A transcriptomic insight into the regulon of the Sinorhizobium meliloti trans-encoded small RNA Sm8

LAGARES ANTONIO; BECKER, ANKE; VALVERDE, CLAUDIO

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

Sociedad Argentina de Microbiología General

Bacterial small regulatory non-coding RNAs (sRNAs) are key players in the fine-tuning of gene expression. Strong evidences of the presence of riboregulatory circuits in the nitrogen-fixing root-nodule symbiont Sinorhizobium meliloti were recently provided by characterization of a functional Hfq protein, the chaperone of sRNA-mRNA interactions, and by the detection of hundreds of sRNAs expressed from all genomic replicons. However, the biological roles of these RNA molecules still remain unknown in S. meliloti. Here, we present the first data on the characterization of the regulon associated with the S. meliloti chromosomally trans-encoded sRNA Sm8. The Sm8 sequence and its predicted secondary structure are highly conserved among -proteobacteria. We have previously reported the construction and initial characterization of S. meliloti 2011 isogenic strains with: i) a deletion of a highly conserved internal Sm8 sequence motif; ii) constitutive sm8-overexpression. Both qRT-PCR and Northern blot assays confirmed that the Sm8 transcript levels were below detection limits in the deletion mutant and approximately 10-fold higher in the overexpression strain than in the wild type. The absence of polar effects on the expression of sm8 flanking genes was also verified. In order to identify genes whose expression is influenced by Sm8, a global transcriptional profiling was carried out using comparative microarray hybridizations of cDNA from the aforementioned strains, upon a short term shift (1 hour) to growth conditions that induce Sm8 accumulation. This approach allowed us to identify a set of genes mostly encoding proteins involved in the regulation of the N2-fixation and respiratory metabolism, whose transcript levels were inversely correlated with Sm8 concentration. Their abundance under different growth conditions was consistently up-regulated in the sm8 mutant and down-regulated in the sm8-overexpressing strain. Validation of the microarray results by qRT-PCR is currently under progress. Interestingly, this set of regulated transcripts markedly overlaps with those comprised in the well-characterized FixJ-associated regulon. The results suggest so far that Sm8 would be a direct or indirect (via FixJ) negative regulator of the expression of this set of genes, either by reducing mRNA transcription rate, by negatively affecting mRNA stability, or both. It is worth mentioning that a symbiotic assay performed with Medicago sativa revealed that sm8- bacteroids have a higher specific N2-fixation activity than wild type bacteroids. The fact that the sm8 promoter itself seems to be regulated by the nitrogen status of the cell (see Ceizel-Borella et al., this meeting), together with the findings reported here, point to a possible role of the Sm8 sRNA in the global control of nitrogen metabolism in Sinorhizobium meliloti.