RcgA and RcgR, Two Novel Proteins Involved in the Conjugative Transfer of Rhizobial Plasmids.

Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.

Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa.

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

A Stringent-Response-Defective Bradyrhizobium diazoefficiens Strain Does Not Activate the Type 3 Secretion System, Elicits an Early Plant Defense Response, and Circumvents NH4NO3-Induced Inhibition of Nodulation.

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N2 in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH4NO3, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system.IMPORTANCE The symbiotic N2 fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N2 fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.

Coinoculation of soybean plants with Bradyrhizobium japonicum and Trichoderma harzianum: Coexistence of both microbes and relief of nitrate inhibition of nodulation.

Coinoculation of plants with mixtures of beneficial microbes sometimes produces synergistic effects. In this study, the effect of soybean coinoculation with the N2-fixing Bradyrhizobium japonicum E109 and the biocontrol fungus Trichoderma harzianum Th5cc was analyzed. Nodulation by E109 was not hampered by Th5cc, which antagonized five out of seven soybean pathogens tested. Furthermore, Th5cc relieved nitrate-inhibition of nodulation, enabling the formation of nodules containing infected cells with bacteroids in the presence of the otherwise inhibitory 10 mM KNO3. Th5cc released micromolar amounts of auxin, and addition of 11 µM indoleacetic acid to soybean plants inoculated with E109 in the absence of Th5cc also induced nodulation in the presence of 10 mM KNO3. Thus, Th5cc may release auxins into the soybean rhizosphere, which hormones might participate in overcoming the nitrate-inhibition of nodulation. Our results suggest that soybean plants coinoculated with these microorganisms might benefit from biocontrol while contributing to soil-nitrogen preservation.

Proteomic Analysis of Rhizobium favelukesii LPU83 in Response to Acid Stress.

Acid soils constitute a severe problem for leguminous crops mainly through a disturbance in rhizobium-legume interactions. Rhizobium favelukesii-an acid-tolerant rhizobium able to nodulate alfalfa-is highly competitive for nodule occupation under acid conditions but inefficient for biologic nitrogen fixation. In this work, we obtained a general description of the acid-stress response of R. favelukesii LPU83 by means of proteomics by comparing the total proteome profiles in the presence or absence of acid stress by nanoflow ultrahigh-performance liquid chromatography coupled to mass spectrometry. Thus, a total of 336 proteins were identified with a significant differential expression, 136 of which species were significantly overexpressed and 200 underexpressed in acidity. An in silico functional characterization with those respective proteins revealed a complex and pleiotropic response by these rhizobia involving components of oxidative phosphorylation, glutamate metabolism, and peptidoglycan biosynthesis, among other pathways. Furthermore, a lower permeability was evidenced in the acid-stressed cells along with several overexpressed proteins related to γ-aminobutyric acid metabolism, such as the gene product of livK, which gene was mutated. This mutant exhibited an acid-sensitive phenotype in agreement with the proteomics results. We conclude that both the γ-aminobutyric acid metabolism and a modified cellular envelope could be relevant to acid tolerance in R. favelukesii.

Soybean-Nodulating Strains With Low Intrinsic Competitiveness for Nodulation, Good Symbiotic Performance, and Stress-Tolerance Isolated From Soybean-Cropped Soils in Argentina.

Soybean is the most important oilseed in the world, cropped in 120-130 million hectares each year. The three most important soybean producers are Argentina, Brazil, and United States, where soybean crops are routinely inoculated with symbiotic N2-fixing Bradyrhizobium spp. This extended inoculation gave rise to soybean-nodulating allochthonous populations (SNAPs) that compete against new inoculant for nodulation, thus impairing yield responses. Competitiveness depends on intrinsic factors contributed by genotype, extrinsic ones determined by growth and environmental conditions, and strain persistence in the soil. To assess these factors in Argentinean SNAPs, we studied 58 isolates from five sites of the main soybean cropping area. BOX-A1R DNA fingerprint distributed these isolates in 10 clades that paralleled the pHs of their original soils. By contrast, reference Bradyrhizobium spp. strains, including those used as soybean-inoculants, were confined to a single clade. More detailed characterization of a subset of 11 SNAP-isolates revealed that five were Bradyrhizobium japonicum, two Bradyrhizobium elkanii, two Rhizobium radiobacter (formerly Agrobacterium tumefaciens), one Bradyrhizobium diazoefficiens, and one Paenibacillus glycanilyticus-which did not nodulate when inoculated alone, and therefore was excluded from further characterization. The remaining subset of 10 SNAP-isolates was used for deeper characterization. All SNAP-isolates were aluminum- and heat-tolerant, and most of them were glyphosate-tolerant. Meanwhile, inoculant strains tested were sensitive to aluminum and glyphosate. In addition, all SNAP-isolates were motile to different degrees. Only three SNAP-isolates were deficient for N2-fixation, and none was intrinsically more competitive than the inoculant strain. These results are in contrast to the general belief that rhizobia from soil populations evolved as intrinsically more competitive for nodulation and less N2-fixing effective than inoculants strains. Shoot:root ratios, both as dry biomass and as total N, were highly correlated with leaf ureide contents, and therefore may be easy indicators of N2-fixing performance, suggesting that highly effective N2-fixing and well-adapted strains may be readily selected from SNAPs. In addition, intrinsic competitiveness of the inoculants strains seems already optimized against SNAP strains, and therefore our efforts to improve nodules occupation by inoculated strains should focus on the optimization of extrinsic competitiveness factors, such as inoculant formulation and inoculation technology.

