RESUMEN
Bacterial surface motility is a complex microbial trait that contributes to host colonization. However, the knowledge about regulatory mechanisms that control surface translocation in rhizobia and their role in the establishment of symbiosis with legumes is still limited. Recently, 2-tridecanone (2-TDC) was identified as an infochemical in bacteria that hampers microbial colonization of plants. In the alfalfa symbiont Sinorhizobium meliloti, 2-TDC promotes a mode of surface motility that is mostly independent of flagella. To understand the mechanism of action of 2-TDC in S. meliloti and unveil genes putatively involved in plant colonization, Tn5 transposants derived from a flagellaless strain that were impaired in 2-TDC-induced surface spreading were isolated and genetically characterized. In one of the mutants, the gene coding for the chaperone DnaJ was inactivated. Characterization of this transposant and newly obtained flagella-minus and flagella-plus dnaJ deletion mutants revealed that DnaJ is essential for surface translocation, while it plays a minor role in swimming motility. DnaJ loss-of-function reduces salt and oxidative stress tolerance in S. meliloti and hinders the establishment of efficient symbiosis by affecting nodule formation efficiency, cellular infection, and nitrogen fixation. Intriguingly, the lack of DnaJ causes more severe defects in a flagellaless background. This work highlights the role of DnaJ in the free-living and symbiotic lifestyles of S. meliloti.
Asunto(s)
Fijación del Nitrógeno , Sinorhizobium meliloti , Fijación del Nitrógeno/genética , Sinorhizobium meliloti/genética , Simbiosis/genética , Medicago sativa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Rhizobia are soil bacteria that can establish a symbiotic association with legumes. As a result, plant nodules are formed on the roots of the host plants where rhizobia differentiate to bacteroids capable of fixing atmospheric nitrogen into ammonia. This ammonia is transferred to the plant in exchange of a carbon source and an appropriate environment for bacterial survival. This process is subjected to a tight regulation with several checkpoints to allow the progression of the infection or its restriction. The type 3 secretion system (T3SS) is a secretory system that injects proteins, called effectors (T3E), directly into the cytoplasm of the host cell, altering host pathways or suppressing host defense responses. This secretion system is not present in all rhizobia but its role in symbiosis is crucial for some symbiotic associations, showing two possible faces as Dr. Jekyll and Mr. Hyde: it can be completely necessary for the formation of nodules, or it can block nodulation in different legume species/cultivars. In this review, we compile all the information currently available about the effects of different rhizobial effectors on plant symbiotic phenotypes. These phenotypes are diverse and highlight the importance of the T3SS in certain rhizobium-legume symbioses.
Asunto(s)
Fabaceae , Rhizobium , Amoníaco/metabolismo , Carbono/metabolismo , Fabaceae/metabolismo , Nitrógeno/metabolismo , Fijación del Nitrógeno/fisiología , Rhizobium/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , Suelo , Simbiosis/fisiología , Sistemas de Secreción Tipo III/metabolismo , Verduras/metabolismoRESUMEN
Bacteria can spread on surfaces to colonize new environments and access more resources. Rhizobia, a group of α- and ß-Proteobacteria, establish nitrogen-fixing symbioses with legumes that rely on a complex signal interchange between the partners. Flavonoids exuded by plant roots and the bacterial transcriptional activator NodD control the transcription of different rhizobial genes (the so-called nod regulon) and, together with additional bacterial regulatory proteins (such as TtsI, MucR or NolR), influence the production of different rhizobial molecular signals. In Sinorhizobium fredii HH103, flavonoids and NodD have a negative effect on exopolysaccharide production and biofilm production. Since biofilm formation and motility are often inversely regulated, we have analysed whether flavonoids may influence the translocation of S. fredii HH103 on surfaces. We show that the presence of nod gene-inducing flavonoids does not affect swimming but promotes a mode of surface translocation, which involves both flagella-dependent and -independent mechanisms. This surface motility is regulated in a flavonoid-NodD1-TtsI-dependent manner, relies on the assembly of the symbiotic type 3 secretion system (T3SS), and involves the participation of additional modulators of the nod regulon (NolR and MucR1). To our knowledge, this is the first evidence indicating the participation of T3SS in surface motility in a plant-interacting bacterium. Interestingly, flavonoids acting as nod-gene inducers also participate in the inverse regulation of surface motility and biofilm formation, which could contribute to a more efficient plant colonisation.
