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Sinorhizobium meliloti can translocate over surfaces. However, little is known about the regulatory mechanisms that control this trait and its relevance for establishing symbiosis with alfalfa plants. To gain insights into this field, we isolated Tn5 mutants of S. meliloti GR4 with impaired surface motility. In mutant strain GRS577, the transposon interrupted the ntrY gene encoding the sensor kinase of the NtrY/NtrX two-component regulatory system. GRS577 is impaired in flagella synthesis and overproduces succinoglycan, which is responsible for increased biofilm formation. The mutant also shows altered cell morphology and higher susceptibility to salt stress. GRS577 induces nitrogen-fixing nodules in alfalfa but exhibits decreased competitive nodulation. Complementation experiments indicate that both ntrY and ntrX account for all the phenotypes displayed by the ntrY::Tn5 mutant. Ectopic overexpression of VisNR, the motility master regulator, was sufficient to rescue motility and competitive nodulation of the transposant. A transcriptome profiling of GRS577 confirmed differential expression of exo and flagellar genes, and led to the demonstration that NtrY/NtrX allows for optimal expression of denitrification and nifA genes under microoxic conditions in response to nitrogen compounds. This study extends our knowledge of the complex role played by NtrY/NtrX in S. meliloti.

Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.

The Sinorhizobium fredii HH103 rkp-3 region has been isolated and sequenced. Based on the similarities between the S. fredii HH103 rkpL, rkpM, rkpN, rkpO, rkpP, and rkpQ genes and their corresponding orthologues in Helicobacter pylori, we propose a possible pathway for the biosynthesis of the S. fredii HH103 K-antigen polysaccharide (KPS) repeating unit. Three rkp-3 genes (rkpM, rkpP, and rkpQ) involved in the biosynthesis of the HH103 KPS repeating unit (a derivative of the pseudaminic acid) have been mutated and analyzed. All the rkp-3 mutants failed to produce KPS and their lipopolysaccharide (LPS) profiles were altered. These mutants showed reduced motility and auto-agglutinated when early-stationary cultures were further incubated under static conditions. Glycine max, Vigna unguiculata (determinate nodule-forming legumes), and Cajanus cajan (indeterminate nodules) plants inoculated with mutants in rkpM, rkpQ, or rkpP only formed pseudonodules that did not fix nitrogen and were devoid of bacteria. In contrast, another indeterminate nodule-forming legume, Glycyrrhiza uralensis, was still able to form some nitrogen-fixing nodules with the three S. fredii HH103 rifampicin-resistant rkp-3 mutants tested. Our results suggest that the severe symbiotic impairment of the S. fredii rkp-3 mutants with soybean, V. unguiculata, and C. cajan is mainly due to the LPS alterations rather than to the incapacity to produce KPS.

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall-degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis.

The microsymbiont of alfalfa, Sinorhizobium meliloti, possesses phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine as major membrane phospholipids, when grown in the presence of sufficient accessible phosphorus sources. Under phosphate-limiting conditions of growth, S. meliloti replaces its phospholipids by membrane lipids that do not contain any phosphorus in their molecular structure and, in S. meliloti, these phosphorus-free membrane lipids are sulphoquinovosyl diacylglycerols (SL), ornithine-containing lipids (OL), and diacylglyceryl-N,N,N-trimethylhomoserines (DGTS). In earlier work, we demonstrated that neither SL nor OL are required for establishing a nitrogen-fixing root nodule symbiosis with alfalfa. We now report the identification of the two structural genes btaA and btaB from S. meliloti required for DGTS biosynthesis. When the sinorhizobial btaA and btaB genes are expressed in Escherichia coli, they cause the formation of DGTS in this latter organism. A btaA-deficient mutant of S. meliloti is unable to form DGTS but can form nitrogen-fixing root nodules on alfalfa, demonstrating that sinorhizobial DGTS is not required for establishing a successful symbiosis with the host plant. Even a triple mutant of S. meliloti, unable to form any of the phosphorus-free membrane lipids SL, OL, or DGTS is equally competitive for nodule occupancy as the wild type. Only under growth-limiting concentrations of phosphate in culture media did mutants that could form neither OL nor DGTS grow to lesser cell densities.

Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the trichloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3' end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.

Rhizobium meliloti strain GRM8 is able to transform ornithine into proline by means of an ornithine cyclodeaminase and, therefore, has the ability to use either of these amino acids as its sole carbon and nitrogen source. By Tn5 insertion mutagenesis we obtained a GRM8 mutant derivative strain (LM1) unable to catabolize either ornithine or proline. DNA hybridization studies showed that the LM1 mutant carries a single Tn5 insertion within a chromosomally located gene that, as deduced from a partial nucleotide sequence, encodes a proline dehydrogenase (ProDH). Enzymatic assays confirmed the lack of ProDH activity in cell extracts of strain LM1 and revealed that production of this enzyme is inducible in the parental strain by proline and ornithine. Ultrastructural nodule microscopy analysis, acetylene reduction assays, and dry-weight determinations of nodulated alfalfa plants showed no obvious defect in the nitrogen fixation process of the ProDH- mutant LM1. However, nodulation tests and competition assays demonstrated that in R. meliloti ProDH is required for nodulation efficiency and competitiveness on alfalfa roots.

The nfe genes located on the large plasmid pRmeGR4b are involved in the nodulation efficiency and competitiveness of Rhizobium meliloti GR4 on alfalfa roots. One hundred twenty-eight base-pairs downstream of nfe2 gene we found an open reading frame designated ORFC, 970 bp long and potentially coding for a 320 amino acid long protein. The amino acid sequence of the putatively encoded ORFC product shows similarity with ornithine cyclodeaminase (OCD) of Agrobacterium tumefaciens an unusual enzyme that converts ornithine into proline. The gene product of ORFC was identified as a 37-kDa protein by in vitro-coupled transcription-translation and in vivo by the T7 RNA polymerase/promoter system. DNA hybridization studies showed that strain GR4 carries a single copy of the ocd-like gene. No homologous sequences to GR4 ORFC DNA were found in other R. meliloti strains or Rhizobium spp. assayed. Furthermore, a GR4 derivative mutant obtained by plasmid disruption of ORFC showed an impaired nodulation efficiency as compared to that of the wild-type strain GR4. Thus, the former locus should be considered a novel nfe gene. We propose to rename the nfe genes, nfe1, 2 and ORFC as nfeA, B, and D, respectively.