Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis

In the biosynthesis of lipochitin oligosaccharides (LCOs) theRhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence ofnodA in thenodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which thenodA, nodB andnodC genes are separately present on different plasmids. Efficient nodulation was only obtained ifnodC was present on a low-copy-number vector. Our results confirm the notion thatnodA ofRhizobium leguminosarum biovarviciae is essential for nodulation onVicia. Surprisingly, replacement ofR. l. bv.viciae nodA by that ofBradyrhizobium sp. ANU289 results in a nodulation-minus phenotype onVicia. Further analysis revealed that theBradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of theR. l. bv.viciae nodF E-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion thatnodA is a commonnod gene should be revised.     

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 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.     

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.     

NoIR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis

The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated. A negative control of plasmid-borne nod gene expression is provided by the NoIR repressor encoded by the chromosomal noIR gene. NoIR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod-box (Kondorosi et al., 1989). We demonstrate here that NoIR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host-specific nod genes located downstream of the n2, n3 and n5 nod-boxes. Thus, the nod genes are differentially regulated by NoIR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation. Furthermore, NoIR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine-tuning mechanism for optimal nodulation, specific to host-plant genotypes. In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C₁₆ fatty acids. Expression of noIR was high both in the free-living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin.     

Characterization of the Rhizobium leguminosarum genes nodLMN involved in efficient host-specific nodulation

Three nodulation genes, nodL, nodM and nodN, were isolated from Rhizobium leguminosarum and their DNA sequences were determined. The three genes are in the same orientation as the previously described nodFE genes and the predicted molecular weights of their products are 20,105 (nodL), 65,795 (nodM) and 18,031 (nodN). Analysis of gene regulation using operon fusions showed that nodL, nodM and nodN are induced in response to flavanone molecules and that this induction is nodD-dependent. In addition, it was shown that the nodM and nodN genes are in one operon which is preceded by a conserved 'nod-box' sequence, whereas the nodL gene is in the same operon as the nodFE genes. DNA hybridizations using specific gene probes showed that strongly homologous genes are present in Rhizobium trifolii but not Rhizobium meliloti or Bradyrhizobium japonicum. A mutation within nodL strongly reduced nodulation of peas, Lens and Lathyrus but had little effect on nodulation of Vicia species. A slight reduction in nodulation of Vicia hirsuta was observed with strains carrying mutations in nodM or nodN.     

The Rhizobium etli argC gene is essential for Arginine biosynthesis and nodulation of Phaseolus vulgaris

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.     

nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.     

Production of root hair deformation factors by Rhizobium meliloti nodulation genes in Escherichia coli: HsnD (NodH) is involved in the plant host-specific modification of the NodABC factor

The role of the hsnD (nodH) gene in the determination of the host-specific nodulation ability of Rhizobium meliloti was studied by expressing the common nodulation genes (nodABC) with or without the hsnD gene in Escherichia coli and testing for biological activity on various leguminous plants. In this way, four categories of plants were established. Upon infection with E. coli carrying the nodABC construct, root hair deformation (Had) was detected on clovers while the hsnD gene was additionally needed for the elicitation of the same response on alfalfa and sweet clover. A weak root hair deformation was seen on siratro by inoculation with E. coli harbouring the nodABC genes and was highly increased when hsnD was also introduced. Cowpea and Desmodium did not respond to any of the E. coli strains constructed. Exudates or cytosolicfractions of the respective E. coli derivatives elicited the same root hair deformation as the intact bacteria. These data indicate that not only the nodABC gene products but also the hsnD product are involved in the synthesis of Had factors. Subclones expressing only the nodA, nodB, or nodC genes or the same genes in pairs (nodAB, nodBC, nodAC) did not provide a compound with activity comparable to the NodABC factor, suggesting that all three genes are required for the production of the Had factor which is active on clover. Coinoculation of alfalfa plants with two strains of E. coli, one carrying the nodABC genes and the other expressing only hsnD, or combining exudates or cytosolic fractions from these strains did not result in root hair deformation on alfalfa. These data indicate that the HsnD protein itself or its product is not an additional alfalfa-specific extracellular signal but more likely is enzymatically involved in the modification of the basic compound determined by the nodABC genes.     

beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis

A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the beta-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in the Bacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. coli enzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.     

Acyl-acyl carrier protein is a donor of fatty acids in the NodA-dependent step in biosynthesis of lipochitin oligosaccharides by rhizobia.

NodA controls transfer of a fatty acid in the biosynthesis of lipochitin oligosaccharides by rhizobia. In an in vitro assay, we used de-N-acetylated chitin oligosaccharides substituted with an O-acetyl moiety as acyl acceptor substrates. We show that acyl-acyl carrier protein is used as a donor in NodA-directed fatty acid transfer.     

