacterial general stress response, comprised of the response regulator TcrX and two extracytoplasmic function sigma factors, EcfG1 and EcfG2. Phenotypic analysis revealed that TcrX is necessary for establishing an effective symbiosis with P. vulgaris. Moreover, the TcrX‑EcfG1‑EcfG2 system is involved in protection against oxidative stress and heat shock and mutants display an increased sensitivity to certain antibiotics targeting cell wall and cell membrane. Moreover, expression analysis using promoterless gusA as a reporter showed that tcrX and ecfG1 expression was induced in the presence of the antibiotics vancomycin and D‑cycloserine. To investigate the interplay between EcfG1 and EcfG2, an extensive transcriptional analysis was conducted. Using transcriptional promoter fusions, we showed that transcription of both sigma factors is positively autoregulated. Moreover, while optimal ecfG2 expression requires the presence of EcfG1, ecfG1 expression appears to be EcfG2‑independent. A microarray analysis was performed to determine the regulons of both sigma factors. We demonstrated that EcfG1 and EcfG2, while partially redundant, control distinct regulons, and identified small RNAs (sRNAs) as novel EcfG targets. We showed that expression of at least one of these sRNAs is under direct EcfG control. The presence of sRNAs in the regulon of an EcfG type sigma factor has not been described previously and adds an extra level of complexity to the regulation of the general stress response in Alpha-proteobacteria. Furthermore, we showed that the stringent response effector molecule (p)ppGpp acts as an important input signal for the general stress response. Based on these results, we propose an integrated model for general stress response regulation in R. etli, in which EcfG1 and EcfG2 function largely independently. Considering the widespread existence of Alpha-proteobacterial genomes with multiple EcfG copies, the results presented here will not only lead to a better understanding of the general stress response in R. etli, but will also contribute to a conceptual paradigm for general stress response regulation by multiple EcfG proteins in Alpha-proteobacteria.In the last part of this dissertation, we studied the role of a GntR‑like transcriptional regulator in stress response and symbiosis. Transcriptional regulators belonging to this superfamily are distributed throughout the bacterial kingdom and have important functions in modulation of gene expression. In a previous genome-wide screening for novel symbiotic genes in R. etli CNPAF512, a mutant affected in a gene encoding a GntR regulator was identified. This mutant showed a significant reduction in nitrogen fixation capacity compared to wild-type R. etli, suggesting a possible role for this regulator during symbiosis. In this work, a defined insertion mutant was constructed and analyzed with regard to its symbiotic phenotype. RT‑qPCR analysis was used to identify candidate target genes of the transcriptional regulator. Although we were not able to confirm the symbiotic phenotype observed with the transposon insertion mutant, analysis of the regulon suggests that this transcriptional regulator might play a role in establishing an effective symbiosis in R. etli.status: publishe