Methods for Studying Swimming and Surface Motilities in Rhizobia.

Rhizobia are soil proteobacteria able to establish a nitrogen-fixing interaction with legumes. In this interaction, rhizobia must colonize legume roots, infect them, and become hosted inside new organs formed by the plants and called nodules. Rhizobial motility, not being essential for symbiosis, might affect the degree of success of the interaction with legumes. Because of this, the study of rhizobial motility (either swimming or surface motility) might be of interest for research teams working on rhizobial symbiotic performance. In this chapter, we describe the protocols we use in our laboratories for studying the different types of motilities exhibited by Sinorhizobium fredii and Sinorhizobium meliloti, as well as for analyzing the presence of flagella in these bacteria. All these protocols might be used (or adapted) for studying bacterial motility in rhizobia.

A New, Nondestructive, Split-Root System for Local and Systemic Plant Responses Studies with Soybean.

Plants use long-distance signaling mechanisms to coordinate their growth and control their interactions, positive or negative, with microbes. Split-root systems (SRS) have been used to study the relevance of both local and systemic plant mechanisms that participate in the control of rhizobia-legume symbioses. In this work we have developed a modification of the standard split-root system (SRS) used with soybean. This modified method, unlike previous systems, operates in hydroponics conditions and therefore is nondestructive and allows for the continuous monitoring of soybean roots throughout the whole experiment.

The underlying process of early ecological and genetic differentiation in a facultative mutualistic Sinorhizobium meliloti population.

The question of how genotypic and ecological units arise and spread in natural microbial populations remains controversial in the field of evolutionary biology. Here, we investigated the early stages of ecological and genetic differentiation in a highly clonal sympatric Sinorhizobium meliloti population. Whole-genome sequencing revealed that a large DNA region of the symbiotic plasmid pSymB was replaced in some isolates with a similar synteny block carrying densely clustered SNPs and displaying gene acquisition and loss. Two different versions of this genomic island of differentiation (GID) generated by multiple genetic exchanges over time appear to have arisen recently, through recombination in a particular clade within this population. In addition, these isolates display resistance to phages from the same geographic region, probably due to the modification of surface components by the acquired genes. Our results suggest that an underlying process of early ecological and genetic differentiation in S. meliloti is primarily triggered by acquisition of genes that confer resistance to soil phages within particular large genomic DNA regions prone to recombination.

Bacterial Molecular Signals in the Sinorhizobium fredii-Soybean Symbiosis.

Sinorhizobium (Ensifer) fredii (S. fredii) is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides), and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system.

Sinorhizobium fredii†HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the Inverted Repeat-Lacking Clade legume Glycyrrhiza uralensis.

In rhizobial species that nodulate inverted repeat-lacking clade (IRLC) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had deoxyribonucleic acid (DNA) contents, cellular sizes and survival rates similar to those of free-living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation.

Structure and biological roles of Sinorhizobium fredii HH103 exopolysaccharide.

Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum.

The Sinorhizobium fredii HH103 lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules.

In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean.

Sinorhizobium fredii HH103 rkp-3 genes are required for K-antigen polysaccharide biosynthesis, affect lipopolysaccharide structure and are essential for infection of legumes forming determinate nodules.

The Sinorhizobium fredii HH103 rkp-3 region has been isolated and sequenced. Based on the similarities between the S. fredii HH103 rkpL, rkpM, rkpN, rkpO, rkpP, and rkpQ genes and their corresponding orthologues in Helicobacter pylori, we propose a possible pathway for the biosynthesis of the S. fredii HH103 K-antigen polysaccharide (KPS) repeating unit. Three rkp-3 genes (rkpM, rkpP, and rkpQ) involved in the biosynthesis of the HH103 KPS repeating unit (a derivative of the pseudaminic acid) have been mutated and analyzed. All the rkp-3 mutants failed to produce KPS and their lipopolysaccharide (LPS) profiles were altered. These mutants showed reduced motility and auto-agglutinated when early-stationary cultures were further incubated under static conditions. Glycine max, Vigna unguiculata (determinate nodule-forming legumes), and Cajanus cajan (indeterminate nodules) plants inoculated with mutants in rkpM, rkpQ, or rkpP only formed pseudonodules that did not fix nitrogen and were devoid of bacteria. In contrast, another indeterminate nodule-forming legume, Glycyrrhiza uralensis, was still able to form some nitrogen-fixing nodules with the three S. fredii HH103 rifampicin-resistant rkp-3 mutants tested. Our results suggest that the severe symbiotic impairment of the S. fredii rkp-3 mutants with soybean, V. unguiculata, and C. cajan is mainly due to the LPS alterations rather than to the incapacity to produce KPS.

Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions.

Rhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.

Sinorhizobium fredii HH103 does not strictly require KPS and/or EPS to nodulate Glycyrrhiza uralensis, an indeterminate nodule-forming legume.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.

