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

Mathieu-Demazière C, Poinsot V, Masson-Boivin C, Garnerone AM, Batut J - BMC Microbiol. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, F-31326 Castanet-Tolosan, France. Jacques.Batut@toulouse.inra.fr.

ABSTRACT

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 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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2′, 3′cAMP effect on Clr-DNA binding and smc02178 expression. (A, B) EMSA assays showing Clr binding to 28-mers oligomers including the wt Clr-box (A) or a mutated version (B), as in Figure 5. Assays were performed in the presence of 1.75 nM oligomers, 200 μM 3′, 5′cAMP and/or 200 μM 2′, 3′cAMP, and 69 μM Clr (for details, see methods). (C) smc02178-lacZ expression was monitored ex planta in S. meliloti 1021 WT and a ΔSpdA strain after addition of M. sativa shoots extract (MS) and/or 7.5 mM 2′, 3′cAMP. *p < 0.03 compared to the wild type.
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Figure 6: 2′, 3′cAMP effect on Clr-DNA binding and smc02178 expression. (A, B) EMSA assays showing Clr binding to 28-mers oligomers including the wt Clr-box (A) or a mutated version (B), as in Figure 5. Assays were performed in the presence of 1.75 nM oligomers, 200 μM 3′, 5′cAMP and/or 200 μM 2′, 3′cAMP, and 69 μM Clr (for details, see methods). (C) smc02178-lacZ expression was monitored ex planta in S. meliloti 1021 WT and a ΔSpdA strain after addition of M. sativa shoots extract (MS) and/or 7.5 mM 2′, 3′cAMP. *p < 0.03 compared to the wild type.

Mentions: Clr is a predicted transcriptional activator of the Crp family [3]. Inspection of the smc02178 promoter region pointed to a short palindromic sequence (TGTTCCGCGGGAAACA) centered ca. 68 bp upstream of the predicted start codon that was a potential binding site for Clr. Accordingly, deletion of this motif abolished activation of the smc02178 promoter by clr in the presence of exogenously provided 3′, 5′cAMP (Figure 5A). In order to directly assess whether this motif was a binding site for the Clr protein, we tested the ability of purified Clr-GST to bind DNA oligomers (28-mers) bracketing the putative Clr-binding motif (Figure 5B) or a mutated version (Figure 5C). We found that Clr induced a retard in oligomer migration that was strictly dependent on the presence of 3′, 5′cAMP, of an intact Clr-box and was Clr concentration-dependent. However, no clear shifted band was observed, irrespectively of the binding and gel electrophoresis conditions tested, which probably reflected dissociation of the Clr/cAMP/DNA complex. Nevertheless we interpreted this as evidence that Clr bound the predicted Clr-box in a 3′, 5′cAMP-dependent manner. 2′, 3′cAMP was unable to promote Clr binding to the Clr-box, at the same concentration as 3′, 5′cAMP. Mixed incubation of the two nucleotides (1/1) with Clr in vitro showed no detectable effect of 2′, 3′cAMP on DNA-binding by Clr (Figure 6A, B).


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

Mathieu-Demazière C, Poinsot V, Masson-Boivin C, Garnerone AM, Batut J - BMC Microbiol. (2013)

2′, 3′cAMP effect on Clr-DNA binding and smc02178 expression. (A, B) EMSA assays showing Clr binding to 28-mers oligomers including the wt Clr-box (A) or a mutated version (B), as in Figure 5. Assays were performed in the presence of 1.75 nM oligomers, 200 μM 3′, 5′cAMP and/or 200 μM 2′, 3′cAMP, and 69 μM Clr (for details, see methods). (C) smc02178-lacZ expression was monitored ex planta in S. meliloti 1021 WT and a ΔSpdA strain after addition of M. sativa shoots extract (MS) and/or 7.5 mM 2′, 3′cAMP. *p < 0.03 compared to the wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222275&req=5

Figure 6: 2′, 3′cAMP effect on Clr-DNA binding and smc02178 expression. (A, B) EMSA assays showing Clr binding to 28-mers oligomers including the wt Clr-box (A) or a mutated version (B), as in Figure 5. Assays were performed in the presence of 1.75 nM oligomers, 200 μM 3′, 5′cAMP and/or 200 μM 2′, 3′cAMP, and 69 μM Clr (for details, see methods). (C) smc02178-lacZ expression was monitored ex planta in S. meliloti 1021 WT and a ΔSpdA strain after addition of M. sativa shoots extract (MS) and/or 7.5 mM 2′, 3′cAMP. *p < 0.03 compared to the wild type.
Mentions: Clr is a predicted transcriptional activator of the Crp family [3]. Inspection of the smc02178 promoter region pointed to a short palindromic sequence (TGTTCCGCGGGAAACA) centered ca. 68 bp upstream of the predicted start codon that was a potential binding site for Clr. Accordingly, deletion of this motif abolished activation of the smc02178 promoter by clr in the presence of exogenously provided 3′, 5′cAMP (Figure 5A). In order to directly assess whether this motif was a binding site for the Clr protein, we tested the ability of purified Clr-GST to bind DNA oligomers (28-mers) bracketing the putative Clr-binding motif (Figure 5B) or a mutated version (Figure 5C). We found that Clr induced a retard in oligomer migration that was strictly dependent on the presence of 3′, 5′cAMP, of an intact Clr-box and was Clr concentration-dependent. However, no clear shifted band was observed, irrespectively of the binding and gel electrophoresis conditions tested, which probably reflected dissociation of the Clr/cAMP/DNA complex. Nevertheless we interpreted this as evidence that Clr bound the predicted Clr-box in a 3′, 5′cAMP-dependent manner. 2′, 3′cAMP was unable to promote Clr binding to the Clr-box, at the same concentration as 3′, 5′cAMP. Mixed incubation of the two nucleotides (1/1) with Clr in vitro showed no detectable effect of 2′, 3′cAMP on DNA-binding by Clr (Figure 6A, B).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, F-31326 Castanet-Tolosan, France. Jacques.Batut@toulouse.inra.fr.

ABSTRACT

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 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.

Show MeSH
Related in: MedlinePlus