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S. meliloti CspA2 CspA4 and CspA5 Native IP followed by RNA-seq

ABSTRACT: Rhizobia are soil bacteria that can associate with some legumes and participate in symbiotic nitrogen fixation. Bacterial CspA family members are small, single stranded nucleic acid binding proteins. Differentiation of rhizobial bacteria from a free-living to symbiotic state within legume root nodules follows a massive re-programming of bacterial gene expression. Here, the role of Sinorhizobium meliloti CspA family members in symbiotic development with Medicago sativa (alfalfa) was investigated. We defined expression patterns of CspA family members, identified CspA interacting RNAs, and investigated phenotypes and transcriptional defects associated with cspA deletion strains. We propose that these proteins affect rhizobial physiology through their global control of the cellular RNA secondary structure strength environment and through specific modulation of small non-coding RNA (sRNA) structures involved in cis-regulation of stress responsive sigma factor expression. This work describes an RNA structure mediated mechanism important for bacterial stress adaptation and symbiotic development within a plant host.

ORGANISM(S): Sinorhizobium meliloti

PROVIDER: GSE102799 | GEO | 2018/08/01

REPOSITORIES: GEO

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Project description:Abiotic stresses disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. In particular, the effect of extracellular acidity on rhizobia has been taken as a model example for analysis because of the economic impact and worldwide distribution of these symbionts in agricultural countries. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0â??6.1. Under such a limiting acid stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a remarkably higher aerobic-respiration rate in acid-stressed rhizobia. Changes in the transcripts encoding enzymes for lipid biosynthesis were also observed, consistent with previous data on rhizobial pH-dependent membrane remodelling. Together with increased energy demands under acidity, proteomic and transcriptomic data revealed that while at pH 7.0 the transport and biosynthesis of cellular compounds were quite active processes, under acid stress most overexpressed markers were associated with protein biosynthesis, macromolecular degradation and/or recycling, and energy metabolism. Within this context, the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Moreover, multivariate analysis of global metabolome data (more than 60 compounds) served to unequivocally correlate the specific-metabolite profiles with the extracellular pH for growth, with strikingly sensitive variations being observed in the rhizobial metabolomes upon extracellular-pH changes of less than 0.5 units. Except for a ca. 120-kb DNA stretch within the pSymA no specific genomic regions were associated with the observed acid-stress responses. Further practical analyses should be focussed on the phenotypic impact and time course of the observed changes during the acid-stress perception and on the search for common responses during the previously described sublethal acid-adaptive processes in rhizobia pH 6.1 vs pH 7

Project description:During the legume-rhizobium symbiosis, free-living soil bacteria known as rhizobia trigger the formation of root nodules. The rhizobia infect these organs and adopt an intracellular lifestyle within the symbiotic nodule cells where they become nitrogen-fixing bacteroids. Several legume lineages enforce their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this differentiation process in Bradyrhizobium sp. ORS285, a symbiont of Aeschynomene spp.. In the absence of BclA, Bradyrhizobium sp. ORS285 proceeds until the intracellular infection of nodule cells but the bacteria cannot differentiate into enlarged polyploid bacteroids and fix nitrogen. The nodule bacteria of the bclA mutant constitute thus an intermediate stage between the free-living soil bacteria and the intracellular nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the ORS285 wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules discriminated nodule-induced genes that are specific to differentiated and nitrogen-fixing bacteroids and others that are activated in the host microenvironment irrespective of bacterial differentiation and nitrogen fixation. These analyses demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied with a first transcriptome switch involving several hundreds of upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving less genes but that are expressed to extremely elevated levels. The transcriptomes further highlighted the dynamics of oxygen and redox regulation of gene expression during nodule formation and we discovered that bclA represses the expression of non-ribosomal peptide synthetase gene clusters suggesting a non-symbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.

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S. meliloti CspA2 CspA4 and CspA5 Native IP followed by RNA-seq

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