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Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti

BACKGROUND: 3′, 5′cAMP signaling in Sinorhizobium meliloti was recently shown to contribute to the autoregulation of legume infection. In planta, three adenylate cyclases CyaD1, CyaD2 and CyaK, synthesizing 3′, 5′cAMP, together with the Crp-like transcriptional regulator Clr and smc02178, a gene of...

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Autores principales: Mathieu-Demazière, Céline, Poinsot, Véréna, Masson-Boivin, Catherine, Garnerone, Anne-Marie, Batut, Jacques
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222275/
https://www.ncbi.nlm.nih.gov/pubmed/24279347
http://dx.doi.org/10.1186/1471-2180-13-268
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author Mathieu-Demazière, Céline
Poinsot, Véréna
Masson-Boivin, Catherine
Garnerone, Anne-Marie
Batut, Jacques
author_facet Mathieu-Demazière, Céline
Poinsot, Véréna
Masson-Boivin, Catherine
Garnerone, Anne-Marie
Batut, Jacques
author_sort Mathieu-Demazière, Céline
collection PubMed
description BACKGROUND: 3′, 5′cAMP signaling in Sinorhizobium meliloti was recently shown to contribute to the autoregulation of legume infection. In planta, three adenylate cyclases CyaD1, CyaD2 and CyaK, synthesizing 3′, 5′cAMP, together with the Crp-like transcriptional regulator Clr and smc02178, a gene of unknown function, are involved in controlling plant infection. RESULTS: Here we report on the characterization of a gene (smc02179, spdA) at the cyaD1 locus that we predicted to encode a class III cytoplasmic phosphodiesterase. First, we have shown that spdA had a similar pattern of expression as smc02178 in planta but did not require clr nor 3′, 5′cAMP for expression. Second, biochemical characterization of the purified SpdA protein showed that, contrary to expectation, it had no detectable activity against 3′, 5′cAMP and, instead, high activity against the positional isomers 2′, 3′cAMP and 2′, 3′cGMP. Third, we provide direct experimental evidence that the purified Clr protein was able to bind both 2′, 3′cAMP and 3′, 5′cAMP in vitro at high concentration. We further showed that Clr is a 3′, 5′cAMP-dependent DNA-binding protein and identified a DNA-binding motif to which Clr binds. In contrast, 2′, 3′cAMP was unable to promote Clr specific-binding to DNA and activate smc02178 target gene expression ex planta. Fourth, we have shown a negative impact of exogenous 2′, 3′cAMP on 3′, 5′cAMP-mediated signaling in vivo. A spdA null mutant was also partially affected in 3′, 5′cAMP signaling. CONCLUSIONS: SpdA is a nodule-expressed 2′, 3′ specific phosphodiesterase whose biological function remains elusive. Circumstantial evidence suggests that SpdA may contribute insulating 3′, 5′cAMP-based signaling from 2′, 3′ cyclic nucleotides of metabolic origin.
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spelling pubmed-42222752014-11-07 Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti Mathieu-Demazière, Céline Poinsot, Véréna Masson-Boivin, Catherine Garnerone, Anne-Marie Batut, Jacques BMC Microbiol Research Article BACKGROUND: 3′, 5′cAMP signaling in Sinorhizobium meliloti was recently shown to contribute to the autoregulation of legume infection. In planta, three adenylate cyclases CyaD1, CyaD2 and CyaK, synthesizing 3′, 5′cAMP, together with the Crp-like transcriptional regulator Clr and smc02178, a gene of unknown function, are involved in controlling plant infection. RESULTS: Here we report on the characterization of a gene (smc02179, spdA) at the cyaD1 locus that we predicted to encode a class III cytoplasmic phosphodiesterase. First, we have shown that spdA had a similar pattern of expression as smc02178 in planta but did not require clr nor 3′, 5′cAMP for expression. Second, biochemical characterization of the purified SpdA protein showed that, contrary to expectation, it had no detectable activity against 3′, 5′cAMP and, instead, high activity against the positional isomers 2′, 3′cAMP and 2′, 3′cGMP. Third, we provide direct experimental evidence that the purified Clr protein was able to bind both 2′, 3′cAMP and 3′, 5′cAMP in vitro at high concentration. We further showed that Clr is a 3′, 5′cAMP-dependent DNA-binding protein and identified a DNA-binding motif to which Clr binds. In contrast, 2′, 3′cAMP was unable to promote Clr specific-binding to DNA and activate smc02178 target gene expression ex planta. Fourth, we have shown a negative impact of exogenous 2′, 3′cAMP on 3′, 5′cAMP-mediated signaling in vivo. A spdA null mutant was also partially affected in 3′, 5′cAMP signaling. CONCLUSIONS: SpdA is a nodule-expressed 2′, 3′ specific phosphodiesterase whose biological function remains elusive. Circumstantial evidence suggests that SpdA may contribute insulating 3′, 5′cAMP-based signaling from 2′, 3′ cyclic nucleotides of metabolic origin. BioMed Central 2013-11-26 /pmc/articles/PMC4222275/ /pubmed/24279347 http://dx.doi.org/10.1186/1471-2180-13-268 Text en Copyright © 2013 Mathieu-Demazière et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Mathieu-Demazière, Céline
Poinsot, Véréna
Masson-Boivin, Catherine
Garnerone, Anne-Marie
Batut, Jacques
Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti
title Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti
title_full Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti
title_fullStr Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti
title_full_unstemmed Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti
title_short Biochemical and functional characterization of SpdA, a 2′, 3′cyclic nucleotide phosphodiesterase from Sinorhizobium meliloti
title_sort biochemical and functional characterization of spda, a 2′, 3′cyclic nucleotide phosphodiesterase from sinorhizobium meliloti
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222275/
https://www.ncbi.nlm.nih.gov/pubmed/24279347
http://dx.doi.org/10.1186/1471-2180-13-268
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