title: The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  Two independent S. meliloti mutants, 2011-3.4 and 1021Deltahfq, 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 1021Deltahfq 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 1021Deltahfq 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, 18008 Granada, Spain.
authors.forename: Omar
authors.initials: O
authors.lastname: Torres-Quesada
authors.forename: Roke I
authors.initials: RI
authors.lastname: Oruezabal
authors.forename: Alexandra
authors.initials: A
authors.lastname: Peregrina
authors.forename: Edgardo
authors.initials: E
authors.lastname: Jofré
authors.forename: Javier
authors.initials: J
authors.lastname: Lloret
authors.forename: Rafael
authors.initials: R
authors.lastname: Rivilla
authors.forename: Nicolás
authors.initials: N
authors.lastname: Toro
authors.forename: José I
authors.initials: JI
authors.lastname: Jiménez-Zurdo
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002244:Carbon / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D035001:Host Factor 1 Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000455:Medicago sativa / Q000382:microbiology*; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009586:Nitrogen Fixation; D010641:Phenotype; D022661:RNA, Untranslated / Q000378:metabolism; D053204:Root Nodules, Plant / Q000382:microbiology; D016415:Sequence Alignment; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013559:Symbiosis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D035001:Host Factor 1 Protein; C061338:NifA protein, Bacteria; D022661:RNA, Untranslated; D014157:Transcription Factors; C066628:FixK protein, Bacteria; D002244:Carbon