Homology of Rhizobium meliloti NodC to polysaccharide polymerizing enzymes.

Rhizobium bacteria form nitrogen-fixing nodules on legume roots. As part of the nodulation process, they secrete Nod factors that are beta-1,4-linked oligomers of N-acetylglucosamine. These factors depend on nodulation (nod) genes, but most aspects of factor synthesis are not yet known. We show here that one gene, nodC, shows striking similarity to genes encoding proteins known to be involved in polysaccharide synthesis in yeast and bacteria, specifically chitin and cellulose synthases, as well as a protein with unknown function in Xenopus embryos, DG42. This similarity is consistent with a role for the NodC protein in the formation of the beta-1,4-linkage in Nod factors.     

The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids

Rhizobia synthesize mono- N -acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation ( nod ) genes, including the nodABC genes present in all rhizobial species. The N -acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti , an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω-1) hydroxylated fatty acid, whereas in Rhizobium tropici , a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA , and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N -acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.     

Transmembrane orientation and receptor-like structure of the Rhizobium meliloti common nodulation protein NodC

The 46.8-kd NodC protein of Rhizobium meliloti is a membrane protein, essential for nodule formation. Gene fusions of nodC to a portion of the λ cI repressor gene were used to define the membrane-anchor domain which is necessary for membrane insertion of the NodC protein into the membrane. The transmembrane orientation of NodC was confirmed by surface-specific radiolabeling and proteolysis experiments. A highly hydrophobic transmembrane-anchor domain was found near the carboxyl terminus, separating a large extracellular domain which contains an unusual cysteine-rich cluster from a short putative intracellular domain. Cross-linking studies showed that the NodC protein exists in the membrane probably as a dimer. The domain structure of the NodC protein shows striking similiarities with cell surface receptors. In nodules of various legumes a truncated form of the NodC protein was detected. The processed NodC was associated with the bacteroids and the amount of this protein increased during nodule development.     

Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosac-charides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three. In vivo radioactive labelling experiments, in which D-[1-¹⁴C]-glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo-chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N-acetyl group on the non-reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O-acetyl substituent on the non-reducing terminal sugar residue (designated NodBCL metabolites). The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP-N-acetyl-D-[U-¹⁴C]-glucosamine was added using thin-layer chromatography. The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites. The same results were obtained when the NodB and NodC proteins were produced separately in two different strains. On the basis of these and other recent results, we propose that NodB is a chitin oilgosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase and, by default, NodA is involved in lipie attachment.     

The Rhizobium, Bradyrhizobium, and Azorhizobium NodC proteins are homologous to yeast chitin synthases.

The nodABC genes of rhizobia are essential for the synthesis of lipo-oligosaccharidic (N-acylated chitin oligomers) nodulation signals. nodC gene products from Rhizobium, Bradyrhizobium, and Azorhizobium exhibit extensive homology with chitin synthases, suggesting that the NodC proteins are involved in the synthesis of the chitin oligomer backbone by catalyzing the beta-1,4-linkage between N-acetyl-D-glucosamine residues.     

High-frequency transformation of Rhizobium meliloti.

Transformation of R factor RP4 and its derivative pRK290 from Escherichia coli to Rhizobium meliloti is reported. The efficiency of transformation was in the range of 10(-5) per viable cell. In addition, chromosomal DNA prepared from one R. meliloti strain resistant to streptomycin was transferred to the isoleucine-valine-requiring mutant susceptible to streptomycin.     

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.     

Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.     

Thymidine incorporation into Rhizobium meliloti.

Thymidine is rapidly catabolized to thymine, beta-aminoisobutyric acid, and carbon dioxide by Rhizobium meliloti cells. The incorporation of labelled thymidine into the DNA of R. meliloti cells can be enhanced by the addition of low concentrations (10-20 micrograms/mL) of deoxyadenosine or other nucleosides (adenosine, uridine, guanosine). However, at high concentrations ( greater than 50 micrograms/mL) these compounds inhibit thymidine incorporation. Conditions to obtain highly radioactive DNA of Rhizobium are described.     

Rhizobium meliloti mutants that overproduce the R. meliloti acidic calcofluor-binding exopolysaccharide.

