The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa.

Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment. Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv- Nod- mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli IlvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv- and Nod-) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa.     

Sinorhizobium meliloti regulator MucR couples exopolysaccharide synthesis and motility

In order to enter symbiosis with its legume partner, Sinorhizobium meliloti requires regulatory systems for the appropriate responses to its environment. For example, motility is required for the chemotactic movement of bacteria toward the compounds released by its host, and exopolysaccharides (EPS) are required for bacterial attachment to the root or for invasion of the infection thread. Previous research has shown that ExoR/ExoS/ChvI as well as the ExpR/Sin quorum-sensing system inversely regulate both motility and EPS production, although the regulation mechanisms were unknown. We were able to attribute the ExpR-mediated regulation of motility to the ability of ExpR to bind a DNA sequence upstream of visN when activated by N-acyl-homoserine lactone. Furthermore, MucR, previously characterized as a regulator of EPS production, also affected motility. MucR inhibited expression of rem encoding an activator of motility gene expression and, consequently, the expression of Rem-regulated genes such as flaF and flgG. Binding of MucR to the rem promoter region was demonstrated and a sequence motif similar to the previously identified MucR binding consensus was identified within this region. The swarming ability of S. meliloti Rm2011 was shown to depend on a functional ExpR/Sin quorum-sensing system and the production of both flagella and EPS. Finally, we propose a model for the coordination of motility and EPS synthesis in S. meliloti.     

A fadD mutant of Sinorhizobium meliloti shows multicellular swarming migration and is impaired in nodulation efficiency on alfalfa roots

Swarming is a form of bacterial translocation that involves cell differentiation and is characterized by a rapid and co-ordinated population migration across solid surfaces. We have isolated a Tn5 mutant of Sinorhizobium meliloti GR4 showing conditional swarming. Swarm cells from the mutant strain QS77 induced on semi-solid minimal medium in response to different signals are hyperflagellated and about twice as long as wild-type cells. Genetic and physiological characterization of the mutant strain indicates that QS77 is altered in a gene encoding a homologue of the FadD protein (long-chain fatty acyl-CoA ligase) of several microorganisms. Interestingly and similar to a less virulent Xanthomonas campestris fadD(rpfB) mutant, QS77 is impaired in establishing an association with its host plant. In trans expression of multicopy fadD restored growth on oleate, control of motility and the symbiotic phenotype of QS77, as well as acyl-CoA synthetase activity of an Escherichia coli fadD mutant. The S. meliloti QS77 strain shows a reduction in nod gene expression as well as a differential regulation of motility genes in response to environmental conditions. These data suggest that, in S. meliloti, fatty acid derivatives may act as intracellular signals controlling motility and symbiotic performance through gene expression.     

Environmental regulation of exopolysaccharide production in Sinorhizobium meliloti

Exopolysaccharide production by Sinorhizobium meliloti is required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners. S. meliloti wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by S. meliloti. The low-phosphate conditions normally found in the soil (1 to 10 microM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by S. meliloti in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.     

Construction and environmental release of a Sinorhizobium meliloti strain genetically modified to be more competitive for alfalfa nodulation

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA- mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492-498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.     

ExpR is not required for swarming but promotes sliding in Sinorhizobium meliloti

Swarming is a mode of translocation dependent on flagellar activity that allows bacteria to move rapidly across surfaces. In several bacteria, swarming is a phenotype regulated by quorum sensing. It has been reported that the swarming ability of the soil bacterium Sinorhizobium meliloti Rm2011 requires a functional ExpR/Sin quorum-sensing system. However, our previous published results demonstrate that strains Rm1021 and Rm2011, both known to have a disrupted copy of expR, are able to swarm on semisolid minimal medium. In order to clarify these contradictory results, the role played by the LuxR-type regulator ExpR has been reexamined. Results obtained in this work revealed that S. meliloti can move over semisolid surfaces using at least two different types of motility. One type is flagellum-independent surface spreading or sliding, which is positively influenced by a functional expR gene mainly through the production of exopolysaccharide II (EPS II). To a lesser extent, EPS II-deficient strains can also slide on surfaces by a mechanism that is at least dependent on the siderophore rhizobactin 1021. The second type of surface translocation shown by S. meliloti is swarming, which is greatly dependent on flagella and rhizobactin 1021 but does not require ExpR. We have extended our study to demonstrate that the production of normal amounts of succinoglycan (EPS I) does not play a relevant role in surface translocation but that its overproduction facilitates both swarming and sliding motilities.     

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.     

Megaplasmid pRme2011a of Sinorhizobium meliloti is not required for viability

We report the curing of the 1,360-kb megaplasmid pRme2011a from Sinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing the sacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.     

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.     

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants.

Nodulation of alfalfa by exoB mutants of Rhizobium meliloti occurred without root hair curling or infection thread formation. nod exoB double mutants had the same nodulation deficiency as single nod mutants. Therefore, all the known nod genes are involved in nodule induction by exoB mutants, which apparently occurs via intercellular invasion.     

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.     

Heterologous complementation of the exopolysaccharide synthesis and carbon utilization phenotypes of Sinorhizobium meliloti Rm1021 polyhydroxyalkanoate synthesis mutants

A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.     

Only one of five groEL genes is required for viability and successful symbiosis in Sinorhizobium meliloti

Many bacterial species contain multiple copies of the genes that encode the chaperone GroEL and its cochaperone, GroES, including all of the fully sequenced root-nodulating bacteria that interact symbiotically with legumes to generate fixed nitrogen. In particular, in Sinorhizobium meliloti there are four groESL operons and one groEL gene. To uncover functional redundancies of these genes during growth and symbiosis, we attempted to construct strains containing all combinations of groEL mutations. Although a double groEL1 groEL2 mutant cannot be constructed, we demonstrate that the quadruple groEL1 groESL3 groEL4 groESL5 and groEL2 groESL3 groEL4 groESL5 mutants are viable. Therefore, like E. coli and other species, S. meliloti requires only one groEL gene for viability, and either groEL1 or groEL2 will suffice. The groEL1 groESL5 double mutant is more severely affected for growth at both 30 degrees C and 40 degrees C than the single mutants, suggesting overlapping functions in stress response. During symbiosis the quadruple groEL2 groESL3 groEL4 groESL5 mutant acts like the wild type, but the quadruple groEL1 groESL3 groEL4 groESL5 mutant acts like the groEL1 single mutant, which cannot fully induce nod gene expression and forms ineffective nodules. Therefore, the only groEL gene required for symbiosis is groEL1. However, we show that the other groE genes are expressed in the nodule at lower levels, suggesting minor roles during symbiosis. Combining our data with other data, we conclude that groESL1 encodes the housekeeping GroEL/GroES chaperone and that groESL5 is specialized for stress response.     

Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation

We show that expression of common nodulation genes in Rhizobium meliloti is under positive as well as negative control. A repressor protein was found to be involved in the negative control of nod gene expression. Whereas the activator NodD protein binds to the conserved cis-regulatory element (nod-box) required for coordinated regulation of nod genes, the repressor binds to the overlapping nodD1 and nodA promoters, at the RNA polymerase binding site. A model depicting the possible interaction of the plant-derived nod gene inducer (luteolin), the NodD and the repressor with the nod promoter elements is presented. Mutants lacking the repressor exhibited delayed nodulation phenotype, indicating that fine tuning of nod gene expression is required for optimal nodulation of the plant host.