The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea.

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium-legume interaction.

Gene SMb21071 of plasmid pSymB is required for osmoadaptation of Sinorhizobium meliloti 1021 and is implicated in modifications of cell surface polysaccharides structure in response to hyperosmotic stress.

Megaplasmid pSymB of the nitrogen-fixing symbiont Sinorhizobium meliloti, implicated in adaptation to hyperosmotic stress, contains 11 gene clusters that apparently encode surface polysaccharides. However, only 2 of these clusters, containing the exo and exp genes, have been associated with the synthesis of the acidic exopolysaccharides succinoglycan and galactoglucan, respectively. The functions of the other 9 clusters remain unsolved. The involvement of one of those regions, pSymB cluster 3, on surface polysaccharide synthesis and its possible implication in osmoadaptation were investigated. In silico analysis of cluster 3 showed that it putatively encodes for the synthesis and transport of a methylated surface polysaccharide. Mutants affected in this cluster were symbiotically effective but showed defects in growth under saline and nonsaline osmotic stress. The gene SMb21071, encoding a putative initiating glycosyltransferase, is transcriptionally induced under hyperosmotic conditions. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and silver staining showed that osmotic stresses changed the profiles of surface polysaccharides of wild-type and mutants strains in different ways. The overall results suggest that cluster 3 is important for growth under saline stress and essential for growth under nonsaline hyperosmotic stress, and it appears to be implicated in maintaining and (or) modifying surface polysaccharides in response to osmotic stress.

Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinate- and indeterminate nodule-forming legumes and overproduce an altered EPS.

Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes.

Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution.

A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes

Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution (AU)

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Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.

A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide.

The pleiotropic phenotype of an auxotrophic purL mutant (SVQ295) of Sinorhizobium fredii HH103 has been investigated. SVQ295 forms colonies that are translucent, produce more slime and absorb less Congo red than those of wild-type strain HH103. SVQ295 did not grow in minimal medium unless the culture was supplemented with thiamin and adenine or with thiamin and AICA-riboside (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), an intermediate of purine biosynthesis. Bacterial cultures supplemented with AICA-riboside or adenine reached the same culture density, although the doubling time of SVQ295 cultures containing AICA-riboside was clearly longer. S. fredii SVQ295 induced pseudonodules on Glycine max and failed to nodulate six different legumes. On Glycyrrhiza uralensis, however, nodules showing nitrogenase activity and containing infected plant cells were formed. SVQ295 showed auto-agglutination when grown in liquid TY medium and its lipopolysaccharide (LPS) electrophoretic profile differed from that of its parental strain HH103-1. In addition, four monoclonal antibodies that recognize the LPS of S. fredii HH103 failed to recognize the LPS produced by SVQ295. In contrast, (1)H-NMR spectra of K-antigen capsular polysaccharides (KPS) produced by SVQ295 and the wild-type strain HH103 were similar. Co-inoculation of soybean plants with SVQ295 and SVQ116 (a nodA mutant derivative of HH103) produced nitrogen-fixing nodules that were only occupied by SVQ116.

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr).

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD- nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.

Alfalfa nodulation by Sinorhizobium fredii does not require sulfated Nod-factors.

Rhizobium strain 042B(s) is able to nodulate both soybean and alfalfa cultivars. We have demonstrated, by mass spectrometry, that the nodulation (Nod) factors produced by this strain are characteristic of those produced by Sinorhizobium fredii, which typically nodulates soybean; they have 3-5 N-acetylglucosamine (GlcNAc) residues, a mono-unsaturated or saturated C16, C18 or C20 fatty-acyl chain, and a (methyl)fucosyl residue on C6 of the reducing-terminal GlcNAc. In order to study Rhizobium strain 042B(s) and its nodulation behaviour further, we introduced an insertion mutation in the noeL gene, which is responsible for the presence of the (methyl)fucose residue on the reducing terminal GlcNAc of the Nod-factors, yielding mutant strain SVQ523. A plasmid (pHM500) carrying nodH, nodP and nodQ, the genes involved in sulfation of Nod-factors on C6 of the reducing-terminal GlcNAc, was introduced into SVQ523, generating SVQ523.pHM500. As expected, strain SVQ523 produces unfucosylated Nod-factors, while SVQ523.pHM500 produces both unfucosylated and unfucosylated sulfated Nod-factors. Plant tests showed that soybean nodulation was reduced if the inoculant (SVQ523.pHM500) produced sulfated Nod-factors. In the Asiatic alfalfa cultivar Baoding, SVQ523 (absence of a substitution at C6) failed to nodulate, but both 042B(s) (fucosyl at C6) and SVQ523.pHM500 (sulfate at C6) formed nodules. In contrast, SVQ523 showed enhanced nodulation capacity with the western alfalfa cultivars ORCA and ARC. These results indicate that Nod-factor sulfation is not a requisite for S. fredii to nodulate alfalfa.