Mutants Of Rhizobium Meliloti Research Articles

The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system.

The fix-2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf-/Fix-) is K+ sensitive and unable to adapt to alkaline pH in the presence of K+. Using directed Tn5 mutagenesis, we delimited a 6kb genomic region in which mutations resulted in both Inf-/Fix- and K+-sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G. The putative PhaABC proteins exhibit homology to the subunits of a Na+/H+ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K+ efflux at alkaline pH after the addition of a membrane-permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K+ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.

Multiple genetic controls on Rhizobium meliloti syrA, a regulator of exopolysaccharide abundance.

Exopolysaccharides (EPS) are produced by a wide assortment of bacteria including plant pathogens and rhizobial symbionts. Rhizobium meliloti mutants defective in EPS production fail to invade alfalfa nodules. Production of EPS in R. meliloti is likely controlled at several levels. We have characterized a new gene of this regulatory circuit. syrA was identified by its ability to confer mucoid colony morphology and by its ability to suppress the colonial phenotype of an exoD mutant. Here we show that syrA encodes a 9-kD hydrophobic protein that has sequence similarity to two other EPS regulatory proteins: ExoX of Rhizobium NGR234 and R. meliloti, and Psi of R. leguminosarum bv. phaseoli. The syrA transcription start site lies 522 nucleotides upstream of a non-canonical TTG start codon. The syrA promoter region is similar to the promoter region of the nodulation regulatory protein, nodD3. We found that in free-living bacteria, syrA expression is activated by the regulatory locus, syrM, but not by nodD3. In planta, syrM is not required for expression of syrA. Instead, expression of the nitrogen fixation (nifHDKE) genes upstream of syrA plays a role. Specific and distinct sets of genetic controls may operate at different times during nodule invasion.

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.

Succinoglycan and galactoglucan

Succinoglycan and galactoglucan, also known as EPS-I and EPS-II, respectively, have much been studied in Rhizobium meliloti isolated from the root nodules of lucerne. These polysaccharides have also been obtained from a number of other bacteria, which have been isolated from diverse sources of soil and water communities. In 1965 Harada et al. isolated from soil a bacterium named Alcaligenes faecalis var. myxogenes which produces a polysaccharide that for the first time was characterized and named as ‘succinoglycan’. Succinoglycans were found to be synthesized normally by strains of Agrobacterium tumefaciens, A. radiobacter and Rhizobium meliloti and were also produced by many strains of Pseudomonas spp. isolated from activated sludge and from other sources. They had component compositions of d-glucose, d-galactose, pyruvate, succinate and acetyl in the ratios 7:1:1:1:1. Because of their useful applications as an emulsion-stabilizing, suspending and thickening agent, succinoglycans were produced industrially by Shell. Galactoglucan-producing bacteria were first encountered in activated sludge in 1974 and could reproducibly be isolated from water sediments and from biofilms in water streams and were identified as Agrobacterium radiobacter and Pseudomonas spp. On isolation of these bacteria, colonies were recognized by their very viscous appearance on agar plates. Polymers were found to have very simple component compositions of d-glucose, d-galactose and pyruvate in the ratios 1:1:1. These highly pyruvylated polymers, which were named ‘galactoglucans’, were structurally identified as having a disaccharide repeating unit of alternatively (1→3)-linked d-glucose and d-galactose residues. Surprisingly, Rhizobium meliloti mutant strains were found, which overproduced galactoglucans instead of normal succinoglycan. The osmotic conditions of the growth media, in which the bacteria were cultivated, were found to have a profound effect on the direction of synthesis of the succinoglycan/galactoglucan oligomeric and polymeric products and it was proposed that these products are related by a common biosynthetic pathway (presented in part at the Beijerinck Centennial Congress in The Hague, The Netherlands, 10–14 December 1995).

MsEnod12A Expression Is Linked to Meristematic Activity During Development of Indeterminate and Determinate Nodules and Roots

During development of nodules and roots, the alfalfa early nodulin gene MsEnod12A is expressed adjacent to the meristem. Using transgenic alfalfa carrying a MsEnod12A promoter-gusA fusion, we investigated the regulation of MsEnod12A expression in mature nodules and roots by GUS assays and reverse transcription-PCR. We found that in alfalfa indeterminate nodules induced by various Fix- Rhizobium meliloti mutants and in spontaneous nodules devoid of rhizobia, MsEnod12A was expressed at the distal end when a persistent meristem was present. However, this gene was not expressed when the meristem was lacking in nodules arrested in development. The MsEnod12A-gusA fusion was introduced into Lotus corniculatus plants that form determinate nodules devoid of a persistent meristem. Using these plants we found MsEnod12A-gusA expression only in young nodules and a disappearance in mature nodules. Moreover, when alfalfa roots were treated with auxins a lateral band of MsEnod12A expression was observed surrounding the club-shaped root apex and coinciding with induced lateral meristematic activities. Thus, in all cases MsEnod12A expression was associated with meristematic activities, suggesting that MsEnod12A plays a role in the differentiation processes of nodule and root cells and that it may serve as molecular tool for analyzing meristem establishment during nodule development.

