A Novel System to Selective Tagging of Sinorhizobium fredii Symbiotic Plasmids.

Conventional systems used to tag and transfer symbiotic plasmids (pSyms) of rhizobial strains are based in mutagenesis with transposons. In those processes, numerous clones must be analyzed to find one of them with the transposon inserted in the pSym. Following this strategy, the insertion might interrupt a gene that can affect the symbiotic phenotype of the bacteria tagged. Here, we have developed a new system based in homologous recombination that generates Sinorhizobium fredii strains with pSyms tagged by the insertion of a suicide vector which harbor a truncated copy of S. fredii HH103 nodZ gene, a mob site, and a kanamycin-resistant gene. When it is introduced by conjugation in a S. fredii strain, the vector integrates in pSym by only one recombination event. This pSym tagged can be transferred in matting experiments to other strains in the presence of a helper plasmid. Following this method, we have tagged several strains and transferred their pSyms to a recipient strain demonstrating the potential of this new system.

A complex regulatory network governs the expression of symbiotic genes in Sinorhizobium fredii HH103.

Introduction: The establishment of the rhizobium-legume nitrogen-fixing symbiosis relies on the interchange of molecular signals between the two symbionts. We have previously studied by RNA-seq the effect of the symbiotic regulators NodD1, SyrM, and TtsI on the expression of the symbiotic genes (the nod regulon) of Sinorhizobium fredii HH103 upon treatment with the isoflavone genistein. In this work we have further investigated this regulatory network by incorporating new RNA-seq data of HH103 mutants in two other regulatory genes, nodD2 and nolR. Both genes code for global regulators with a predominant repressor effect on the nod regulon, although NodD2 acts as an activator of a small number of HH103 symbiotic genes. Methods: By combining RNA-seq data, qPCR experiments, and b-galactosidase assays of HH103 mutants harbouring a lacZ gene inserted into a regulatory gene, we have analysed the regulatory relations between the nodD1, nodD2, nolR, syrM, and ttsI genes, confirming previous data and discovering previously unknown relations. Results and discussion: Previously we showed that HH103 mutants in the nodD2, nolR, syrM, or ttsI genes gain effective nodulation with Lotus japonicus, a model legume, although with different symbiotic performances. Here we show that the combinations of mutations in these genes led, in most cases, to a decrease in symbiotic effectiveness, although all of them retained the ability to induce the formation of nitrogen-fixing nodules. In fact, the nodD2, nolR, and syrM single and double mutants share a set of Nod factors, either overproduced by them or not generated by the wild-type strain, that might be responsible for gaining effective nodulation with L. japonicus.

Rhizobial Exopolysaccharides: Genetic Regulation of Their Synthesis and Relevance in Symbiosis with Legumes.

Rhizobia are soil proteobacteria able to engage in a nitrogen-fixing symbiotic interaction with legumes that involves the rhizobial infection of roots and the bacterial invasion of new organs formed by the plant in response to the presence of appropriate bacterial partners. This interaction relies on a complex molecular dialogue between both symbionts. Bacterial N-acetyl-glucosamine oligomers called Nod factors are indispensable in most cases for early steps of the symbiotic interaction. In addition, different rhizobial surface polysaccharides, such as exopolysaccharides (EPS), may also be symbiotically relevant. EPS are acidic polysaccharides located out of the cell with little or no cell association that carry out important roles both in free-life and in symbiosis. EPS production is very complexly modulated and, frequently, co-regulated with Nod factors, but the type of co-regulation varies depending on the rhizobial strain. Many studies point out a signalling role for EPS-derived oligosaccharides in root infection and nodule invasion but, in certain symbiotic couples, EPS can be dispensable for a successful interaction. In summary, the complex regulation of the production of rhizobial EPS varies in different rhizobia, and the relevance of this polysaccharide in symbiosis with legumes depends on the specific interacting couple.

Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis.

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l-glycero-l-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing ß-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.

Sinorhizobium fredii HH103 RirA Is Required for Oxidative Stress Resistance and Efficient Symbiosis with Soybean.

