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.     

Immunogold localization of the NodC and NodA proteins of Rhizobium meliloti.

Monospecific, polyclonal antibodies to the nodC and nodA gene products of Rhizobium meliloti were used in combination with immunogold labeling and transmission electron microscopy to localize the NodC and NodA proteins in cultures of R. meliloti. Both NodC and NodA were detected in the cytoplasm and cell envelope in thin sections of free-living rhizobia treated with luteolin, a known inducer of nod gene expression; however, only NodC was detected on cell surfaces when immunolabeling was performed with intact induced cells. In view of biochemical data characterizing NodC as an outer membrane protein with a large extracellular domain, the pattern of immunolabeling on thin sections suggests that NodC is produced on free cytoplasmic ribosomes prior to assembly in the membrane. The pattern of NodA labeling on thin sections is consistent with biochemical data detecting NodA in both soluble and membrane fractions of NodA-overexpressing strains of R. meliloti.     

Nucleotide sequence of Rhizobium meliloti nodulation genes

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.     

Modular structure of the Rhizobium meliloti DctB protein

Abstract To investigate the modular structure of the Rhizobium meliloti dicarboxylic acid sensor protein, DctB, three truncated DctB proteins (DctB4, DctB5 and DctB4G) were constructed, overproduced in Escherichia coli and purified. The DctB4G protein was composed of 446 amino acids of the DctB C-terminus and displayed strong autophosphorylation activity in vitro. This activity was sustained when a further 120 amino acids at the N-terminus of the polypeptide were deleted (DctB5). This protein which has an intact transmitter domain exhibits specific but inefficient phospho-transfer capabilities. Removal of 58 amino acids from the DctB4G C-terminus which included blocks F and G2 of the transmitter domain, rendered the resultant protein (DctB4) incompetent in autophosphorylation. Phosphorylation activity was restored to DctB4 through intramolecular complementation with DctB. Therefore, it would appear that the R. meliloti DctB protein is active as a dimer (or higher order oligomer). Furthermore, the intramolecular complementation experiments indicate that the amino acids 171–291, a predicted periplasmic stretch, play an important role in the dimerization process.     

Localization of nodulation gene on the chromosome of Rhizobium meliloti

Using N-methyl-N'-nitro-N-nitrosoguanidine mutant RM54 of Rhizobium meliloti L5-30 defective in the nodulation process (Nod-) and in the biosynthesis of adenine was obtained. Nod- phenotype of this mutant was not caused by the auxotrophic mutation. The nod gene is located on the chromosome. The wild type strain of R. meliloti and Nod- mutant RM54 harbour two indigenous plasmids having a molecular weight of 90 Mdal and about 300 Mdal.     

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.     

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.     

In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation.

Early stages of nodulation involve the exchange of signals between the bacterium and the host plant. Bacterial nodulation (nod) genes are required for Rhizobium spp. to synthesize lipooligosaccharide morphogens, termed Nod factors. The common nod genes encode enzymes that synthesize the factor core structure, which is modified by host-specific gene products. Here we show direct in vitro evidence that Rhizobium meliloti NodH, a host-specific nodulation gene, catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the terminal 6-O position of Nod factors, and we show substrate requirements for the reaction. Our results indicate that polymerization of the chitooligosaccharide backbone likely precedes sulfation and that sulfation is not absolutely dependent on the presence or the particular structure of the N-acyl modification. NodH sulfation provides a tool for the enzymatic in vitro synthesis of novel Nod factors, or putative Nod factors intermediates, with high specific activity.     

Primary structure of the DNA-binding protein HRm from Rhizobium meliloti

The amino acid sequence of protein HRm, a DNA-binding HU-type protein of 90 residues (Mr 9303), isolated from Rhizobium meliloti, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at arginine and aspartic acid residues. The comparison of the primary structure of protein HRm with that of other HU-type proteins shows that two short sequences, of 7 and 6 residues respectively, located in the median part of the molecule, appear highly conserved and may be important in the function of the protein.     

Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti.

A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.     

Rhizobium nodulation protein NodC is an important determinant of chitin oligosaccharide chain length in Nod factor biosynthesis.

Synthesis of chitin oligosaccharides by NodC is the first committed step in the biosynthesis of rhizobial lipochitin oligosaccharides (LCOs). The distribution of oligosaccharide chain lengths in LCOs differs between various Rhizobium species. We expressed the cloned nodC genes of Rhizobium meliloti, R. leguminosarum bv. viciae, and R. loti in Escherichia coli. The in vivo activities of the various NodC proteins differed with respect to the length of the major chitin oligosaccharide produced. The clearest difference was observed between strains with R. meliloti and R. loti NodC, producing chitintetraose and chitinpentaose, respectively. In vitro experiments, using UDP-[14C]GlcNAc as a precursor, show that this difference reflects intrinsic properties of these NodC proteins and that it is not influenced by the UDP-GlcNAc concentration. Analysis of oligosaccharide chain lengths in LCOs produced by a R. leguminosarum bv. viciae nodC mutant, expressing the three cloned nodC genes mentioned above, shows that the difference in oligosaccharide chain length in LCOs of R. meliloti and R. leguminosarum bv. viciae is due only to nodC. The exclusive production of LCOs which contain a chitinpentaose backbone by R. loti strains is not due to NodC but to end product selection by Nod proteins involved in further modification of the chitin oligosaccharide. These results indicate that nodC contributes to the host specificity of R. meliloti, a conclusion consistent with the results of several studies which have shown that the lengths of the oligosaccharide backbones of LCOs can strongly influence their activities on host plants.     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

Influence of the herbicide phosphinothricin on growth and nodulation capacity of Rhizobium meliloti

  Rhizobium meliloti proved to be sensitive to low concentrations of the herbicide phosphinothricintripeptide (PTT) and its active ingredient phosphinothricin (PT), which was formerly assumed to be non-toxic for most of the bacteria analysed. Growth was more strongly reduced in sterile synthetic media and less reduced in sterile soil; in unsterile soil only a transient growth reduction was detectable. Sensitivity was also observed in five out of eight other species analysed. In all sensitive species tested, spontaneous resistances to PT occurred. Under sterile conditions, PTT and PT reduced rhizobial nodulation rates of PT-resistant alfalfa plants drastically; however, nitrogen fixation in the few nodules that arose was unaffected. Because of the small number of nodules, the overall fixation rate was strongly diminished. In unsterile soil, nodulation and nitrogen fixation rates were not changed, possibly because of the rapid degradation of PTT and PT in the soil. Using a herbicide as model substance it could be demonstrated that the sensitivity of R. meliloti to chemical additives in the soil can be detected by analysing its growth rate, and that even a weak impact can influence its nodulation capacity. R. meliloti has proven to be a fast, easy and sensitive detection system for bacteriostatic components present in the soil. Received: 12 April 1996 / Received revision: 15 July 1996 / Accepted: 18 July 1996     

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Transmembrane homodimerization of receptor-like protein tyrosine phosphatases

Receptor-like protein tyrosine phosphatases (RPTPs) are type I integral membrane proteins. Together with protein tyrosine kinases, RPTPs regulate the phosphotyrosine levels in the cell. Studies of two RPTPs, CD45 and PTPalpha, have provided strong evidence that dimerization leads to inactivation of the receptors, and that the dimerization of PTPalpha involves interactions in the transmembrane domain (TMD). Using the TOXCAT assay, a genetic approach for analyzing TM interactions in Escherichia coli membranes, we show that the TMD of RPTPs interact in the membrane, albeit to different extents. Using fusion proteins of TMDs, we also observe an equilibrium between monomer and dimer in sodium dodecyl sulfate (SDS) micelles. Through a mutational study of the DEP1 TMD, we demonstrate that these interactions are specific. Taken together, our results define a subset of the RPTP family in which TM homodimerization may act as a mediator of protein function.