Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and consequences for nodule development.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide. Mutants that fail to synthesize this exopolysaccharide are also unable to nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp. strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The background genetics and nod genes of Rhizobium sp. strain NGR234 are retained, but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. These exo genes were replaced with genes required for the synthesis of succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic manipulation, the ability of these hybrids to synthesize exopolysaccharide was restored, but the structure was that of succinoglycan and not that of Rhizobium sp. strain NGR234. The replacement genes were contained on a cosmid which encoded the entire known R. meliloti exo gene cluster, with the exception of exoB. Cosmids containing smaller portions of this exo gene cluster did not restore exopolysaccharide production. The presence of succinoglycan was indicated by staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant containing the R. meliloti exo genes produced multimers of the succinoglycan repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer. The deletion mutant therefore appeared to lack a function that affects the multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234 background. Although these hybrid strains produced succinoglycan, they were still able to induce the development of an organized nodule structure on L. leucocephala. The resulting nodules did not fix nitrogen, but they did contain infection threads and bacteroids within plant cells. This clearly demonstrated that a heterologous acidic exopolysaccharide structure was sufficient to enable nodule development to proceed beyond the developmental barrier imposed on mutants of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic exopolysaccharide.     

Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum.     

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.     

Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses.

To investigate the role of dicarboxylate transport in nitrogen-fixing symbioses between Rhizobium and tropical legumes, we made a molecular genetic analysis of the bacterial transport system in Rhizobium sp. NGR234. This braod host range strain fixes nitrogen in association with evolutionarily divergent legumes. Two dicarboxylate transport systems were cloned from Rhizobium NGR234. One locus was chromosomally located, whereas the other was carried on the symbiotic plasmid (pSym) and contained a dctA carrier protein gene, which was analyzed in detail. Although the DNA and derived amino acid sequences of the structural gene were substantially homologous to that of R. meliloti, its promoter sequences was quite distinct, and the upstream sequence also exhibited no homology to dctB, which is found at this position in R. meliloti. A site-directed internal deletion mutant in dctA of NGR234 exhibited a (unique) exclusively symbiotic phenotype that could grow on dicarboxylates ex planta, but could not fix nitrogen in planta. This phenotype was found for tested host plants of NGR234 with either determinate- or indeterminate-type nodules, confirming for the first time that symbiosis-specific uptake of dicarboxylates is a prerequisite for nitrogen fixation in tropical legume symbioses.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

An evolutionary hot spot: the pNGR234b replicon of Rhizobium sp. strain NGR234

Rhizobium sp. strain NGR234 has an exceptionally broad host range and is able to nodulate more than 112 genera of legumes. Since the overall organization of the NGR234 genome is strikingly similar to that of the narrow-host-range symbiont Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the obvious question is why are the spectra of hosts so different? Study of the early symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide obvious answers. Yet, both rhizobia also possess second megaplasmids that bear, among many other genes, those that are involved in the synthesis of extracellular polysaccharides (EPSs). EPSs are involved in fine-tuning symbiotic interactions and thus may help answer the broad- versus narrow-host-range question. Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open reading frames (ORFs). Comparisons revealed 19 conserved gene clusters with high similarity to R. meliloti, suggesting that a minimum of 28% (158 ORFs) of the genetic information may have been acquired from a common ancestor. The largest conserved cluster carried the exo and exs genes and contained 31 ORFs. In addition, nine highly conserved regions with high similarity to Agrobacterium tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain MAFF303099, as well as two conserved clusters that are highly homologous to similar regions in the plant pathogen Erwinia carotovora, were identified. Altogether, these findings suggest that >/==" BORDER="0">40% of the pNGR234b genes are not strain specific and were probably acquired from a wide variety of other microbes. The presence of 26 ORFs coding for transposases and site-specific integrases supports this contention. Surprisingly, several genes involved in the degradation of aromatic carbon sources and genes coding for a type IV pilus were also found.     

Two genes that regulate exopolysaccharide production in Rhizobium meliloti.

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R. leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP. The effect of exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence. The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes. exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background. exoX mutants produced increased levels of succinoglycan. However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background. exoF did not affect the expression of exoP. Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition. We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.     

Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae.

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.     

Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges

Genetically, Rhizobium sp. strain NGR234 and R. fredii USDA257 are closely related. Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257. What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains. The two bacteria nodulated P. andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested. Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae. Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae. On Vigna spp. (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the "cowpea" (V. unguiculata) miscellany. USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera). If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234. The only exceptions were with Apios americana, Glycine max, and G. soja. Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes. Relationships between the ability to nodulate and the origin of the host were not apparent. As both P. andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward.     

Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction.

A fix region of Rhizobium meliloti 41 involved both in symbiotic nodule development and in the adsorption of bacteriophage 16-3 was delimited by directed Tn5 mutagenesis. Mutations in this DNA region were assigned to four complementation units and were mapped close to the pyr-2 and pyr-29 chromosomal markers. Phage inactivation studies with bacterial cell envelope preparations and crude lipopolysaccharides (LPS) as well as preliminary characterization of LPS in the mutants indicated that these genes are involved in the synthesis of a strain-specific LPS. Mutations in this DNA region resulted in a Fix- phenotype in AK631, an exopolysaccharide (EPS)-deficient derivative of R. meliloti 41; however, they did not influence the symbiotic efficiency of the parent strain. An exo region able to restore the EPS production of AK631 was isolated and shown to be homologous to the exoB region of R. meliloti SU47. By generating double mutants, we demonstrated that exo and lps genes determine similar functions in the course of nodule development, suggesting that EPS and LPS may provide equivalent information for the host plant.     