Members of Rhizobiaceae contain a homologue of the iron-responsive regulatory protein RirA. In different bacteria, RirA acts as a repressor of iron uptake systems under iron-replete conditions and contributes to ameliorate cell damage during oxidative stress. In Rhizobium leguminosarum and Sinorhizobium meliloti, mutations in rirA do not impair symbiotic nitrogen fixation. In this study, a rirA mutant of broad host range S. fredii HH103 has been constructed (SVQ780) and its free-living and symbiotic phenotypes evaluated. No production of siderophores could be detected in either the wild-type or SVQ780. The rirA mutant exhibited a growth advantage under iron-deficient conditions and hypersensitivity to hydrogen peroxide in iron-rich medium. Transcription of rirA in HH103 is subject to autoregulation and inactivation of the gene upregulates fbpA, a gene putatively involved in iron transport. The S. fredii rirA mutant was able to nodulate soybean plants, but symbiotic nitrogen fixation was impaired. Nodules induced by the mutant were poorly infected compared to those induced by the wild-type. Genetic complementation reversed the mutant's hypersensitivity to H2O2, expression of fbpA, and symbiotic deficiency in soybean plants. This is the first report that demonstrates a role for RirA in the Rhizobium-legume symbiosis.

Sinorhizobium fredii Strains HH103 and NGR234 Form Nitrogen Fixing Nodules With Diverse Wild Soybeans (Glycine soja) From Central China but Are Ineffective on Northern China Accessions.

Sinorhizobium fredii indigenous populations are prevalent in provinces of Central China whereas Bradyrhizobium species (Bradyrhizobium japonicum, B. diazoefficiens, B. elkanii, and others) are more abundant in northern and southern provinces. The symbiotic properties of different soybean rhizobia have been investigated with 40 different wild soybean (Glycine soja) accessions from China, Japan, Russia, and South Korea. Bradyrhizobial strains nodulated all the wild soybeans tested, albeit efficiency of nitrogen fixation varied considerably among accessions. The symbiotic capacity of S. fredii HH103 with wild soybeans from Central China was clearly better than with the accessions found elsewhere. S. fredii NGR234, the rhizobial strain showing the broadest host range ever described, also formed nitrogen-fixing nodules with different G. soja accessions from Central China. To our knowledge, this is the first report describing an effective symbiosis between S. fredii NGR234 and G. soja. Mobilization of the S. fredii HH103 symbiotic plasmid to a NGR234 pSym-cured derivative (strain NGR234C) yielded transconjugants that formed ineffective nodules with G. max cv. Williams 82 and G. soja accession CH4. By contrast, transfer of the symbiotic plasmid pNGR234a to a pSym-cured derivative of S. fredii USDA193 generated transconjugants that effectively nodulated G. soja accession CH4 but failed to nodulate with G. max cv. Williams 82. These results indicate that intra-specific transference of the S. fredii symbiotic plasmids generates new strains with unpredictable symbiotic properties, probably due to the occurrence of new combinations of symbiotic signals.

Studies of rhizobial competitiveness for nodulation in soybean using a non-destructive split-root system.

Split-root systems (SRS) constitute an appropriate methodology for studying the relevance of both local and systemic mechanisms that participate in the control of rhizobia-legume symbioses. In fact, this kind of approach allowed to demonstrate the autoregulation of nodulation (AON) systemic response in soybean in the 1980s. In SRS, the plant main root is cut and two lateral roots that emerge from the seedlings after root-tip removal are confined into separate compartments. After several days of growth, these plants have two separate roots that can be inoculated with the same or with different bacteria, at the same or at different times. In this work, we have used a non-destructive SRS to study two different competitiveness relations between rhizobial strains in soybean roots. One of them is the competition for nodulation between two soybean-nodulating rhizobia: the slow-grower Bradyrhizobium japonicum USDA110 and the fast-grower Sinorhizobium fredii HH103. The second is the competitive blocking of S. fredii 257DH4 nodulation in the American soybean Osumi by Sinorhizobium fredii USDA257, which is unable to nodulate American soybeans. Our results showed that the competitiveness relationships studied in this work are mitigated or even avoided when the competitive strains are spatially separated in different compartments containing half-roots from the same plant, suggesting that competitive relations are more related to local plant responses. In our opinion, split-root systems are an appropriate approach to further study competitive relations among rhizobial strains.

Exopolysaccharide Production by Sinorhizobium fredii HH103 Is Repressed by Genistein in a NodD1-Dependent Manner.

