Search

In the biosynthesis of lipochitin oligosaccharides (LCOs) the Rhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence of nodA in the nodABCIJ 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 the nodA, nodB and nodC genes are separately present on different plasmids. Efficient nodulation was only obtained if nodC was present on a low-copy-number vector. Our results confirm the notion that nodA of Rhizobium leguminosarum biovar viciae is essential for nodulation on Vicia. Surprisingly, replacement of R. l. by viciae nodA by that of Bradyrhizobium sp. ANU289 results in a nodulation-minus phenotype on Vicia. Further analysis revealed that the Bradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of the R. l. by, viciae nodFE-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion that nodA is a common nod gene should be revised.

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.

Signal molecules which determine the host specificity of rhizobia for particular leguminous plants include flavonoids secreted by the host plant roots and the bacterial lipo-chitin oligosaccharides (LCOs). This presentation will mainly focuss on the role of the LCOs. For instance, a highly unsaturated fattyl moiety of LCOs plays a major role in the specificity of rhizobia which associate with indeterminate-nodulating plants. The genes nodF (acyl carrier protein), nodE (β-ketoacylsynthase) and nodA (acyl transferase) are involved in the production of LCOs containing such a special fatty acids. For determinate nodulating plants a fucosyl residue (located at the reducing N-acetylglucosamine residue of LCOs) plays a major role in determining specificity. The nodZ gene which is present in various rhizobia such as Bradyrhizobium japonicum, Rhizobium loti and R.etli, is a fucosyltransferase involved in the addition of this fucose (Lopez-Lara et al, 1996). The role of other determinants of host specificity, such as nodX and nolL will be discussed.

Thin-layer chromatographic analysis of extracts of D-[1-14C]glucosamine-labelled rhizobia was used to analyze the effects of nodI, nodJ, and nodT on secretion of lipochitin oligosaccharide (LCO) signal molecules. Secretion was analyzed by comparing quantities of radiolabelled LCOs present in the cellular and spent growth medium fractions. A second rapid and sensitive method was introduced to estimate the secreted LCO fractions by using D-[1-14C]glucosamine-labelled cells grown in medium supplemented with chitinase. At various times after induction of LCO synthesis, the quantity of degradation products of LCOs was compared with the amount of nondegraded LCOs. In wild-type strains of Rhizobium leguminosarum biovars viciae and trifolii the nodI and nodJ genes (but not the nodT gene) strongly enhance the secretion of LCOs during the first 5 h after the induction of LCO synthesis. In LCO-overproducing strains the enhancement of secretion was observed only during the first 3 h after induction. At times later than 5 h after induction, a significant influence of the presence of the nodI and nodJ genes on LCO secretion was detectable neither in the wild type nor in LCO-overproducing strains. By using plasmids in which the nodI and nodJ genes are cloned separately under control of a flavonoid-inducible promoter, it was shown that both genes are needed for a wild-type level of LCO secretion. Therefore, these results demonstrate that nodI and nodJ play a role in determining the efficiency of LCO secretion.

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosaccharides. 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 oligosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase and, by default, NodA is involved in lipid attachment.

All tested strains of the genera Rhizobium,Bradyrhizobium. and Azorhizobium are able to produce lipo-oligosaccharide signals upon activation of the flavonoid-inducible nod genes. R.leguminosarum was chosen as a model system to study the biochemical function of the Nod proteins in the synthesis of the lipooligosaccharides. In R.leguminosarum bv. viciae, the operons nodABC and nodFEL encode for the Nod proteins which are minimally required for synthesis of the wild type lipo-oligosaccharides and have therefore been studied in most detail. The NodE protein determines host-specificity by its involvement in the synthesis of highly unsaturated fatty acid moieties. The role of the lipo-oligosaccharides in the host plant will be emphasized. Results are presented which tempt us to believe that lipo-oligosaccharides, similar to those produced by rhizobia, are naturally occurring in the uninfected plant

In a Sym plasmid-cured strain of R.leguminosarum the presence of the NodA,B,C,F,E,L proteins is sufficient for production and secretion of the wild type lipo-oligosaccharides. Using PCR technology we have constructed plasmids of various incompatibility groups (IncW, IncP, IncQ) which contain the separated single genes, under control of a common promoter (nodA- or T7-derived) and a synthetic ribosomal binding site. Rhizobium strains harbouring various combinations of the single nod gene-containing plasmids were tested for the production of lipooligosaccharides and the results show that: (i) The absence of either of the NodA, NodB or NodC proteins prevents the production of lipo-oligosaccharide molecules, (ii) absence of NodF or NodE proteins prevents the production of lipooligosaccharides which contain a highly unsaturated (C18:4) fatty acyl moiety, (iii) absence of the NodL protein prevents the addition of the O-acetyl group on the non-reducing terminal sugar. The biochemical function of these Nod proteins is further studied by the analysis of biosynthetic intermediates of the lipooligosaccharides in strains producing the various combinations of Nod proteins. Furthermore, we have set up assays for in vitro biochemical activity of the Nod proteins at present focusing on NodA, NodB, NodC and NodL. The obtained results suggest that NodC functions as an N-acetylglucosaminyltransferase involved in the synthesis of the sugar backbone whereas NodA and NodB are involved in the de-N-acetylation and/or acylation of the non-reducing terminal Nacetylglucosamine. The NodL protein is an O-acetyl transferase which can use non-O-acetylated lipo-oligosaccharides or chitin oligomers as substrates.

A previously described (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988) Sym plasmid-dependent, naringenin-inducible 50-kilodalton protein of Rhizobium leguminosarum biovar viciae is further characterized in this paper. The protein was overproduced by constructing a strain containing multiple copies of the R. meliloti nodD gene, which facilitated its purification. An antiserum was used to screen Tn5 insertion mutants located in the pRL1JI region found to be responsible for the production of the 50-kilodalton protein. These inserts define a new nod locus left of the nod genes identified previously. Mutations in this region affect the nodulation ability in a way which is dependent on the bacterial background as well as on the host plant. The mutants nodulate normally in a strain RBL1532 (R. leguminosarum biovar viciae strain 248, cured of its Sym plasmid) background on all three tested host plant species. In contrast, in a strain RBL5045 (R. leguminosarum biovar trifolii strain RCR5, cured of its Sym plasmid) background, nodulation on Vicia sativa is severely impaired, whereas nodulation on Vicia hirsuta and Trifolium subterraneum is apparently unaltered.

The region of the Rhizobium leguminosarum biovar viciae Sym plasmid pRL1JI, responsible for the production and secretion of a previously described 50-kilodalton protein (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988), was cloned and its nucleotide sequence was determined. A new nod gene, nodO, preceded by a poorly conserved nod box, was identified and its transcriptional start site was determined. Comparison of its predicted protein product with the N-terminal amino acid sequence of the isolated secreted protein showed that nodO is the structural gene of this protein, although the nucleotide sequence predicted a protein only 30,002 daltons in size. This comparison also showed that the secreted protein is not the product of N-terminal processing of a larger precursor. A conventional N-terminal signal sequence was not detected in the NodO protein. The NodO protein has significant homology with a part (residues 720 to 920) of the hemolysin protein (HlyA) of Escherichia coli. Analysis of the transcriptional regulation of the nodO gene revealed that, in contrast with other nod promoters in this species, activity of the nodO promoter is greatly enhanced in the presence of multiple copies of the nodD gene.