Isolation and characterization of Azospirillum lipoferum locus that complements Rhizobium meliloti dctA and dctB mutations.

A DNA probe containing the structural gene for dicarboxylate transport (dct A) of Rhizobium meliloti hybridized strongly with the fragments of Azospirillum lipoferum genomic DNA. A genomic library of A. lipoferum was screened for the dct A gene by complementation of a dct A mutant of Rhizobium meliloti. A recombinant cosmid, p37D, capable of restoring growth of the dct A mutant on dicarboxylates was isolated and found to hybridize to the dctA probe. The ability of p37D to complement the dct B mutant of R. meloliti indicated that dct A and dct B genes in A. lipoferum may be organized adjacent to each other.

Nodulin gene expression in effective root nodules of white sweetclover (Melilotus albaDesr.) and in ineffective nodules elicited by mutant strains ofRhizobium meliloti

Fifteen nodulins and several nodule-stimulated gene products were expressed in effective, nitrogen-fixing root nodules of white sweetclover (Melilotus alba Desr. cv. U389), as determined by two-dimensional gel electrophoresis of in vitro translation products. The number and gel position of eight leghaemoglobin (Lb) products, as well as a product tentatively identified as nodule-stimulated glutamine synthetase (GS), was similar to previous reports of alfalfa (Medicago sativa L. cv. Iroquois) nodulins. Three mutants of Rhizobium meliloti, including an exoH mutant, a lipopolysaccharide mutant, and a nifH mutant, elicited ineffective sweetclover nodules blocked at empty (bacteria-free), partially infected, or fully infected stages of nodule development, respectively. In these ineffective nodules, the nodulin Nma30 and nodule-stimulated NSTma42 were expressed early in development, while a group of four nodulins and two nodule-stimulated products were intermediate in order of expression. Lb, GS and the late nodulin Nmal2a were expressed later, following infection. The exoH mutant, Rm7154, appeared to be a leaky mutant, as a small percentage of the plants developed nitrogen-fixing nodules about 4 weeks after inoculation. The sequential expression of a large number of nodulins and nodule-stimulated products, as well as the availability of sweetclover nodulation mutants indicates that sweetclover is a useful diploid system for analysis of host genes essential to the Rhizobium/legume symbiosis.

A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti

The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection. In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions. In the present study, we have used a thin-layer chromatographic screening method to identify a novel R. meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone. Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents. As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells. Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.

The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for "respiratory" nitrate reduction ex planta and for nitrogen fixation during symbiosis.

We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix-) and "respiratory" nitrate reduction (Rnr-). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr- and Fix- phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Cc11 and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multisubunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.

Molecular architecture of a galactoglucan from Rhizobium meliloti

Rhizobium meliloti mutants produce a linear, acidic exopolysaccharide with alternating galactopyranosyl and glucopyranosyl units having (1 → 3) linkages. It has a 4,6- O-pyruvic cyclic acetal group on the α-galactosyl and 6- O-acetyl ester group on the β-glucosyl units. X-ray diffraction patterns from polycrystalline and well oriented specimens of its potassium salt indicate that the polymer forms a 2-fold helix of pitch 15.89 Å. The three-dimensional structure has been determined and refined by using the X-ray intensities and the linked-atom least-squares technique. The details of the antiparallel packing arrangement of two helices in an orthorhombic unit cell, a = 14.49, b = 9.79, and c = 15.89 Å, reveal that each disaccharide repeating unit is associated with one potassium and three water molecules. The helices are interconnected by a series of ⋯ COO − ⋯ K + ⋯ W ⋯ COO − ⋯ interactions. Both pyruvyl and acetyl groups, which are on the periphery of the helix, are involved in the association of the polysaccharide chains and thus appear to be an integral component of the galactoglucan in the nodule invasion process.

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 N2 (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.

The regulation of exopolysaccharide production is important at two levels of nodule development in Rhizobium meliloti.

We show that two exopolysaccharide overproducing Tn5 mutants of Rhizobium meliloti, exoR and exoS, have distinct symbiotic defects. While the exoR mutant is unable to colonize nodules, the exoS mutant retains that ability but varies in its ability to produce nitrogen-fixing nodules. We correlate these defects with different degrees of exopolysaccharide overproduction and growth impairment. We further show that the exoR mutant is able to enter developing infection threads but is unable to invade nodule cells. The exoR mutant gives rise to spontaneous pseudorevertants containing second-site suppressor mutations that decrease exopolysaccharide synthesis. These pseudorevertants form nitrogen-fixing nodules. Although the suppressor mutations have the opposite effect on exopolysaccharide production compared to the exoS::Tn5 mutation, they consistently map to the exoS::Tn5 region and belong to the same genetic complementation group as defined by transposon insertion mutations. The effect of the suppressor mutations on exopolysaccharide production is correlated with effects on the expression of exo genes involved in exopolysaccharide synthesis. Finally, we provide evidence that the exoR gene is not required for the regulation of exopolysaccharide synthesis by ammonia.

