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.     

The exoD gene of Rhizobium meliloti encodes a novel function needed for alfalfa nodule invasion

During the symbiotic interaction between alfalfa and the nitrogen-fixing bacterium Rhizobium meliloti, the bacterium induces the formation of nodules on the plant roots and then invades these nodules. Among the bacterial genes required for nodule invasion are the exo genes, involved in production of an extracellular polysaccharide, and the ndv genes, needed for production of a periplasmic cyclic glucan. Mutations in the exoD gene result in altered exopolysaccharide production and in a nodule invasion defect. In this work we show that the stage of symbiotic arrest of exoD mutants is similar to that of other exo and ndv mutants. However, the effects of exoD mutations on exopolysaccharide production and growth on various media are different from the effects of other exo and ndv mutations. Finally, exoD mutations behave differently from other exo mutations in their ability to be suppressed or complemented extracellularly. The results suggest that exoD represents a new class of Rhizobium genes required for nodule invasion, distinct from the other exo genes and the ndv genes. We discuss models for the function of exoD.     

Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula

The nitrogen-fixing rhizobial symbiont Sinorhizobium meliloti 1021 produces acidic symbiotic exopolysaccharides that enable it to initiate and maintain infection thread formation on host legume plants. The exopolysaccharide that is most efficient in mediating this process is succinoglycan (exopolysaccharide I [EPSI]), a polysaccharide composed of octasaccharide repeating units of 1 galactose and 7 glucose residues, modified with succinyl, acetyl, and pyruvyl substituents. Previous studies had shown that S. meliloti 1021 mutants that produce increased levels of succinoglycan, such as exoR mutants, are defective in symbiosis with host plants, leading to the hypothesis that high levels of succinoglycan production might be detrimental to symbiotic development. This study demonstrates that increased succinoglycan production itself is not detrimental to symbiotic development and, in fact, enhances the symbiotic productivity of S. meliloti 1021 with the host plant Medicago truncatula cv. Jemalong A17. Increased succinoglycan production was engineered by overexpression of the exoY gene, which encodes the enzyme responsible for the first step in succinoglycan biosynthesis. These results suggest that the level of symbiotic exopolysaccharide produced by a rhizobial species is one of the factors involved in optimizing the interaction with plant hosts.     

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.     

CbrA is a stationary-phase regulator of cell surface physiology and legume symbiosis in Sinorhizobium meliloti

Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that plays a critical role in promoting symbiosis with its host legume, alfalfa (Medicago sativa). We performed a transposon mutagenesis and screened for mutants with altered succinoglycan production and a defect in symbiosis. In this way, we identified a putative two-component histidine kinase associated with a PAS sensory domain, now designated CbrA (calcofluor-bright regulator A). The cbrA::Tn5 mutation causes overproduction of succinoglycan and results in increased accumulation of low-molecular-weight forms of this exopolysaccharide. Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype through increased expression of exo genes required for succinoglycan biosynthesis and modification. Interestingly, CbrA-dependent regulation of exo and exs genes is observed almost exclusively during stationary-phase growth. The cbrA::Tn5 mutant also has an apparent cell envelope defect, based on increased sensitivity to a number of toxic compounds, including the bile salt deoxycholate and the hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode putative inner membrane ABC transporters with a high degree of similarity to lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which is required for lipopolysaccharide transport, while ExsE is a member of the eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in transport of very long-chain fatty acids, which are a unique component of the lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in regulating the lipopolysaccharide or lipoprotein components of the cell envelope.     

The Sinorhizobium meliloti stringent response affects multiple aspects of symbiosis

Sinorhizobium meliloti and host legumes enter into a nitrogen-fixing, symbiotic relationship triggered by an exchange of signals between bacteria and plant. S. meliloti produces Nod factor, which elicits the formation of nodules on plant roots, and succinoglycan, an exopolysaccharide that allows for bacterial invasion and colonization of the host. The biosynthesis of these molecules is well defined, but the specific regulation of these compounds is not completely understood. Bacteria control complex regulatory networks by the production of ppGpp, the effector molecule of the stringent response, which induces physiological change in response to adverse growth conditions and can also control bacterial development and virulence. Through detailed analysis of an S. meliloti mutant incapable of producing ppGpp, we show that the stringent response is required for nodule formation and regulates the production of succinoglycan. Although it remains unknown whether these phenotypes are connected, we have isolated suppressor strains that restore both defects and potentially identify key downstream regulatory genes. These results indicate that the S. meliloti stringent response has roles in both succinoglycan production and nodule formation and, more importantly, that control of bacterial physiology in response to the plant and surrounding environment is critical to the establishment of a successful symbiosis.     

Mesorhizobium loti produces nodPQ-dependent sulfated cell surface polysaccharides

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.     

The ExpR/Sin quorum-sensing system controls succinoglycan production in Sinorhizobium meliloti

Sinorhizobium meliloti is a gram-negative soil bacterium capable of forming a symbiotic nitrogen-fixing relationship with its plant host, Medicago sativa. Various bacterially produced factors are essential for successful nodulation. For example, at least one of two exopolysaccharides produced by S. meliloti (succinoglycan or EPS II) is required for nodule invasion. Both of these polymers are produced in high- and low-molecular-weight (HMW and LMW, respectively) fractions; however, only the LMW forms of either succinoglycan or EPS II are active in nodule invasion. The production of LMW succinoglycan can be generated by direct synthesis or through the depolymerization of HMW products by the action of two specific endoglycanases, ExsH and ExoK. Here, we show that the ExpR/Sin quorum-sensing system in S. meliloti is involved in the regulation of genes responsible for succinoglycan biosynthesis as well as in the production of LMW succinoglycan. Therefore, quorum sensing, which has been shown to regulate the production of EPS II, also plays an important role in succinoglycan biosynthesis.     

Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

H protein of bacteriophage 16-3 and RkpM protein of Sinorhizobium meliloti 41 are involved in phage adsorption

The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.     

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.     

The ++Sinorhizobium meliloti lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.     

Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.     

Mutational analysis of the Sinorhizobium meliloti short-chain dehydrogenase/reductase family reveals substantial contribution to symbiosis and catabolic diversity

The short-chain dehydrogenase/reductase (SDR) family is one of the largest and most ubiquitous protein families in bacterial genomes. Despite there being a few well-characterized examples, the substrate specificities or functions of most members of the family are unknown. In this study, we carried out a large-scale mutagenesis of the SDR gene family in the alfalfa root nodule symbiont Sinorhizobium meliloti. Subsequent phenotypic analysis revealed phenotypes for mutants of 21 of the SDR-encoding genes. This brings the total number of S. meliloti SDR-encoding genes with known function or associated phenotype to 25. Several of the mutants were deficient in the utilization of specific carbon sources, while others exhibited symbiotic deficiencies on alfalfa (Medicago sativa), ranging from partial ineffectiveness to complete inability to form root nodules. Five of the mutants had both symbiotic and carbon utilization phenotypes. These results clearly demonstrate the importance of the SDR family in both symbiosis and saprotrophy, and reinforce the complex nature of the interaction of S. meliloti with its plant hosts. Further analysis of the genes identified in this study will contribute to the overall understanding of the biology and metabolism of S. meliloti in relation to its interaction with alfalfa.     

Second Site Mutations Specifically Suppress the Fix(-) Phenotype of Rhizobium Meliloti Ndvf Mutations on Alfalfa: Identification of a Conditional Ndvf-Dependent Mucoid Colony Phenotype

Rhizobium meliloti mutants carrying ndvF insertion or deletion mutations induce nodules on alfalfa which contain very few infected cells and fail to fix N(2) (Fix(-)). We have characterized five independent second site mutations (designated sfx) which completely suppress the Fix(-) phenotype of ndvF mutants on Medicago sativa but not on another R. meliloti host Melilotus alba. Genetic mapping and phenotypic analysis revealed that the suppressor mutations sfx-1, sfx-4 and sfx-5 mapped to a single locus which was distinct from another locus defined by the sfx-2 and sfx-3 mutations. Tn5-mob-mediated conjugal mapping experiments showed that the sfx-1 locus was located clockwise from trp-33 on the R. meliloti chromosome and a detailed cotransduction map of this region was generated. To clone the sfx-1 locus, we prepared a cosmid library from total DNA obtained from an sfx-1, ndvF deletion strain. From this library, a cosmid pTH56, which converted Fix(-) ndvF mutants to Fix(+), was isolated. Southern blot analysis provided direct physical evidence that the insert DNA in plasmid pTH56 was contiguous with the sfx-1 region. On low osmolarity glutamate-yeast extract-mannitol-salts medium (GYM) agar medium, ndvF insertion and deletion mutants were found to have a mucoid colony phenotype, as opposed to the dry colony phenotype of the wild-type strain. This phenotype was shown to be dependent on the exoB and expE genes required for synthesis of exopolysaccharide II in R. meliloti but not to be dependent on genes required exclusively for the synthesis of the succinoglycan or exopolysaccharide I. Transduction of either sfx-1 or sfx-2 or transfer of the cosmid pTH56 into the ndvF mutants restored them to a wild-type dry colony phenotype. The mucoid phenotype is not responsible for the Fix(-) phenotype of ndvF mutants as the Fix(-), ndvF exp double mutants can be complemented to Fix(+) by introducing plasmids which carry only the wild-type ndvF genes.     

Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.     

Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti.

Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa. Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits. These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate. Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T. L. Reuber and G. C. Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway. We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R. meliloti and have identified several new genes. In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence. In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.     

Auxotrophy accounts for nodulation defect of most Sinorhizobium meliloti mutants in the branched-chain amino acid biosynthesis pathway

Some Sinorhizobium meliloti mutants in genes involved in isoleucine, valine, and leucine biosynthesis were previously described as being unable to induce nodule formation on host plants. Here, we present a reappraisal of the interconnection between the branched-chain amino acid biosynthesis pathway and the nodulation process in S. meliloti. We characterized the symbiotic phenotype of seven mutants that are auxotrophic for isoleucine, valine, or leucine in two closely related S. meliloti strains, 1021 and 2011. We showed that all mutants were similarly impaired for nodulation and infection of the Medicago sativa host plant. In most cases, the nodulation phenotype was fully restored by the addition of the missing amino acids to the plant growth medium. This strongly suggests that auxotrophy is the cause of the nodulation defect of these mutants. However, we confirmed previous findings that ilvC and ilvD2 mutants in the S. meliloti 1021 genetic background could not be restored to nodulation by supplementation with exogenous amino acids even though their Nod factor production appeared to be normal.