The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa.

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.

Polychlorinated biphenyl rhizoremediation by Pseudomonas fluorescens F113 derivatives, using a Sinorhizobium meliloti nod system to drive bph gene expression.

Rhizoremediation of organic chemicals requires high-level expression of biodegradation genes in bacterial strains that are excellent rhizosphere colonizers. Pseudomonas fluorescens F113 is a biocontrol strain that was shown to be an excellent colonizer of numerous plant rhizospheres, including alfalfa. Although a derivative of F113 expressing polychlorinated biphenyl (PCB) biodegradation genes (F113pcb) has been reported previously, this strain shows a low level of bph gene expression, limiting its rhizoremediation potential. Here, a high-level expression system was designed from rhizobial nod gene regulatory relays. Nod promoters were tested in strain F113 by using beta-galactosidase transcriptional fusions. This analysis showed that nodbox 4 from Sinorhizobium meliloti has a high level of expression in F113 that is dependent on an intact nodD1 gene. A transcriptional fusion of a nodbox cassette containing the nodD1 gene and nodbox 4 fused to a gfp gene was expressed in the alfalfa rhizosphere. The bph operon from Burkholderia sp. strain LB400 was cloned under the control of the nodbox cassette and was inserted as a single copy into the genome of F113, generating strain F113L::1180. This new genetically modified strain has a high level of BphC activity and grows on biphenyl as a sole carbon and energy source at a growth rate that is more than three times higher than that of F113pcb. Degradation of PCBs 3, 4, 5, 17, and 25 was also much faster in F113L::1180 than in F113pcb. Finally, the modified strain cometabolized PCB congeners present in Delor103 better than strain LB400, the donor of the bph genes used.

MucR and mucS activate exp genes transcription and galactoglucan production in Sinorhizobium meliloti EFB1.

When grown under standard conditions, Sinorhizobium meliloti EFB1 simultaneously produces two acidic exopolysaccharides, succinoglycan and galactoglucan, yielding very mucoid colonies. In this strain, MucR is essential for galactoglucan synthesis. A mutation in the mucS gene resulted in less mucoid colonies than in the wild-type EFB1. This mucS- strain was complemented to the wild-type phenotype by the cloned mucS gene, indicating that mucS is necessary for a wild-type level of galactoglucan production. Reverse transcription-polymerase chain reaction analysis of exp genes, which encode the pathway for galactoglucan production, in EFB1 and in the mutants affected in mucS, mucR, and both genes simultaneously, showed that MucS is a transcriptional activator of the exp genes but does not affect its own transcription. Furthermore, MucR is necessary for mucS transcriptional activation. As introduction of a cloned mucS gene in a mucR- strain yielded colonies less mucoid than the wild type, MucR could also activate exp genes transcription through other pathways. Deletion analysis of the expE promoter showed a region important for transcription and MucS activation. This region, containing a palindrome, is present in the putative expA, expC, expD, and expE promoters but not in the mucS promoter, suggesting that it is the target for MucS. A mucR-mucS- mutant, which does not produce galactoglucan, was impaired in competitive nodulation of alfalfa in soil microcosms, indicating another possible role for this exopolysaccharide in symbiosis.