Asunto(s)
Rhizobium , Sinorhizobium fredii , Proteínas Bacterianas/metabolismo , Flavonoides/metabolismo , Flavonoides/farmacología , Regulación Bacteriana de la Expresión Génica , Plantas/metabolismo , Rhizobium/metabolismo , Sinorhizobium fredii/metabolismo , Simbiosis/fisiología , Sistemas de Secreción Tipo III/metabolismoRESUMEN
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.
Asunto(s)
Glycine max/microbiología , Lipopolisacáridos/química , Sinorhizobium fredii/química , Simbiosis , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Antígenos de Superficie/inmunología , Proteínas Bacterianas/genética , Conformación de Carbohidratos , Espectroscopía de Resonancia Magnética con Carbono-13 , Epítopos/inmunología , Lipopolisacáridos/inmunología , Espectroscopía de Protones por Resonancia Magnética , Sinorhizobium fredii/genética , Sinorhizobium fredii/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Azúcares Ácidos/químicaRESUMEN
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.
Asunto(s)
Fabaceae , Raíces de Plantas , Polisacáridos Bacterianos/metabolismo , Rhizobium/metabolismo , Simbiosis/fisiología , Fabaceae/metabolismo , Fabaceae/microbiología , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiologíaRESUMEN
Sinorhizobium fredii HH103 RifR is a broad host-range rhizobial strain able to nodulate with soybean and Lotus burttii, but it is ineffective with L. japonicus. Here, we study the role of the HH103 RifR SyrM protein in the regulation of gene expression and its relevance in symbiosis with those three legumes. RNAseq analyses show that HH103 SyrM is an important transcriptional regulator not only in the presence of inducer flavonoids but also in its absence. Lack of SyrM increases Nod factors production and decreases genistein-mediated repression of exopolysaccharide production in HH103. In symbiosis, mutation of syrM partially impaired interaction with soybean but improves effectiveness with L. burttii and extends the host-rage to L. japonicus Gifu. In addition, HH103 syrM mutants enter in both Lotus species by infection threads, whereas HH103 uses the more primitive intercellular infection to enter into L. burttii roots These symbiotic phenotypes were previously observed in two other HH103 mutants affected in symbiotic regulators, nodD2 and nolR, revealing that in S. fredii HH103 numerous transcriptional regulators finely modulate symbiotic gene expression.
Asunto(s)
Genes Bacterianos/genética , Glycine max/microbiología , Lotus/microbiología , Nodulación de la Raíz de la Planta/genética , Simbiosis/genética , Proteínas Bacterianas/metabolismo , Silenciador del Gen , Especificidad del Huésped/genética , Mutación , Fenotipo , Raíces de Plantas/metabolismo , Rhizobium/genética , Sinorhizobium fredii/genéticaRESUMEN
The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis.
Asunto(s)
Lotus , Sinorhizobium fredii , Sinorhizobium , Proteínas Bacterianas/genética , Nodulación de la Raíz de la Planta , Sinorhizobium fredii/genética , Simbiosis , Sistemas de Secreción Tipo IIIRESUMEN
Sinorhizobium fredii HH103 RifR , a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR . Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involved.
