Characterization of a partner switching system regulating c-di-GMP levels in Sinorhizobium meliloti. Implication in the synthesis of a novel exopolysaccharide

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 04-02-2016Esta tesis tiene embargado el acceso al texto completo hasta el 04-08-2017Sinorhizobium meliloti produce un β-D-glucano de enlaces alternos (1→3) (1→4) (ML β-glucano) en respuesta a altos niveles de diguanilato cíclico (di-GMP-c). Dos proteínas, BgsB y BgsA, son las responsables de la síntesis, siendo BgsA la glucano sintasa que sensa dichos niveles de di-GMP-c. La transcripción de los genes bgsBA depende del regulador global ExpR, perteneciente al sistema de quorum sensing Sin/ExpR. Este estudio se centra en la caracterización de un operón que regula la síntesis del ML β-glucano en S. meliloti. Dicho operón fue identificado a raíz de un análisis fenotípico de mutantes afectados en el metabolismo del di-GMP-c. De los seis genes que lo constituyen, SMb20447 codifica para una proteína anotada como diguanilato ciclasa/fosfodiesterasa. Se demostró que esta proteína es activa como diguanilato ciclasa y que induce la síntesis del ML β-glucano. A diferencia de lo observado con los genes bgsBA, la transcripción del operón de estudio no es dependiente de ExpR, formando así un sistema de regulación adicional. Al menos tres de las proteínas codificadas en este operón conforman un sistema de “partner switching” semejante al que existe en Bacillus subtilis, el cual se encarga de regular respuestas frente a stress a través de los factores de transcripción σB y σF. Mediante un abordaje genético, hemos determinado que las proteínas SMb20450 y SMb20451, que presentan dominios característicos de fosfatasas y serin-quinasas respectivamente, modulan la actividad represora de SMb20452, una proteína con dominio STAS presente en factores anti-anti-sigma y en transportadores de sulfato. Este sistema regula la actividad diguanilato ciclasa de SMb20447 a nivel posttranscripcional, a través de una posible interacción con SMb20452. El operón parece estar bien conservado en especies de la familia de las Rizobiáceas que también presentan los genes bgsBA, por lo que el sistema de regulación del ML β-glucano en estas bacterias podría ser parecido. En cuanto a su papel biológico, el ML β-glucano es necesario durante el proceso de adhesión a las raíces de alfalfa. Sin embargo, su producción de forma constitutiva no supone ninguna ventaja en relación a este tipo de adhesión. El ML β-glucano no participa en la interacción simbiótica con la planta, ya que no sustituye las funciones desempeñadas por los exopolisacáridos succinoglicano (EPS I) o galactoglucano (EPS II). Un análisis transcriptómico global mostró que el operón SMb20447-SMb20452 podría regular otros procesos como la producción de EPS II y sideróforos, adaptación a condiciones microaeróbicas, o transporte de cationes.Sinorhizobium meliloti synthesizes a mixed-linked (1→3) (1→4)-β-D-glucan (ML β-glucan) in response to high levels of cyclic diguanylate (c-di-GMP). Two proteins, BgsA and BgsB, are required for the synthesis, being BgsA the glucan synthase sensing c-di-GMP levels. The transcription of the bgsBA operon is dependent on the global regulator ExpR, which also forms part of the quorum sensing system Sin/ExpR. This study is focused on the characterization of an operon regulating the synthesis of the ML-β glucan in S. meliloti. The operon was identified as a consequence of a screening of mutants affected in c-di-GMP metabolism. Among the six genes that constitute the operon, SMb20447 codes for a protein annotated as a diguanylate cyclase/phosphodiesterase. We demonstrated this protein is active as a diguanylate cyclase, and triggers the synthesis of the ML β-glucan. Unlike the bgsBA genes, the transcription of the operon is not dependent on ExpR, thus forming and additional regulatory system. At least three of the gene products in the operon seem to form a partner switching system that resembles the one regulating Bacillus subtilis general stress response through sigma factors σB and σF. Using a genetic approach we determined that the proteins SMb20450 and SMb20451, which present putative phosphatase and kinase effector domains respectively, modulate the repressor activity of SMb20452, a STAS (sulphate transporter and anti-sigma antagonist) domain protein. The system regulates the diguanylate cyclase activity of SMb20447 at the posttranscriptional level, probably through a direct interaction with SMb20452. The operon is well conserved in bacteria from the Rhizobiaceae family that present the bgsBA genes, indicating a similar role in these putative ML β-glucan producers. Regarding its biological role, the ML β-glucan proved to be very important for the attachment to alfalfa roots. However, its constitutive expression does not provide any advantage in relation to such attachment. This novel EPS did not present a symbiotic function, since it could not substitute either succynoglican (EPS I) nor galactoglucan (EPS II). Global transcriptomic analysis revealed that the SMb20447-SMb20452 operon might regulate other processes like galactoglucan (EPS II) and siderophore production, adaptation to microaerobic environments, or cation transpor

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

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

Plant responses to inoculation of <i>Glycine max</i> cv. Osumi with <i>S. fredii</i> SMH12 and derivatives.