The acidic Calcofluor-binding exopolysaccharide of Rhizobium meliloti Rm1021 plays one or more critical roles in nodule invasion and possibly in nodule development. Two loci, exoR and exoS, that affect the regulation of synthesis of this exopolysaccharide were identified by screening for derivatives of strain Rm1021 that formed mucoid colonies that fluoresced extremely brightly under UV light when grown on medium containing Calcofluor. The exopolysaccharide produced in large quantities by the exoR95::Tn5 and exoS96::Tn5 strains was indistinguishable from that produced by the parental strain Rm1021, and its synthesis required the function of at least the exoA, exoB, and exoF genes. Both the exoR and exoS loci were located on the chromosome, and the exo96::Tn5 mutation was 84% linked to the trp-33 mutation by phi M12 transduction. Synthesis of the Calcofluor-binding exopolysaccharide by strain Rm1021 was greatly stimulated by starvation for ammonia. In contrast, the exoR95::Tn5 mutant produced high levels of exopolysaccharide regardless of the presence or absence of ammonia in the medium. The exoS96::Tn5 mutant produced elevated amounts of exopolysaccharide in the presence of ammonia, but higher amounts were observed after starvation for ammonia. The presence of either mutation increased the level of expression of exoF::TnphoA and exoP::TnphoA fusions (TnphoA is Tn5 IS50L::phoA). Analyses of results obtained when alfalfa seedlings were inoculated with the exoR95::Tn5 strain indicated that the mutant strain could not invade nodules. However, pseudorevertants that retained the original exoR95::Tn5 mutation but acquired unlinked suppressors so that they produced an approximately normal amount of exopolysaccharide were able to invade nodules and fix nitrogens. The exoS95::Tn5 strain formed Fix+ nodules, although some minor variability was observed.     

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.     

Phosphoglucose isomerase mutant of Rhizobium meliloti.

A mutant strain of complex phenotype was selected in Rhizobium meliloti after nitrosoguanidine mutagenesis. It failed to grow on mannitol, sorbitol, fructose, mannose, ribose, arabitol, or xylose, but grew on glucose, maltose, gluconate, L-arabinose, and many other carbohydrates. Assay showed the enzyme lesion to be in phosphoglucose isomerase (pgi), and revertants, which were of normal growth phenotype, contained the enzyme again. Nonpermissive substrates such as fructose and xylose prevented growth on permissive ones such as L-arabinose, and in such situations there was high accumulation of fructose 6-phosphate. The mutant strain had about 20% as much exopolysaccharide as the parent. Nitrogen fixation by whole plants was low and delayed when the mutant strain was the inoculant.     

alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti.

A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth.     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Succinate dehydrogenase mutant of Rhizobium meliloti.

A succinate dehydrogenase mutant strain of Rhizobium meliloti was isolated after nitrosoguanidine mutagenesis. It failed to grow on succinate, glutamate, acetate, pyruvate, or arabinose but grew on glucose, sucrose, fructose, and other carbohydrates. The mutant strain showed delayed nodulation of lucerne plants, and the nodules were white and ineffective. A spontaneous revertant strain of normal growth phenotype induced red and effective nodules.     

Electrophoretic separation of the three Rhizobium meliloti replicons.

The megaplasmids and the chromosome from the bacterium Rhizobium meliloti 1021 were separated in preparative quantities by using transverse alternating-field gel electrophoresis. The genetic content of each electrophoretically separated band was determined by Southern hybridization with replicon-specific probes and by comparison with Agrobacterium tumefaciens transconjugants harboring either pSym-a or pSym-b megaplasmids. Pulsed-field gel electrophoresis analyses of PacI (5'-TTAATTAA-3') and SwaI (5'-ATTTAAAT-3') digests of the whole genome and of the separated replicons were used to calculate genome sizes in two R. meliloti strains. In these strains, PacI digestion yielded only four fragments for the entire genome. The sizes of the PacI fragments from R. meliloti 1021 in megabase pairs (Mb) were 3.32 +/- 0.30, 1.42 +/- 0.13, 1.21 +/- 0.10, and 0.55 +/- 0.08, for a total genome size of 6.50 +/- 0.61 Mb. Southern hybridization with replicon-specific probes assigned one PacI fragment to the chromosome of R. meliloti 1021, one to pRme1021a, and two to pRme1021b. PacI digestion of A. tumefaciens pTi-cured, pSym transconjugants confirmed these assignments. In agreement with PacI data, the addition of the six SwaI fragments from R. meliloti 1021 gave a genome size of 6.54 +/- 0.43 Mb. pRme1021a was calculated to be 1.42 +/- 0.13 Mb, 1.34 +/- 0.09 Mb, and 1.38 +/- 0.12 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021a, respectively. pRme1021b was calculated to be 1.76 +/- 0.18 Mb, 1.65 +/- 0.10 Mb, and 1.74 +/- 0.13 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021B, respectively. The R. meliloti 1021 chromosome was calculated to be 3.32 +/- 0.30 Mb, 3.55 +/- 0.24 Mb, and 3.26 +/- 0.46 Mb on the basis of PacI data, SwaI data, and the migration of uncut chromosome, respectively.