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

BMC Microbiology

Jacques Batut¹  Anne-Marie Garnerone¹  Catherine Masson-Boivin¹  Véréna Poinsot²  Céline Mathieu-Demazière¹ 

[1] CNRS, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR2594, F-31326 Castanet-Tolosan, France;Laboratoire IMRCP UMR UPS/CNRS 5623, Université Paul Sabatier, Toulouse, Cedex 31062, France

关键词: Crp;    RNA degradation;    Phosphodiesterase;    2′, 3′cAMP;    3′, 5′cAMP;    Sinorhizobium;   

Others  :  1142604
DOI  :  10.1186/1471-2180-13-268

 received in 2013-07-25, accepted in 2013-11-11,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Mathieu-Demazière et al.; licensee BioMed Central Ltd.

【 图 表 】

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