Induction by Bradyrhizobium diazoefficiens of Different Pathways for Growth in D-mannitol or L-arabinose Leading to Pronounced Differences in CO2 Fixation, O2 Consumption, and Lateral-Flagellum Production.

Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, constitutes the basic input in one of the most prominent inoculant industries worldwide. This bacterium may be cultured with D-mannitol or L-arabinose as carbon-plus-energy source (C-source) with similar specific growth rates, but with higher biomass production with D-mannitol. To better understand the bacterium's carbon metabolism, we analyzed, by liquid chromatography and tandem mass spectrometry (MS), the whole set of proteins obtained from cells grown on each C-source. Among 3,334 proteins identified, 266 were overproduced in D-mannitol and 237 in L-arabinose, but among these, only 22% from D-mannitol cultures and 35% from L-arabinose cultures were annotated with well defined functions. In the D-mannitol-differential pool we found 19 enzymes of the pentose-phosphate and Calvin-Benson-Bassham pathways and accordingly observed increased extracellular-polysaccharide production by D-mannitol grown bacteria in a CO2-enriched atmosphere. Moreover, poly-3-hydroxybutyrate biosynthesis was increased, suggesting a surplus of reducing power. In contrast, the L-arabinose-differential pool contained 11 enzymes of the L-2-keto-3-deoxyarabonate pathway, 4 enzymes for the synthesis of nicotinamide-adenine dinucleotide from aspartate, with those cultures having a threefold higher O2-consumption rate than the D-mannitol cultures. The stoichiometric balances deduced from the modeled pathways, however, resulted in similar O2 consumptions and ATP productions per C-mole of substrate. These results suggested higher maintenance-energy demands in L-arabinose, which energy may be used partly for flagella-driven motility. Since B. diazoefficiens produces the lateral-flagella system in only L-arabinose, we calculated the O2-consumption rates of a lafR::Km mutant devoid of lateral flagella cultured in L-arabinose or D-mannitol. Contrary to that of the wild-type, the O2-consumption rate of this mutant was similar on both C-sources, and accordingly outcompeted the wild-type in coculture, suggesting that the lateral flagella behaved as parasitic structures under these conditions. Proteomic data are available via ProteomeXchange with identifier PXD008263.

Analysis of two polyhydroxyalkanoate synthases in Bradyrhizobium japonicum USDA 110.

Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic redundancy, only phaC1 and phaC2 were expressed at significant rates, while PHA accumulation in stationary-phase cultures was impaired only in the ΔphaC1 mutant. Meanwhile, the ΔphaC2 mutant produced more PHA than the wild type under this condition, and surprisingly, the phaC3 transcript increased in the ΔphaC2 background. A double mutant, the ΔphaC2 ΔphaC3 mutant, consistently accumulated less PHA than the ΔphaC2 mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the ΔphaC1 mutant and increased in the ΔphaC2 mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a ΔphaC1 ΔphaC2 double mutant, to study the B. japonicum PHA requirements for survival, competition for nodulation, and plant growth promotion. All mutants, as well as the wild type, survived for 60 days in a carbon-free medium, regardless of their initial PHA contents. When competing for nodulation against the wild type in a 1:1 proportion, the ΔphaC1 and ΔphaC1 ΔphaC2 mutants occupied only 13 to 15% of the nodules, while the ΔphaC2 mutant occupied 81%, suggesting that the PHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation.

Soybean seed lectin prevents the accumulation of S-adenosyl methionine synthetase and the S1 30S ribosomal protein in Bradyrhizobium japonicum under C and N starvation.

Soybean lectin (SBL) participates in the recognition between Bradyrhizobium japonicum and soybean although its role remains unknown. To search for changes in the proteome in response to SBL, B. japonicum USDA 110 was incubated for 12 h in a C- and N-free medium with or without SBL (10 µg ml(-1)), and the soluble protein profiles were compared. Two polypeptides, S-adenosyl-methionine synthetase (MetK) and the 30S ribosomal protein S1 (RpsA), were found only in the fractions from rhizobia incubated without SBL. Transcript levels of metK and rpsA were not correlated with polypeptide levels, indicating that there was regulation at translation. In support of this proposal, the 5' translation initiation-region of rpsA mRNA contained folding elements as those involved in regulation of its translation in other species. Disappearance of MetK and RpsA from the soluble protein fractions of SBL-treated rhizobia suggests that SBL might have attenuated the nutritional stress response of B. japonicum.

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean.

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.

Lack of galactose or galacturonic acid in Bradyrhizobium japonicum USDA 110 exopolysaccharide leads to different symbiotic responses in soybean.

