Caracterización del rnoma del endosimbionte diazotrófico sinorhizobium melilotiaproximación a la función de la chaperona de rna hfq y de los ribo-reguladores smr7c y smr15c1/c2

Resumen

Traditionally, bacterial genomes are divided into regions containing genes (ORFs) and intergenic regions (IGRs), apparently without assigned annotation. The transcriptome initially is consisted for the mRNAs that are translated into protein and abundance transcripts which are not translated, such as rRNAs and tRNAs. Post-genomic research has revealed in the last years an unexpected abundance of transcripts which are not translated into proteins, called small non-coding RNAs (sRNAs). Therefore, the central dogma of biology which assumes that the final product of gene expression is the protein is currently invalid. Bacterial sRNAs are being recognized as novel widespread regulators of gene expression in response to environmental signals. This whole new set of transcripts that are not translated into protein is known such RNome. Most of the new riboregulators identified correspond to trans-sRNA. These RNAs may exert its function through two mechanisms: base pairing with the target mRNA or binding to proteins by altering their activity and thus producing a direct effect on transcription and translation. This class of transcripts frequently binds to the RNA chaperone Hfq, which assess them on their regulation process. In our group, we are working with the endosymbiont Sinorhizobium meliloti, which is capable of conducting effective symbiosis with legume plants and form structures on the roots called nodule, where the bacteria fixes atmospheric nitrogen into differentiated and intracellular form, the bacteroid. The sequenced strain 1021 genome consists of three replicons, a chromosome and two symbiotic plasmids PsymA and PsymB. Previously at this work, the S. meliloti RNome consisted in 54 tRNAs, three loci rRNA, the ribozymes/group II introns and the cromosomal sRNA tmRNA. The first chapter of this Thesis presents a novel approach which integrates a genome-wide computational method of IGRs of S. meliloti and seven alpha-proteobacterial related species and RNA-Seq of RNAs that are targeted by the common bacterial Sm-like protein Hfq. We have used eQRNA and RNAz as complementary predictive tools to identified non-coding transcripts in the bacterial genome. This approach identified 10 RNAs: the house-keeping genes of RNasa P, signal recognition particle (SRP) RNA and 6S, and seven novel sRNAs (Smr7C, Smr9C, Smr14C2, Smr15C1/C2, Smr45C and Smr35B). Northern and RACE experiments confirm which the novel sRNAs are located into independent transcriptional units and differentially expressed, predicting such trans-sRNAs. Therefore, we have analyzed the conservation pattern of these sRNAs in ¿-proteobacteria. The covariance models have identified six families of non-coding transcripts with members present in phylogenetically related plant-interacting bacteria and animal pathogens of the order of the Rhizobiales, some occurring with high levels of paralogy in individual genomes (e.g. alphar14 and alphar15). In silico and experimental evidences predict differential regulation of paralogous sRNAs in S. meliloti 1021. The distribution patterns of these sRNA families suggest major contributions of vertical inheritance and extensive ancestral duplication events to the evolution of sRNAs in plant-interacting bacteria. High-throughput analysis of cDNA libraries collected from cells grown on exponential and stationary phase revealed that the major population of RNAs that bind to Hfq are the mRNAs. Indeed, this study identified 460 sRNAs belong to trans and cis-antisense classes, most of them without any annotation on the S. meliloti genome, probably because Hfq binds an specific RNA population. Chapter two focusses in the study of the chaperone Hfq, which at this moment was unclear in the endosymbiont. S. meliloti. Hfq protein is small compared to the Enterobacteriaceae, one of the best studied, although similar to those identified in other alpha-proteobacteria. The study of the regulatory role of Hfq in bacteria was performed by analysis of Dhfq deletion mutant, both in free-living bacteria and in symbiosis, and the impact of mutation on the transcriptome. In free-living state, the mutant shows a delay growth that predicts the pleiotropic effect of the loss of Hfq observed in other bacteria. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Dhfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria Transcriptomic profiling of 1021Dhfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. RNA-Seq of Hfq-binding population revealed that 40 % of transcripts differentially regulated in the hfq mutant depend directly of the chaperone, including transport and fix/nif genes that support the observed phenotype in the mutant. The last chapter presents the first functional characterization of the homologs Smr15C1 and Smr15C2, which are part of the sRNA family of paralogs alphar15 and the chromosomal single-copy transcript Smr7C. The two alphar15 sRNAs are very similar in sequence and structure, but they have divergent expression that suggests non- hierarchical mechanisms of regulation. Therefore, the covariance models identified a third copy located on pSymA Smr15A. Smr7C shows a typical trans-sRNA structure with several hairpin-loops in its primary transcript and a smaller processed form derived from the primary one. Therefore, our results have revealed that the three sRNAs are dispensable for the symbiotic interaction of S. meliloti with M. sativa and accumulate differentially for post-transcriptional regulation of transport proteins through mechanisms dependent (Smr15C1/C2) or independent (Smr7C) of the chaperone Hfq. Smr15C1/C2 probably affect the translation of several ABC transporters involved on nitrogen/carbon uptake, which represents a multi-target regulation of sRNAs. Smr7C can act as a positive regulator of rhtA mRNA, a siderophore transporter of rizobactine.