Genetic analysis of carbamoylphosphate synthesis in Rhizobium meliloti 104A14

We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase. We describe here the molecular and genetic analysis of the R. meliloti genes coding for carbamoylphosphate synthetase. Plasmids that complement the mutations were isolated from a R. meliloti gene bank. Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R. meliloti chromosome, carA and carB. Genetic complementation between the plasmids and mutants demonstrated a single complementation group for carA, but two overlapping complementation groups for carB. The cloned R. meliloti genes hybridize to the corresponding E. coli carA and carB genes which encode the two subunits of carbamoylphosphate synthetase. Transposon Tn5 mutagenesis was used to localize the carA and carB genes on the cloned R. meliloti DNA. The cloned R. meliloti carA and carB genes were unable to complement E. coli carA or carB mutants alone or in combination. We speculate on the mechanism of the unusual pattern of genetic complementation at the R. meliloti carB locus.     

The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.     

Identification of genes relevant to symbiosis and competitiveness in Sinorhizobium meliloti using signature-tagged mutants

Sinorhizobium meliloti enters an endosymbiosis with alfalfa plants through the formation of nitrogen-fixing nodules. In order to identify S. meliloti genes required for symbiosis and competitiveness, a method of signature-tagged mutagenesis was used. Two sets, each consisting of 378 signature-tagged mutants with a known transposon insertion site, were used in an experiment in planta. As a result, 67 mutants showing attenuated symbiotic phenotypes were identified, including most of the exo, fix, and nif mutants in the sets. For 38 mutants in genes previously not described to be involved in competitiveness or symbiosis in S. meliloti, attenuated competitiveness phenotypes were tested individually. A large part of these phenotypes was confirmed. Moreover, additional symbiotic defects were observed for mutants in several novel genes such as infection deficiency phenotypes (ilvI and ilvD2 mutants) or delayed nodulation (pyrE, metA, thiC, thiO, and thiD mutants).     

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.     

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions.

Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.     

Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity.

The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia. Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species. These are encoded by the glnA and glnII genes, respectively. Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source. For two auxotrophs that lacked any detectable GS activity, R. meliloti DNA of the mutated region was cloned and partially characterized. Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity. One of the cloned regions was identified as glnII. An R. meliloti glnII mutant and an R. meliloti glnA glnII double mutant were constructed. Both formed effective nodules on alfalfa. This is unlike the B. japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop. However, the R. meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.     

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 glutamate synthase: cloning and initial characterization of the glt locus.

The genetic locus glt, encoding glutamate synthase from Rhizobium meliloti 1021, was selected from a pLAFR1 clone bank by complementation of the R. meliloti 41 Glt- mutant AK330. A fragment of cloned DNA complementing this mutant also served to complement the Escherichia coli glt null mutant PA340. Complementation studies using these mutants suggested that glutamate synthase expression requires two complementation groups present at this locus. Genomic Southern analysis using a probe of the R. meliloti 1021 glt region showed a close resemblance between R. meliloti 1021, 41, and 102f34 at glt, whereas R. meliloti 104A14 showed many differences in restriction fragment length polymorphism patterns at this locus. R. meliloti 102f34, but not the other strains, showed an additional region with sequence similarity to glt. Insertion alleles containing transposable kanamycin resistance elements were constructed and used to derive Glt- mutants of R. meliloti 1021 and 102f34. These mutants were unable to assimilate ammonia and were Nod+ Fix+ on alfalfa seedlings. The mutants also showed poor or no growth on nitrogen sources such as glutamate, aspartate, arginine, and histidine, which are utilized by the wild-type parental strains. Strains that remained auxotrophic but grew nearly as well as the wild type on these nitrogen sources were readily isolated from populations of glt insertion mutants, indicating that degradation of these amino acids is negatively regulated in R. meliloti as a result of disruptions of glt.     

Functional analysis of Sinorhizobium meliloti genes involved in biotin synthesis and transport

External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.     

Roles for riboflavin in the Sinorhizobium-alfalfa association

Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes. The ribBA and ribC loci complemented corresponding E. coli rib mutants. S. meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin. Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain. No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N. These results support the importance of extracellular riboflavin for alfalfa root colonization by S. meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.     

Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene.

The gene encoding Rhizobium meliloti isocitrate dehydrogenase (ICD) was cloned by complementation of an Escherichia coli icd mutant with an R. meliloti genomic library constructed in pUC18. The complementing DNA was located on a 4.4-kb BamHI fragment. It encoded an ICD that had the same mobility as R. meliloti ICD in nondenaturing polyacrylamide gels. In Western immunoblot analysis, antibodies raised against this protein reacted with R. meliloti ICD but not with E. coli ICD. The complementing DNA fragment was mutated with transposon Tn5 and then exchanged for the wild-type allele by recombination by a novel method that employed the Bacillus subtilis levansucrase gene. No ICD activity was found in the two R. meliloti icd::Tn5 mutants isolated, and the mutants were also found to be glutamate auxotrophs. The mutants formed nodules, but they were completely ineffective. Faster-growing pseudorevertants were isolated from cultures of both R. meliloti icd::Tn5 mutants. In addition to lacking all ICD activity, the pseudorevertants also lacked citrate synthase activity. Nodule formation by these mutants was severely affected, and inoculated plants had only callus structures or small spherical structures.     

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.     

Genetic analysis of a region of the Rhizobium meliloti pSym plasmid specifying catabolism of trigonelline, a secondary metabolite present in legumes.

Genes controlling the catabolism of trigonelline, a secondary metabolite that is often present in legumes, are located on the pSym megaplasmid of Rhizobium meliloti. To investigate the role of bacterial trigonelline catabolism in the Rhizobium-legume symbiosis, we identified and characterized the R. meliloti RCR2011 genetic loci (trc) controlling trigonelline catabolism. Tn5-B20 mutagenesis showed that the trc region is a continuous DNA segment of 9 kb located 4 kb downstream of the nifAB and fdxN genes. Trc mutants fell into two classes according to their phenotype and location: (i) mutants carrying Tn5-B20 insertions in the right-hand part of the trc region were incapable of growing on trigonelline as the sole carbon and/or nitrogen source, and (ii) insertions in the left-hand part of the trc region resulted in delayed growth on trigonelline as the sole carbon and/or nitrogen source. No significant defect in nodule formation or nitrogen fixation was detected for mutants of either class. Screening of a set of R. meliloti strains from various geographical origins showed that all of these strains are able to catabolize trigonelline and show sequence homology between their megaplasmids and a trc probe.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Sinorhizobium meliloti genes involved in tolerance to the antimicrobial peptide protamine

The innate resistance of plants and animals to microbial infection is mediated in part by small cationic peptides with antimicrobial activity. We assessed the susceptibility of the alfalfa symbiont Sinorhizobium meliloti to the model antimicrobial peptide protamine. Twenty-one Tn5-induced mutants showing increased sensitivity to protamine were isolated, and nine were further characterized in detail. These nine mutants carried distinct transposon insertions that affected a total of seven different genes. Three of these genes are involved in exopolysaccharide and beta-(1,2)-glucan biosynthesis (exoT, exoU and ndvB), three other genes are implicated in nitrogen metabolism, such as a putative dyhidropyrimidinase, hutU and ureF, and the last gene exhibited similarity to the ATP binding cassette family of membrane transporters. Symbiotic defects ranging from severe to moderate were displayed by some of the protamine-hypersensitive mutants suggesting that S. meliloti possess active mechanisms to counteract hypothetical cationic peptides that may be produced by its host plant.