High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234.

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

Molecular basis of symbiosis between Rhizobium and legumes.

Access to mineral nitrogen often limits plant growth, and so symbiotic relationships have evolved between plants and a variety of nitrogen-fixing organisms. These associations are responsible for reducing 120 million tonnes of atmospheric nitrogen to ammonia each year. In agriculture, independence from nitrogenous fertilizers expands crop production and minimizes pollution of water tables, lakes and rivers. Here we present the complete nucleotide sequence and gene complement of the plasmid from Rhizobium sp. NGR234 that endows the bacterium with the ability to associate symbiotically with leguminous plants. In conjunction with transcriptional analyses, these data demonstrate the presence of new symbiotic loci and signalling mechanisms. The sequence and organization of genes involved in replication and conjugal transfer are similar to those of Agrobacterium, suggesting a recent lateral transfer of genetic information.

Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 nod factors.

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a N-linked methyl group on the glucosaminyl terminus. Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.

Organization of host-inducible transcripts on the symbiotic plasmid of Rhizobium sp. NGR234.

In a systematic approach to identify genes involved in the early steps of the legume-Rhizobium symbiosis, we studied transcription patterns of symbiotic plasmid-borne loci. A competitive hybridization procedure was used to identify DNA restriction fragments carrying genes whose expression is enhanced by plant root exudates or by purified flavonoids. Fragments containing induced genes were then located on the physical map of the 500 kb pNGR234a. New inducible loci as well as previously described genes were identified and their time course of induction determined. After initial induction, transcription of loci such as nodABC and the host-specificity genes nodSU decreased to undetectable levels 24 h after incubation with purified flavonoids. In contrast, expression of other loci is detectable only after several hours of induction. Surprisingly, many genes remained transcribed in the nodD1- mutant suggesting the presence of other flavonoid-dependent activators in NGR234. The hsnl region, which is involved in host specificity, was shown to carry several inducible but independently regulated transcripts. Sequencing analysis revealed several open reading frames whose products, based on sequence similarities, may be involved in L-fucose metabolism and its adjunction to the Nod factors.

Subtraction hybridisation and shot-gun sequencing: a new approach to identify symbiotic loci.

Traditionally, new loci involved in the Rhizobium-legume symbiosis have been identified by transposon mutagenesis and/or complementation. Wide dispersal of the symbiotic loci in Rhizobium species NGR234, as well as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques. As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, subtractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of most nodulation genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmid library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonoids. To target genes not present in the closely related strain R. fredii USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation. Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins. Amongst these, a symbiotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.

Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp. strain NGR234 to nodulate Leucaena spp.

Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod-box promoter from NGR234 are sufficient to confer this extended host-range phenotype to L. leucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod-box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5'-end of nodS of USDA257. Consequently, the deduced NodS protein of USDA257 is shorter than its counterpart from NGR234, and the distance between the nod-box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod-box and extends into the upstream leader sequence. Transcriptional fusions with both nod-boxes confirmed that the promoter from NGR234 is flavonoid-inducible, and that the nod-box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS-containing Xhol fragment as hybridization probe. Flavonoid-induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRI fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives of Bradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild-type, leucaena-nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species.