Physicochemical properties of ‘Exogel’ exocellular β(1–4)- d-glucuronan from Rhizobium meliloti strain M5N1 C.S. (NCIMB 40472)

The Rhizobium meliloti mutant strain M5N1 C.S. (NCIMB 40472) produces a polyuronic acid as β(1 → 4)- d-glucuronan with molecular weight between 1·5 × 10 5 and 3 × 10 5. With a p K o of about 3 and L p ⋍ 110 A ̊ , this polyelectrolyte is comparable with alginates and pectins. In solution, acetyl content and high external salt concentrations favour aggregation. At high concentrations the polysaccharide forms thermoreversible or thermally stable gels according to the nature of the counterion, the polymer concentration and the ionic strength.

Identification and cloning of a cyclic beta-(1-->3), beta-(1-->6)-D-glucan synthesis locus from Bradyrhizobium japonicum.

A cosmid clone complementing a cyclic beta-(1-->2)-glucan biosynthesis (ndvB) mutant of Rhizobium meliloti was isolated from a Bradyrhizobium japonicum gene library. This clone specified synthesis of beta-(1-->3), beta-(1-->6)-linked glucans in R. meliloti. The complemented strain was osmotically tolerant and symbiotically competent on alfalfa. Thus, beta-(1-->3), beta-(1-->6)-glucans can substitute functionally for beta-(1-->2)-glucans in R. meliloti.

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.

A Rhizobium meliloti mutant, lacking a functional γ-aminobutyrate (GABA) bypass, is defective in glutamate catabolism and symbiotic nitrogen fixation

A Rhizobium meliloti mutant, lacking a functional γ-aminobutyrate (GABA) bypass, is defective in glutamate catabolism and symbiotic nitrogen fixation

A Rhizobium meliloti mutant, lacking a functional γ-aminobutyrate (GABA) bypass, is defective in glutamate catabolism and symbiotic nitrogen fixation

A Rhizobium meliloti mutant, CMF1 2:38, was isolated which was specifically defective in the degradation of glutamate as sole carbon and nitrogen source. Biochemical analysis of CMF1 2:38 revealed a reduction in succinic semialdehyde dehydrogenase (SSDH) activity, the third enzyme of the γ-aminobutyrate (GABA) bypass. Evidence is presented which suggests that the Tn 5-induced mutation in CMF1 2:38 exists in a regulatory gene governing the expression of both NAD and NADP-linked SSDH activity. CMF1 2:38 nodulated alfalfa plants, but was reduced in its nitrogen fixation activity and biomass accumulating ability relative to the wild-type strain. The results presented in this study indicate that the GABA bypass is a major mechanism of glutamate degradation in R. meliloti CMF1 and that glutamate catabolism via this pathway may play an important role in the symbiotic nitrogen fixation process.

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.

Rhizobium meliloti mutants with decreased DAHP synthase activity are sensitive to exogenous tryptophan and phenylalanine and form ineffective nodules.

We isolated two Tn5-generated mutants of Rhizobium meliloti whose growth was inhibited by rich medium or by exogenous tryptophan or phenylalanine. These mutants, Rm7479 and Rm7480, belonged to the same genetic complementation group. The mutant locus could not be found on either indigenous megaplasmid but was localized on the chromosome. The mutants formed ineffective nodules on alfalfa plants. They invaded nodules within infection threads and were released into plant cells enclosed within peribacteroid membranes, but once released into the plant cells they failed to differentiate into mature bacteroids. The mutants demonstrated a decrease in total 2-keto-3-deoxy-D-arabino-heptonic acid 7-phosphate synthase (DAHP synthase) activity, which is the first committed step in aromatic biosynthesis. Wild-type genes were isolated that complemented in one case or suppressed in another case, all three mutant phenotypes: growth on rich medium, symbiotic effectiveness, and DAHP synthase activity. Each mutant strain gave rise to linked second-site suppressor mutations that restored growth on rich medium. The suppressor mutants showed restoration of near wild-type DAHP synthase levels. One of the suppressor strains restored effective symbiosis while the other did not. Genetic complementation experiments showed that growth on rich medium, DAHP synthase activity, and effective symbiosis were all affected by the same genetic lesion. These results suggest that normal flux of metabolites through the aromatic biosynthesis pathway is essential for bacteroid development.

Enhanced Levels of Chalcone Synthase in Alfalfa Nodules Induced by a Fix‾ Mutant ofRhizobium meliloti

Enhanced Levels of Chalcone Synthase in Alfalfa Nodules Induced by a Fix‾ Mutant of<i>Rhizobium meliloti</i>