Asunto(s)
Lotus/microbiología , Enfermedades de las Plantas/microbiología , Sinorhizobium fredii/fisiología , Especificidad del Huésped , Mutación , Raíces de Plantas/microbiología , Sinorhizobium fredii/genética , Sinorhizobium fredii/aislamiento & purificaciónRESUMEN
Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing symbiotic associations with clover plants (Trifolium spp.). Surface polysaccharides, transport systems, and extracellular components synthesized by this bacterium are required for both the adaptation to changing environmental conditions and successful infection of host plant roots. The pssZ gene located in the Pss-I region, which is involved in the synthesis of extracellular polysaccharide, encodes a protein belonging to the group of serine/threonine protein phosphatases. In this study, a comparative transcriptomic analysis of R. leguminosarum bv. trifolii wild-type strain Rt24.2 and its derivative Rt297 carrying a pssZ mutation was performed. RNA-Seq data identified a large number of genes differentially expressed in these two backgrounds. Transcriptome profiling of the pssZ mutant revealed a role of the PssZ protein in several cellular processes, including cell signalling, transcription regulation, synthesis of cell-surface polysaccharides and components, and bacterial metabolism. In addition, we show that inactivation of pssZ affects the rhizobial ability to grow in the presence of different sugars and at various temperatures, as well as the production of different surface polysaccharides. In conclusion, our results identified a set of genes whose expression was affected by PssZ and confirmed the important role of this protein in the rhizobial regulatory network.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Rhizobium leguminosarum/genética , Transcriptoma , Proteínas Bacterianas/genética , Polisacáridos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Rhizobium leguminosarum/enzimología , Rhizobium leguminosarum/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Glycine max/genética , Glycine max/microbiología , Estrés Oxidativo/genética , Sinorhizobium fredii/genética , Simbiosis/genética , Fabaceae/genética , Fabaceae/microbiología , Genes Bacterianos/genética , Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Fijación del Nitrógeno/genética , Rhizobium leguminosarum/genética , Sideróforos/genética , Sinorhizobium meliloti/genética , Transcripción Genética/genéticaRESUMEN
Reversible phosphorylation is a key mechanism that regulates many cellular processes in prokaryotes and eukaryotes. In prokaryotes, signal transduction includes two-component signaling systems, which involve a membrane sensor histidine kinase and a cognate DNA-binding response regulator. Several recent studies indicate that alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) also play an essential role in regulation of many different processes in bacteria, such as growth and cell division, cell wall biosynthesis, sporulation, biofilm formation, stress response, metabolic and developmental processes, as well as interactions (either pathogenic or symbiotic) with higher host organisms. Since these enzymes are not DNA-binding proteins, they exert the regulatory role via post-translational modifications of their protein targets. In this review, we summarize the current knowledge of STKs and STPs, and discuss how these enzymes mediate gene expression in prokaryotes. Many studies indicate that regulatory systems based on Hanks-type STKs and STPs play an essential role in the regulation of various cellular processes, by reversibly phosphorylating many protein targets, among them several regulatory proteins of other signaling cascades. These data show high complexity of bacterial regulatory network, in which the crosstalk between STK/STP signaling enzymes, components of TCSs, and the translational machinery occurs. In this regulation, the STK/STP systems have been proved to play important roles.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Fosfoproteínas Fosfatasas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glycine max/microbiología , Lotus/microbiología , Regulón/genética , Sinorhizobium fredii/fisiología , Simbiosis , Proteínas Bacterianas/genética , Flavonoides/metabolismo , Fijación del Nitrógeno , Nodulación de la Raíz de la Planta , Análisis de Secuencia de ARN , Sinorhizobium fredii/genéticaRESUMEN
Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.
Asunto(s)
Lotus/microbiología , Polisacáridos Bacterianos/metabolismo , Sinorhizobium fredii/fisiología , Simbiosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Especificidad del Huésped , Lotus/citología , Lotus/fisiología , Mutación , Fenotipo , Nodulación de la Raíz de la Planta , Raíces de Plantas/citología , Raíces de Plantas/microbiología , Raíces de Plantas/fisiología , Polisacáridos Bacterianos/química , Purinas/metabolismo , Pirimidinas/metabolismo , Sinorhizobium fredii/citología , Sinorhizobium fredii/genéticaRESUMEN
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.
Asunto(s)
Genoma Bacteriano , Glycine max/microbiología , Sinorhizobium fredii/genética , Genes Bacterianos , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Raíces de Plantas/microbiología , Polisacáridos Bacterianos/genética , Percepción de Quorum , Sinorhizobium fredii/fisiología , Simbiosis/genéticaRESUMEN
(1) Background: Some rhizobia, such as Rhizobium tropici CIAT 899, activate nodulation genes when grown under osmotic stress. This work aims to determine whether this phenomenon also takes place in Sinorhizobium fredii HH103. (2) Methods: HH103 was grown with and without 400 mM mannitol. ß-galactosidase assays, nodulation factor extraction, purification and identification by mass spectrometry, transcriptomics by RNA sequencing, motility assays, analysis of acyl-homoserine lactones, and indole acetic acid quantification were performed. (3) Results: Non-ionic osmotic stress induced the production of nodulation factors. Forty-two different factors were detected, compared to 14 found in the absence of mannitol. Transcriptomics indicated that hundreds of genes were either activated or repressed upon non-ionic osmotic stress. The presence of 400 mM mannitol induced the production of indole acetic acid and acyl homoserine lactones, abolished swimming, and promoted surface motility. (4) Conclusions: In this work, we show that non-ionic stress in S. fredii HH103, caused by growth in the presence of 400 mM mannitol, provokes notable changes not only in gene expression but also in various bacterial traits, including the production of nodulation factors and other symbiotic signals.
