Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816.

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.     

Rhizobium sp. BR816 produces a complex mixture of known and novel lipochitooligosaccharide molecules

Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development. We report here the LCO structures of the broadhost range strain Rhizobium sp. BR816. The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue. A second acetyl group can be present on the penultimate N-acetylglucosamine from the nonreducing terminus. Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.     

The nifA gene of Rhizobium meliloti is oxygen regulated.

Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels. Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC. The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1. Aerobic induction of nifA can also occur during nitrogen starvation at low pH, but in this case induction is dependent on ntrC and does not lead to P1 activation. The data indicate that reduced oxygen tension is potentially a major trigger for symbiotic activation of nitrogen fixation in Rhizobium species.     

The Rhizobium leguminosarum nodulation gene nodF encodes a polypeptide similar to acyl-carrier protein and is regulated by nodD plus a factor in pea root exudate

The DNA sequence of ~3.5 kb of the nodulation (nod) region of the Rhizobium leguminosarum symbiotic plasmid pRL1JI was determined. Three open reading frames were identified; genes corresponding to these have been called nodD, nodE and nodF.nodD is adjacent to nodA and is transcribed in the opposite direction. The nodF and nodE genes are downstream of, and transcribed in the same direction as, nodD with 667 nucleotides between nodD and nodF and three nucleotides separating nodF and nodE. The induction of the nodFE operon requires the nodD gene product and a component present in plant root exudate. Regions of DNA sequence preceding nodF are similar to those preceding nodA; these sequences may be involved in the regulation of the expression of nodA and nodF. Analysis of nodD revealed an amino acid sequence similar to the predicted DNA-binding domain of known DNA-binding proteins. A protein comparison of the nodF protein showed it to be similar to the acyl-carrier protein from Escherichia coli and barley, especially around the pantothenate-binding region and on this basis it is thought that this protein may be involved in an acyl transfer reaction.     

Inactivation of the nodH gene in Sinorhizobium sp. BR816 enhances symbiosis with Phaseolus vulgaris L

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.     

Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD 1 is preceded by nolE, a gene whose product is secreted from the cytoplasm

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia. Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences. Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.