<p>Data represent means ± sd of six soybean jars. Each jar contained two soybean plants. Determinations were made six weeks after inoculation. Mutant <i>nodD1</i> and the lactonase strain parameters were individually compared with the parental strain SMH12 parameters by using the Mann-Whitney non-parametric test. Values tagged by * are significantly different at the level α = 5%.</p><p>Plant responses to inoculation of <i>Glycine max</i> cv. Osumi with <i>S. fredii</i> SMH12 and derivatives.</p

Visualization of the soybean root colonization.

<p>A. Epifluorescence microscopy analysis of the colonization of the soybean rhizosphere by gfp-tagged bacteria [SMH12, SVQ648, SMH12 (pME6863)]. Roots were visualized 7 days after inoculation. 1. Proximal root. 2. Lateral roots. Bar, 100 µm. B. Scanning microscopy analysis of the colonization of the soybean rhizosphere by SMH12, SVQ648 and SMH12 (pME6863). Roots were visualized 7 days after inoculation. 1. Proximal root. 2. Lateral roots. Bar, 5 µm. SMH12: wild-type, SVQ648: <i>nodD1</i> mutant, SMH12 (pME6863): lactonase strain.</p

Biofilm structure of <i>S. fredii</i> SMH12 and derivatives on glass surfaces: reconstruction of the Z-stacks and measure of the surface coverage.

<p>Main fluorescence value of the wild-type strain was arbitrarily given a value of 100. Averages and standard deviations of five randomized optical fields per strain corresponding to two independent experiments are shown. The asterisks indicate a significant different at the level α = 5% with respect to wild-type strain by using the Mann-Whitney non-parametrical test. Left side corresponds to cultures without flavonoids. Right side corresponds to cultures with inducing flavonoid. A. SMH12. B. SVQ648. C. SMH12 (pME6863). D. SMH12 (pME6000). Bar, 20 µm. SMH12: wild-type, SVQ648: <i>nodD1</i> mutant, SMH12 (pME6863): lactonase strain. SMH12 (pME6000): carrying the empty plasmid.</p

β-galactosidase activity obtained using an adapted assay with <i>A. tumefaciens</i> NT1 (pZLR4) as bioreporter and grown in the presence of supernatants from biofilm cultures (1% v/v).

<p>Data are the mean ± SD of three independent experiments performed in triplicate.</p><p>n: percentage of induction of each supernatant with respect to SMH12 without flavonoids, defined as 100%.</p><p>Each β-galactosidase activity using biofilm supernatant was individually compared to that obtained in SMH12 without flavonoids by using the Mann-Whitney non-parametrical test. Numbers on the percentage of induction column followed by * are significantly different at the level α = 5%.</p><p>β-galactosidase activity obtained using an adapted assay with <i>A. tumefaciens</i> NT1 (pZLR4) as bioreporter and grown in the presence of supernatants from biofilm cultures (1% v/v).</p

Biofilm structure of <i>S. fredii</i> SMH12 and derivatives on glass surfaces: reconstruction of the XY-axis, XZ-axis and YZ-axis. The top corresponds to cultures without flavonoids.

<p>The bottom corresponds to cultures with inducing flavonoid. A. SMH12. B. SVQ648. C. SMH12 (pME6863). D. SMH12 (pME6000). Bar, 20 µm. SMH12: wild-type, SVQ648: <i>nodD1</i> mutant, SMH12 (pME6863): lactonase strain. SMH12 (pME6000): carrying the empty plasmid.</p

Adhesion of <i>S. fredii</i> SMH12 and derivatives on polystyrene surfaces.

<p>Biofilms were measured as the amount of crystal violet absorbed by the biofilm formed on multi-well plates and determined by absorbance at 570 nm after de-staining with ethanol (see methods). Absorbance of the wild-type strain was arbitrarily given a value of 1. Averages and standard deviations of eight replicas per strain corresponding to five independent experiments are shown. The asterisks indicate a significant different at the level α = 5% with respect to wild-type strain by using the Mann-Whitney non-parametrical test. A. Dark gray bars correspond to experiments performed without flavonoids, white to experiments with umbelliferone and light grey bars to experiments with genistein. B. Dark gray bars correspond to experiments performed without flavonoids and light grey bars to experiments with genistein. 3-oxo-C8-HSL and C8-HSL are used at 5.5 µM. C14-HSL is used at 55 µM. SMH12: wild-type, SVQ648: <i>nodD1</i> mutant, SMH12 (pME6863): lactonase strain. SMH12 (pME6000): carrying the empty plasmid.</p

Quantitative RT-PCR analysis of the expression of <i>traI</i> and <i>nodA</i> from <i>S. fredii</i> SMH12 and derivatives from biofilm cultures.

<p>Expression data shown are the mean (± standard deviation of the mean) for three biological replicates. Expression was calculated relative to the expression without flavonoids of the wild-type strain by using the Mann-Whitney non-parametrical test. The asterisks indicate a significant different at the level α = 5%. White bars: biofilm cultures without flavonoids. Gray bars: biofilm cultures with genistein. A. <i>traI</i> relative expression. B. <i>nodA</i> relative expression. SMH12: wild-type, SVQ648: <i>nodD1</i> mutant, SMH12 (pME6863): lactonase strain.</p