In the rhizobia-legume symbiotic interaction, bacterial surface polysaccharides, such as exopolysaccharide (EPS), lipopolysaccharide (LPS), K-antigen polysaccharide (KPS) or cyclic glucans (CG), appear to play crucial roles either acting as signals required for the progression of the interaction and/or preventing host defence mechanisms. The symbiotic significance of each of these polysaccharides varies depending on the specific rhizobia-legume couple. In this work we show that the production of exopolysaccharide by Sinorhizobium fredii HH103, but not by other S. fredii strains such as USDA257 or NGR234, is repressed by nod gene inducing flavonoids such as genistein and that this repression is dependent on the presence of a functional NodD1 protein. In agreement with the importance of EPS for bacterial biofilms, this reduced EPS production upon treatment with flavonoids correlates with decreased biofilm formation ability. By using quantitative RT-PCR analysis we show that expression of the exoY2 and exoK genes is repressed in late stationary cultures of S. fredii HH103 upon treatment with genistein. Results presented in this work show that in S. fredii HH103 EPS production is regulated just in the opposite way than other bacterial signals such as Nod factors and type 3 secreted effectors: it is repressed by flavonoids and NodD1 and enhanced by the nod repressor NolR. These results are in agreement with our previous observations showing that lack of EPS production by S. fredii HH103 is not only non-detrimental but even beneficial for symbiosis with soybean.

The Sinorhizobium fredii HH103 Genome: A Comparative Analysis With S. fredii Strains Differing in Their Symbiotic Behavior With Soybean.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

NopM and NopD are rhizobial nodulation outer proteins: identification using LC-MALDI and LC-ESI with a monolithic capillary column.

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLC-MS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD.

Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA.

Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean / La inactivación del gen rhcJ de Sinorhizobium fredii HH103 elimina la secreción de las proteínas externas de nodulación (Nops) y disminuye la capacidad de simbiosis con la soja

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA (AU)

Se ha propuesto que las proteínas externas de nodulación (Nops) impiden la nodulación efectiva de Sinorhizobium fredii USDA257 con las sojas americanas. S. fredii HH103 nodula de forma natural tanto con las sojas asiáticas (no comercializadas) como con las americanas (comercializadas). La inactivación del gen rhcJ de HH103, que pertenece a la agrupación génica tts (secreción de tipo III), anuló la secreción de Nops y redujo la capacidad simbiótica de esta bacteria con las dos variedades de soja. Las cepas HH103 y USDA257 de S. fredii, que sólo nodula sojas asiáticas, mostraron perfiles SDS-PAGE diferentes de Nop, lo cual sugiere que estas cepas podrían secretar distintos conjuntos de Nops. Cuando las cepas USDA257 y HH103 fueron inoculadas conjuntamente, la capacidad de nodulación de esta última cepa con el cultivar americano Williams de soja se redujo significativamente. Estos resultados indican que las Nops secretadas por S. fredii pueden actuar como factores simbióticos tanto positivos como negativos dependiendo de la cepa-cultivar rizobiana. Se detectaron también diferencias entre la expresión mediada por flavonoides del gen rhcJ y del nodA. Además, una de las Nops secretadas por la cepa HH103 fue identificada como NopA (AU)

NolR regulates diverse symbiotic signals of Sinorhizobium fredii HH103.

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Str(r)) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysaccharide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.

The effect of FITA mutations on the symbiotic properties of Sinorhizobium fredii varies in a chromosomal-background-dependent manner.

nodD1 of Sinorhizobium fredii HH103, which is identical to that of S. fredii USDA257 and USDA191, repressed its own expression. Spontaneous flavonoid-independent transcription activation (FITA) mutants of S. fredii HH103 M (=HH103 RifR pSym::Tn 5-Mob) showing constitutive expression of nod genes were isolated. No differences were found among soybean cultivar Williams plants inoculated with FITA mutants SVQ250 or SVQ253 or with the parental strain HH103M. Soybean plants inoculated with mutant SVQ255 formed more nodules, and those inoculated with mutant SVQ251 had symptoms of nitrogen starvation. Sequence analyses showed that all of the FITA mutants carried a point mutation in their nodD1 coding region. Mutants SVQ251 and SVQ253 carried the same mutation, but only the former was symbiotically impaired, which indicated the presence of an additional mutation elsewhere in the genome of mutant SVQ251. Mutants SVQ251 and SVQ255 were outcompeted by the parental strain for nodulation of soybean cultivar Williams. The symbiotic plasmids of mutants SVQ251 and SVQ255 (pSym251 and pSym255, respectively) and that (pSymHH103M) of the parental strain were transferred to pSym-cured derivatives of S. fredii USDA192 and USDA193 (USDA192C and USDA193C, respectively). Soybean responses to inoculation with S. fredii USDA192C and USDA193C transconjugants carrying pSym251 and pSymHH103M were not significantly different, whereas more nodules were formed after inoculation with transconjugants carrying pSym255. Only transconjugant USDA192C(pSym255) produced a significant increase in soybean dry weight.