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

Regulation and symbiotic significance of nodulation outer proteins secretion in Sinorhizobium fredii HH103

In this work we show that the Sinorhizobium fredii HH103 ttsI gene is essential for the expression of the tts genes and secretion of nodulation outer proteins (Nops). Moreover, we demonstrate for the first time, to our knowledge, that the nod box preceding ttsI is necessary for Nops secretion. TtsI is responsible for the transcriptional activation of nopX, nopA, rhcJ and rhcQ. We confirm that the S. fredii HH103 ttsI gene is activated by NodD1 and repressed by NolR. In contrast, NodD2 is not involved in the regulation of ttsI expression. Despite the dependence of expression of both ttsI and nodA on NodD1 and flavonoids, clear differences in the capacity of some flavonoids to activate these genes were found. The expression of the ttsI and nodA genes was also sensitive to differences in the pH of the media. Secretion of Nops in the ttsI mutant could not be complemented with a DNA fragment containing the ttsI gene and its nod box, but it was restored when a plasmid harbouring the ttsI, rhcC2 and y4xK genes was transferred to the mutant strain. The symbiotic effect of Nops secretion was host-dependent but independent of the type of nodule formed by the host legume. Nops are beneficial in the symbiosis with Glycine max and Glycyrrhiza uralensis, and detrimental in the case of the tropical legume Erythrina variegata

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

Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899

Background: Transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes-five-and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants. Methods: The three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.). Results: Phenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions. Conclusions: We report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene-nodD4-might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresse

The symbiotic biofilm of Sinorhizobium fredii SMH12, necessary for successful colonization and symbiosis of glycine max cv osumi, is regulated by quorum sensing systems and inducing Flavonoids via NodD1

Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation) are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis

Rice and bean AHL-mimic quorum-sensing signals specifically interfere with the capacity to form biofilms by plant-associated bacteria

Many bacteria regulate their gene expression in response to changes in their population density in a process called quorum sensing (QS), which involves communication between cells mediated by small diffusible signal molecules termed autoinducers. n-acyl-homoserine-lactones (AHLs) are the most common autoinducers in proteobacteria. QS-regulated genes are involved in complex interactions between bacteria of the same or different species and even with some eukaryotic organisms. Eukaryotes, including plants, can interfere with bacterial QS systems by synthesizing molecules that interfere with bacterial QS systems. In this work, the presence of AHL-mimic QS molecules in diverse Oryza sativa (rice) and Phaseolus vulgaris (bean) plant-samples were detected employing three biosensor strains. A more intensive analysis using biosensors carrying the lactonase enzyme showed that bean and rice seed-extract contain molecules that lack the typical lactone ring of AHLs. Interestingly, these molecules specifically alter the QS-regulated biofilm formation of two plant-associated bacteria, Sinorhizobium fredii SMH12 and Pantoea ananatis AMG501, suggesting that plants are able to enhance or to inhibit the bacterial QS systems depending on the bacterial strain. Further studies would contribute to a better understanding of plant–bacteria relationships at the molecular level

Transferencia y expresión de los plásmidos simbióticos pJB5JI y pRtr5a en diversas estirpes de Rhizobium

Las bacterias de los géneros Rhizobium y Bradyrhizobium pueden infectar las raíces de muchas leguminosas y dar lugar a la formación de unas estructuras semejantes a tumores que reciben el nombre de nódulos

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

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 conlas 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.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.Ministerio de Ciencia e Innovación BIO2011-30229-C02-0

Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant–microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis

NolR Regulates Diverse Symbiotic Signals of Sinorhizobium fredii HH103

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Strr ) 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.Comisión Interministerial de Ciencia y Tecnología (CICYT), Gobierno de España BIO99-0614-C03 y BOS2002-04164-C03-0