Exopolysaccharide (EPS) and lipopolysaccharide (LPS) from Bradyrhizobium japonicum are important for infection and nodulation of soybean (Glycine max), although their roles are not completely understood. To better understand this, we constructed mutants in B. japonicum USDA 110 impaired in galactose or galacturonic acid incorporation into the EPS without affecting the LPS. The derivative LP 3010 had a deletion of lspL-ugdH and produced EPS without galacturonic acid whereas LP 3013, with an insertion in exoB, produced EPS without galactose. In addition, the strain LP 3017, with both mutations, had EPS devoid of both galactosides. The missing galactosides were not replaced by other sugars. The defects in EPS had different consequences. LP 3010 formed biofilms and nodulated but was defective in competitiveness for nodulation; and, inside nodules, the peribacteroid membranes tended to fuse, leading to the merging of symbiosomes. Meanwhile, LP 3013 and LP 3017 were unable to form biofilms and produced empty pseudonodules but exoB suppressor mutants were obtained when LP 3013 plant inoculation was supplemented with wild-type EPS. Similar phenotypes were observed with all these mutants in G. soja. Therefore, the lack of each galactoside in the EPS has a different functional effect on the B. japonicum-soybean symbiosis.

Soybean Lectin Enhances Biofilm Formation by Bradyrhizobium japonicum in the Absence of Plants.

Soybean lectin (SBL) purified from soybean seeds by affinity chromatography strongly bound to Bradyrhizobium japonicum USDA 110 cell surface. This lectin enhanced biofilm formation by B. japonicum in a concentration-dependent manner. Presence of galactose during biofilm formation had different effects in the presence or absence of SBL. Biofilms were completely inhibited in the presence of both SBL and galactose, while in the absence of SBL, galactose was less inhibitory. SBL was very stable, since its agglutinating activity of B. japonicum cells as well as of human group A+ erythrocytes was resistant to preincubation for one week at 60 degrees C. Hence, we propose that plant remnants might constitute a source of this lectin, which might remain active in soil and thus favor B. japonicum biofilm formation in the interval between soybean crop seasons.

The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation.

The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.

Strain selection for improvement of Bradyrhizobium japonicum competitiveness for nodulation of soybean.

A Bradyrhizobium japonicum USDA 110-derived strain able to produce wider halos in soft-agar medium than its parental strain was obtained by recurrent selection. It was more chemotactic than the wild type towards mannitol and three amino acids. When cultured in minimal medium with mannitol as a single carbon-source, it had one thick subpolar flagellum as the wild type, plus several other flagella that were thinner and sinusoidal. Root adsorption and infectivity in liquid media were 50-100% higher for the selected strain, but root colonization in water-unsaturated vermiculite was similar to the wild type. A field experiment was then carried out in a soil with a naturalized population of 1.8 x 10(5) soybean-nodulating rhizobia g of soil(-1). Bradyrhizobium japonicum strains were inoculated either on the soybean seeds or in the sowing furrows. Nodule occupation was doubled when the strains were inoculated in the sowing furrows with respect to seed inoculation (significant with P<0.05). On comparing strains, nodule occupation with seed inoculation was 6% or 10% for the wild type or selected strains, respectively, without a statistically significant difference, while when inoculated in the sowing furrows, nodule occupation increased to 12% and 22%, respectively (differences significant with P<0.05).

Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots.

The exopolysaccharide (EPS) is an extracellular molecule that in Bradyrhizobium japonicum affects bacterial efficiency to nodulate soybean. Culture conditions such as N availability, type of C-source, or culture age can modify the amount and composition of EPS. To better understand the relationship among these conditions for EPS production, we analyzed their influence on EPS in B. japonicum USDA 110 and its derived mutant DeltaP22. This mutant has a deletion including the 3' region of exoP, exoT, and the 5' region of exoB, and produces a shorter EPS devoid of galactose. The studies were carried out in minimal media with the N-source at starving or sufficient levels, and mannitol or malate as the only C-source. Under N-starvation there was a net EPS accumulation, the levels being similar in the wild type and the mutant with malate as the C-source. By contrast, the amount of EPS diminished in N-sufficient conditions, being poyhydroxybutyrate accumulated with culture age. Hexoses composition was the same in both N-situations, either with mannitol or malate as the only C-source, in contrast to previous observations made with different strains. This result suggests that the change in EPS composition in response to the environment is not general in B. japonicum. The wild type EPS composition was 1 glucose:0.5 galactose:0.5 galacturonic acid:0.17 mannose. In DeltaP22 the EPS had no galactose but had galacturonic acid, thus indicating that it was not produced from oxidation of UDP-galactose. Infectivity was lower in DeltaP22 than in USDA 110. When the mutant infectivity was compared between N-starved or N-sufficient cultures, the N-starved were not less infective, despite the fact that the amounts of altered EPS produced by this mutant under N-starvation were higher than in N-sufficiency. Since this altered EPS does not bind soybean lectin, the interaction of EPS with this protein was not involved in increasing DeltaP22 infectivity under N-starvation.