Deciphering the Symbiotic Significance of Quorum Sensing Systems of Sinorhizobium fredii HH103

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.Spanish Ministerio de Economía y Competitividad (MINECO) BIO2016-78409-

Diversity of Sinorhizobium (Ensifer) meliloti Bacteriophages in the Rhizosphere of Medicago marina: Myoviruses, Filamentous and N4-Like Podovirus

Using different Sinorhizobium meliloti strains as hosts, we isolated eight new virulent phages from the rhizosphere of the coastal legume Medicago marina. Half of the isolated phages showed a very narrow host range while the other half exhibited a wider host range within the strains tested. Electron microscopy studies showed that phages M_ort18, M_sf1.2, and M_sf3.33 belonged to the Myoviridae family with feature long, contractile tails and icosaedral head. Phages I_sf3.21 and I_sf3.10T appeared to have filamentous shape and produced turbid plaques, which is a characteristic of phages from the Inoviridae family. Phage P_ort11 is a member of the Podoviridae, with an icosahedral head and a short tail and was selected for further characterization and genome sequencing. P_ort11 contained linear, double-stranded DNA with a length of 75239 bp and 103 putative open reading frames. BLASTP analysis revealed strong similarities to Escherichia phage N4 and other N4-like phages. This is the first report of filamentous and N4-like phages that infect S. melilot

The Sinorhizobium fredii HH103 double-edged sword

Rhizobia are soil proteobacteria able to stablish an efficient symbiosis with legume plants (Poole et al., 2018). In this interaction, bacteria infect the plant roots and penetrate inside through the root hairs. Simultaneously to the infection process, plant develop new organs called nodules, generally located on roots, which hosted the rhizobial cells. Once rhizobia are located in the nodules, they invade the plant cells and differentiate into bacteroids, a morphological and physiological state able to fix the atmospheric nitrogen to ammonium, which is supplied to the plant (Tsyganova et al., 2017). This interaction requires a complex and coordinated molecular signals interchange between two partners, since this event determine the susceptible plants to be nodulate by a specific rhizobium and therefore the success of the process (Oldroyd 2013). One of these signals are the molecules called Nod Factors, lipochitooligosaccharides secreted by the bacteria in response to the plant signals and detected by the plant receptors. Several bacterial regulators finely regulate these molecules; however, their overproduction produces changes in the host specificity and the effectiveness of the infection process.Motivation: Since the host specificity is determined by the bacterial and plant signals, the alteration of some of them could modify the bacterial host range and even increase the efficiency with other plants.Methods: All the experiments were carried out with Sinorhizobium fredii HH103, a rhizobial strain isolated from China and natural symbiont of soybean (Glycine max cv. Williams).Gene expression analysis were carried out by RNA-seq and validated by RT-qPCR.Nod Factors were extracted from the supernatant culture and analysed by HPLC-HRMS/MS.Plant assays with Glycine max cv. Williams, Lotus burttii and L. japonicus Gifu were carried out in Leonard jars.Infection mode analyses were carried out by epifluorescence microscopy.Results: The mutation of any regulator that finely regulates the Nod Factor production causes an increase of Nod Factor gene expression, among other changes in the gene expression pattern, and in consequence an overproduction of these molecules. These changes provoke a partial impairment in symbiosis with soybean, its natural host, but on the other hand improve the nodulation effectiveness with L. burttii and allow the gaining the nodulation capacity with L. japonicus Gifu, where the wild type strain is not able to stablish an effective symbiosis. The infection mode analysis revealed that these mutants switched the infection way from intercellular infection, a primitive mode, to infection threads formation, more evolved way.Conclusions: S. fredii HH103 has evolved with its natural host, soybean, to improve their symbiotic performance even though it could diminish or abolish the nodulation effectivity with other legume plants

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.Andalucian Government Grant No. P11-CVI-7500Spanish Government Grant Nos. BIO2013-42801-P and BIO2016-78409-REuropean Regional Development Funds (ERDF)VPPI (V Plan Propio de Investigación) of University of Seville

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-glyc-ero-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 b-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its a-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.Fil: Di Lorenzo, Flaviana. Università degli Studi di Napoli Federico II; ItaliaFil: Speciale, Immacolata. Università degli Studi di Napoli Federico II; ItaliaFil: Silipo, Alba. Università degli Studi di Napoli Federico II; ItaliaFil: Alías Villegas, Cynthia. Universidad de Sevilla; EspañaFil: Acosta Jurado, Sebastián. Universidad de Sevilla; EspañaFil: Rodríguez Carvajal, Miguel Ángel. Universidad de Sevilla; EspañaFil: Dardanelli, Marta Susana. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Biotecnología Ambiental y Salud - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Palmigiano, Angelo. Consiglio Nazionale delle Ricerche; ItaliaFil: Garozzo, Domenico. Consiglio Nazionale delle Ricerche; ItaliaFil: Ruiz Sainz, José Enrique. Universidad de Sevilla; EspañaFil: Molinaro, Antonio. University of Naples Federico II; ItaliaFil: Vinardell, José María. Universidad de Sevilla; Españ

Plant growth promotion in cereal and leguminous agricultural important plants: From microorganism capacities to crop production

Plant growth-promoting rhizobacteria (PGPR) are free-living bacteria which actively colonize plant roots, exerting beneficial effects on plant development. The PGPR may (i) promote the plant growth either by using their own metabolism (solubilizing phosphates, producing hormones or fixing nitrogen) or directly affecting the plant metabolism (increasing the uptake of water and minerals), enhancing root development, increasing the enzymatic activity of the plant or “helping” other beneficial microorganisms to enhance their action on the plants; (ii) or may promote the plant growth by suppressing plant pathogens. These abilities are of great agriculture importance in terms of improving soil fertility and crop yield, thus reducing the negative impact of chemical fertilizers on the environment. The progress in the last decade in using PGPR in a variety of plants (maize, rice, wheat, soybean and bean) along with their mechanism of action are summarized and discussed here

Surface Motility Regulation of Sinorhizobium fredii HH103 by Plant Flavonoids and the NodD1, TtsI, NolR, and MucR1 Symbiotic Bacterial Regulators

Bacteria can spread on surfaces to colonize new environments and access more resources. Rhizobia, a group of α- and β-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