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.     

Nod factor signaling genes and their function in the early stages of Rhizobium infection

A lipochitosaccharide-based signal molecule that is secreted by Rhizobium, named Nod factor (NF), induces root nodule formation in legumes. This molecule is also essential for the establishment of bacterial infection. Genetic analyses in the legume species Lotus japonicus and Medicago truncatula have led to the identification of many components of the NF signaling cascade. At least three of these genes do not function exclusively in the Rhizobium symbiosis but are also essential for the formation of mycorrhiza, an endosymbiosis found in many higher plant species. Recent studies have advanced our understanding of the functions of NF signaling genes in the Rhizobium infection process and the extent to which these genes are unique to legumes.     

Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems

The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.     

nodT,a positively-acting cultivar specificity determinant controlling nodulation of Trifolium subterraneum by Rhizobium leguminosarum biovar trifolii

Rhizobium leguminosarum biovar trifolii strain TA1 nodulates a range of Trifolium plants including red, white and subterranean clovers. Nitrogen-fixing nodules are promptly initiated on the tap roots of these plants at the site of inoculation. In contrast to these associations, strain TA1 has a Nod^- phenotype on a particular cultivar of subterranean clover called Woogenellup (A.H. Gibson, Aust J Agric Sci 19: (1968) 907–918) where it induces rare, poorly developed, slow-to-appear and ineffective lateral root nodules. By comparing the nodulation gene region of strain TA1 with that of another R. leguminosarum bv. trifolii strain ANU843, which is capable of efficiently nodulating cv. Woogenellup, we have shown that the nodT gene (B.P. Surin et al., Mol Microbiol 4: (1990) 245–252) is essential for nodulation on cv. Woogenellup. The nodT gene is naturally absent in strain TA1. A cosmid clone spanning the entire nodulation gene region of strain TA1 was capable of conferring nodulation ability to R.l. bv. trifolii strains deleted for nodulation genes, but only on cultivars of subterranean clovers nodulated by strain TA1. This shows that cultivar recognition events are, in part, determined by genes in the nodulation region of strain TA1. Complementation studies also indicated that strain TA1 contains negatively-acting genes located on the Sym plasmid and elsewhere, which specifically block nodulation of cv. Woogenellup.     

Identification of two fatty acids in a group determinant ofChlamydia trachomatis

Chlamydia trachomatis releases a hydrophobic, group-reactive antigenic substance that can be isolated from culture media by a new method developed in this laboratory. Octyl-Sepharose binds the free, hydrophobic antigen but not the antigen complexed with specific antibody. Analysis by GLC, of derivatives of samples, demonstrated the presence of C₁₇ and C₁₈₁ fatty acids and showed they were complexed with specific antibody. TLC analyses demonstrated a glycolipid band unique to preparations of isolated antigen. Neither analyses demonstrated similar material in uninfected media. It is suggested that the lipids may be complexed with a polysaccharide heteropolymer containing a form of the unusual hexose, gulose.     

Natural variation in host-specific nodulation of pea is associated with a haplotype of the SYM37 LysM-type receptor-like kinase

Rhizobium leguminosarum bv. viciae, which nodulates pea and vetch, makes a mixture of secreted nodulation signals (Nod factors) carrying either a C18:4 or a C18:1 N-linked acyl chain. Mutation of nodE blocks the formation of the C18:4 acyl chain, and nodE mutants, which produce only C18:1-containing Nod factors, are less efficient at nodulating pea. However, there is significant natural variation in the levels of nodulation of different pea cultivars by a nodE mutant of R. leguminosarum bv. viciae. Using recombinant inbred lines from two pea cultivars, one which nodulated relatively well and one very poorly by the nodE mutant, we mapped the nodE-dependent nodulation phenotype to a locus on pea linkage group I. This was close to Sym37 and PsK1, predicted to encode LysM-domain Nod-factor receptor-like proteins; the Sym2 locus that confers Nod-factor-specific nodulation is also in this region. We confirmed the map location using an introgression line carrying this region. Our data indicate that the nodE-dependent nodulation is not determined by the Sym2 locus. We identified several pea lines that are nodulated very poorly by the R. leguminosarum bv. viciae nodE mutant, sequenced the DNA of the predicted LysM-receptor domains of Sym37 and PsK1, and compared the sequences with those derived from pea cultivars that were relatively well nodulated by the nodE mutant. This revealed that one haplotype (encoding six conserved polymorphisms) of Sym37 is associated with very poor nodulation by the nodE mutant. There was no such correlation with polymorphisms at the PsK1 locus. We conclude that the natural variation in nodE-dependent nodulation in pea is most probably determined by the Sym37 haplotype.