RESUMEN
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.
RESUMEN
Sinorhizobium fredii HH103 is a fast-growing rhizobial strain that is able to nodulate legumes that develop determinate nodules, e.g., soybean, and legumes that form nodules of the indeterminate type. Here we present the genome of HH103, which consists of one chromosome and five plasmids with a total size of 7.22 Mb.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Genoma Bacteriano , Sinorhizobium fredii/genética , Cromosomas Bacterianos , Datos de Secuencia Molecular , Plásmidos , Análisis de Secuencia de ADN , Sinorhizobium fredii/aislamiento & purificación , Sinorhizobium fredii/fisiología , Glycine max/microbiología , SimbiosisRESUMEN
Rhizobial NodD proteins and appropriate flavonoids induce rhizobial nodulation gene expression. In this study, we show that the nodD1 gene of Sinorhizobium fredii HH103, but not the nodD2 gene, can restore the nodulation capacity of a double nodD1/nodD2 mutant of Rhizobium tropici CIAT 899 in bean plants (Phaseolus vulgaris). S. fredii HH103 only induces pseudonodules in beans. We have also studied whether the mutation of different symbiotic regulatory genes may affect the symbiotic interaction of HH103 with beans: ttsI (the positive regulator of the symbiotic type 3 protein secretion system), and nodD2, nolR and syrM (all of them controlling the level of Nod factor production). Inactivation of either nodD2, nolR or syrM, but not that of ttsI, affected positively the symbiotic behavior of HH103 with beans, leading to the formation of colonized nodules. Acetylene reduction assays showed certain levels of nitrogenase activity that were higher in the case of the nodD2 and nolR mutants. Similar results have been previously obtained by our group with the model legume Lotus japonicus. Hence, the results obtained in the present work confirm that repression of Nod factor production, provided by either NodD2, NolR or SyrM, prevents HH103 to effectively nodulate several putative host plants.
RESUMEN
Quorum sensing (QS) is a bacterial cell-to-cell signaling mechanism that collectively regulates and synchronizes behaviors by means of small diffusible chemical molecules. In rhizobia, QS systems usually relies on the synthesis and detection of N-acyl-homoserine lactones (AHLs). In the model bacterium Sinorhizobium meliloti functions regulated by the QS systems TraI-TraR and SinI-SinR(-ExpR) include plasmid transfer, production of surface polysaccharides, motility, growth rate and nodulation. These systems are also present in other bacteria of the Sinorhizobium genus, with variations at the species and strain level. In Sinorhizobium fredii NGR234 phenotypes regulated by QS are plasmid transfer, growth rate, sedimentation, motility, biofilm formation, EPS production and copy number of the symbiotic plasmid (pSym). The analysis of the S. fredii HH103 genomes reveal also the presence of both QS systems. In this manuscript we characterized the QS systems of S. fredii HH103, determining that both TraI and SinI AHL-synthases proteins are responsible of the production of short- and long-chain AHLs, respectively, at very low and not physiological concentrations. Interestingly, the main HH103 luxR-type genes, expR and traR, are split into two ORFs, suggesting that in S. fredii HH103 the corresponding carboxy-terminal proteins, which contain the DNA-binding motives, may control target genes in an AHL-independent manner. The presence of a split traR gene is common in other S. fredii strains.
RESUMEN
Sinorhizobium fredii HH103 secretes through the type III secretion system at least eight nodulation outer proteins (Nops), including the effector NopP. These proteins are necessary for an effective nodulation of soybean. In this work, we show that expression of the nopP gene depended on flavonoids and on the transcriptional regulators NodD1 and TtsI. Inactivation of nopP led to an increase in the symbiotic capacity of S. fredii HH103 to nodulate Williams soybean. In addition, we studied whether Nops affect the expression of the pathogenesis-related genes GmPR1, GmPR2, and GmPR3 in soybean roots and shoots. In the presence of S. fredii HH103, expression of pathogenesis-related (PR) gene PR1 was induced in soybean roots 4 days after inoculation and it increased 8 days after inoculation. The absence of Nops provoked a higher induction of PR1 in both soybean roots and shoots, suggesting that Nops function early, diminishing plant defense responses during rhizobial infection. However, the inactivation of nopP led to a decrease in PR1 expression. Therefore, the absence of NopP or that of the complete set of Nops seems to have opposite effects on the symbiotic performance and on the elicitation of soybean defense responses.