Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia.

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.

Construction and environmental release of a Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodulation.

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA- mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492-498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.

Sinorhizobium meliloti putA gene regulation: a new model within the family Rhizobiaceae.

Proline dehydrogenase (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. In Sinorhizobium meliloti, as in other microorganisms, the putA gene is transcriptionally activated in response to proline. In Rhodobacter capsulatus, Agrobacterium, and most probably in Bradyrhizobium, this activation is dependent on an Lrp-like protein encoded by the putR gene, located immediately upstream of putA. Interestingly, sequence and genetic analysis of the region upstream of the S. meliloti putA gene did not reveal such a putR locus or any other encoded transcriptional activator of putA. Furthermore, results obtained with an S. meliloti putA null mutation indicate the absence of any proline-responsive transcriptional activator and that PutA serves as an autogenous repressor. Therefore, the model of S. meliloti putA regulation completely diverges from that of its Rhizobiaceae relatives and resembles more that of enteric bacteria. However, some differences have been found with the latter model: (i) S. meliloti putA gene is not catabolite repressed, and (ii) the gene encoding for the major proline permease (putP) does not form part of an operon with the putA gene.

Multicopy vectors carrying the Klebsiella pneumoniae nifA gene do not enhance the nodulation competitiveness of Sinorhizobium meliloti on alfalfa.

It has been reported that Sinorhizobium meliloti strains harboring IncQ and IncP multicopy vectors containing constitutively expressed Klebsiella pneumoniae nifA exhibit an increase in nodulation competitiveness on alfalfa (J. Sanjuan and J. Olivares, Mol. Plant-Microbe Interact. 4:365-369, 1991). In our efforts to understand the mechanisms involved, in this work, we have found that the observed enhancement on nodulation competitiveness by IncQ derivatives carrying K. pneumoniae nifA was not dependent on the plasmid-borne nifA activity but on the sensitivity of nonresistant strains to the streptomycin carried over from growth cultures. Furthermore, it was also determined that the nifA of K. pneumoniae on an IncP vector does not have an effect on competitiveness.

Characterization of a Rhizobium meliloti proline dehydrogenase mutant altered in nodulation efficiency and competitiveness on alfalfa roots.

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.

Serine residue 45 of nodulation protein NodF from Rhizobium leguminosarum bv. viciae is essential for its biological function.

A system for testing the role of the Rhizobium nodF gene in the production of host-specific lipochitin oligosaccharides and in nodulation was developed. We show that a mutant nodF gene, in which the codon for serine residue 45 was changed to that for threonine, still expresses NodF, which, however, is no longer functional.