Molecular analysis of an essential gene upstream of rpoN in Rhizobium NGR234

Rhizobium sp. NGR234 is a broad-host range strain. The rpoN gene of this organism encodes a sigma factor which is a primary co-regulator of endosymbiosis. We characterized the locus upstream of rpoN, and identified a contiguous open reading frame, here termed ORF1. DNA sequence analysis of this ORF showed that it encoded a polypeptide highly conserved with a corresponding ORF of Rhizobium meliloti. The gene product contained two ATP/GTP binding pockets. Codon usage in the ORF and the nitrogenase operon nifKDH of NGR234 was similar. Although we used a non-transposable cassette flanked by appropriate sized DNA fragments, we were unable to isolate site-directed mutants in the ORF, whose ATP/GTP binding protein product is thus probably of essential biological function. ORF1 and rpoN exhibited conserved linkage among diverse rhizobia, and in Azotobacter vinelandii. Intragenomic and interspecific homology studies confirmed directly that ORF1 (NGR234) belonged to a large family of ATP-binding protein genes.     

Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes

Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc., into two Nod- Rhizobium mutants as well as into Agrobacterium tumefaciens. Subsequently, cosmid clones of pMPIK3030a were mobilized into Nod+ Rhizobium that cannot nodulate the chosen hosts. Nodule development was monitored by examining the ultrastructure of nodules formed by the transconjugants. pMPIK3030a could complement Nod- and Nif- deletions in R. leguminosarum and R. meliloti as well as enable A. tumefaciens to nodulate. Three non-overlapping sets of cosmids were found that conferred upon a slow-growing Rhizobium species, as well as on R. loti and R. meliloti, the ability to nodulate Psophocarpus and Vigna, thus pointing to the existence of three sets of host-specificity genes. Recipients harboring these hsn regions had truly broadened host-range since they could nodulate both their original hosts as well as MPIK3030 hosts.     

紫云英根瘤菌107菌株exo基因簇非连锁突变位点的克隆

用鸟枪法从3株紫云英根瘤菌107菌株的胞外多糖合成缺陷变种（Exo-）NA－05、NA－07和NA－08中克隆获得含有107菌株exo基因及Tn5的exo::Tn5片段。以pRK415为载体构建107菌株EcoRI酶切后DNA片段的部分基因库,用exo::Tn5做探针原位杂交得到一个阳性克隆。该克隆的外源片段4．2kb能恢复3个变种的多糖表型及结瘤固氮能力。酶切分析和Southern杂交表明,3株变种中Tn5插入位点相近。     

Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.     

Host range,RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10R. fredii strains withRhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. TheR. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated byR. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and commonnod genes,nodDABC; the soybean cultivar specificity gene,nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence fromR. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes,nolBTUVWX, was monomorphic. Using antisera against whole cells of threeR. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a singleR. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences amongR. fredii strains were as great as those between NGR234 andR. fredii, our results confirm that NGR234 has a distinctly wider host range thanR. fredii.     

Two genes that regulate exopolysaccharide production in Rhizobium sp. strain NGR234: DNA sequences and resultant phenotypes.

Two closely linked genes involved in the regulation of exopolysaccharide (EPS) production in Rhizobium sp. strain NGR234, exoX and exoY, were sequenced, and their corresponding phenotypes were investigated. Inhibition of EPS synthesis occurred in wild-type strains when extra copies of exoX were introduced, but only when exoY had been deleted or mutated or was present at a lower copy number. Normal EPS synthesis occurred in Rhizobium sp. when both exoX and exoY were introduced on the same replicon. Surprisingly, the presence of multiple copies of exoY in exoY:: Tn5 mutants of NGR234 adversely affected cellular growth. This was apparent when exoY was introduced into exoY mutants on IncP1 vectors, where the copy number was approximately 10, but was not apparent when present on much larger R-prime plasmids with lower copy numbers (approximately 3 per cell). Multiple copies of exoX did not adversely affect cellular growth of any strain. The exoX gene appeared analogous, in size and phenotype, to a previously described Rhizobium leguminosarum biovar phaseoli EPS gene, psi (D. Borthakur and A.W.B. Johnston, Mol. Gen. Genet. 207:149-154, 1987), and the deduced ExoX and Psi shared strikingly similar secondary structures. Despite this, ExoX and Psi showed little homology at the primary amino acid level, except for a central region of 18 amino acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS biosynthesis. The presence of a multicopy exoX in Rhizobium meliloti and R. fredii similarly abolished EPS biosynthesis in these species.     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.     

Rhizobium sp. strain NGR234 NodZ protein is a fucosyltransferase.

Rhizobium sp. strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents. Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells. Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid. Crude extracts of this transconjugant possess fucosyltransferase activity. Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors. Pentameric chitin is the preferred substrate. These data suggest that fucosylation occurs before acylation of the Nod factors.