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BackgroundThe bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored.ResultsTwo independent S. meliloti mutants, 2011-3.4 and 1021Δhfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Δhfq 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. 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 1021Δhfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein.ConclusionsOur results support that the S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.

Structural and biochemical features suggest that the almost ubiquitous bacterial YbeY protein may serve catalytic and/or Hfq-like protective functions central to small RNA (sRNA)-mediated regulation and RNA metabolism. We have biochemically and genetically characterized the YbeY ortholog of the legume symbiont Sinorhizobium meliloti (SmYbeY). Co-immunoprecipitation (CoIP) with a FLAG-tagged SmYbeY yielded a poor enrichment in RNA species, compared to Hfq CoIP-RNA uncovered previously by a similar experimental setup. Purified SmYbeY behaved as a monomer that indistinctly cleaved single- and double-stranded RNA substrates, a unique ability among bacterial endoribonucleases. SmYbeY-mediated catalysis was supported by the divalent metal ions Mg2+, Mn2+ and Ca2+, which influenced in a different manner cleavage efficiency and reactivity patterns, with Ca2+ specifically blocking activity on double-stranded and some structured RNA molecules. SmYbeY loss-of-function compromised expression of core energy and RNA metabolism genes, whilst promoting accumulation of motility, late symbiotic and transport mRNAs. Some of the latter transcripts are known Hfq-binding sRNA targets and might be SmYbeY substrates. Genetic reporter and in vitro assays confirmed that SmYbeY is required for sRNA-mediated down-regulation of the amino acid ABC transporter prbA mRNA. We have thus discovered a bacterial endoribonuclease with unprecedented catalytic features, acting also as gene silencing enzyme.