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The Sinorhizobium meliloti RNA chaperone Hfq mediates symbiosis of S. meliloti and alfalfa.

ABSTRACT: There exist commonalities between symbiotic Sinorhizobium meliloti and pathogenic Brucella bacteria in terms of extensive gene synteny and the requirements for intracellular survival in their respective hosts. The RNA chaperone Hfq is essential for virulence for several bacterial groups, including Brucella; however, its role in S. meliloti has not been investigated. Our studies of an S. meliloti loss-of-function hfq mutant have revealed that Hfq plays a key role in the establishment of the symbiosis between S. meliloti and its host Medicago sativa. S. meliloti Hfq is involved in controlling the population density under a free-living state and affects the growth parameters and nodulation. An hfq mutant poorly colonizes the infection threads that are necessary for the bacteria to invade the developing nodule. An hfq mutant is severely impaired in its ability to invade plant cells within the nodule, which leads to the formation of small, ineffective nodules unable to fix nitrogen. In culture, the hfq mutant did not accumulate transcripts of nifA, which encodes a key regulator necessary for nitrogen fixation. Hfq may be involved in regulation of several proteins relevant to hfq mutant phenotypes. The crucial role of Hfq in symbiosis suggests that small regulatory RNAs are important for its interactions with its plant host.

SUBMITTER: Barra-Bily L

PROVIDER: S-EPMC2832522 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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The Sinorhizobium meliloti RNA chaperone Hfq mediates symbiosis of S. meliloti and alfalfa.

Barra-Bily Lise L Pandey Shree P SP Trautwetter Annie A Blanco Carlos C Walker Graham C GC

Journal of bacteriology 20100115 6

There exist commonalities between symbiotic Sinorhizobium meliloti and pathogenic Brucella bacteria in terms of extensive gene synteny and the requirements for intracellular survival in their respective hosts. The RNA chaperone Hfq is essential for virulence for several bacterial groups, including Brucella; however, its role in S. meliloti has not been investigated. Our studies of an S. meliloti loss-of-function hfq mutant have revealed that Hfq plays a key role in the establishment of the symbi ...[more]

PMID: 20081033

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Project description: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 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.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.

Project description:Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.

Dataset Information

The Sinorhizobium meliloti RNA chaperone Hfq mediates symbiosis of S. meliloti and alfalfa.

Publications

The Sinorhizobium meliloti RNA chaperone Hfq mediates symbiosis of S. meliloti and alfalfa.

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