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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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SpdA is expressed in planta, independently of clr. Expression of a spdA-lacZ reporter gene fusion in S. meliloti 1021 [A-C] and clr mutant [D-F], in infection threads (A, D), young nodules (7 dpi) (B, E) and mature nodules (14 dpi) (C, F) of M. sativa. (G) spdA-lacZ expression was monitored ex planta in S. meliloti 1021 strain after addition of 5 mM 3′, 5′cAMP or water as a negative control. smc02178-lacZ was used as a control.
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Figure 2: SpdA is expressed in planta, independently of clr. Expression of a spdA-lacZ reporter gene fusion in S. meliloti 1021 [A-C] and clr mutant [D-F], in infection threads (A, D), young nodules (7 dpi) (B, E) and mature nodules (14 dpi) (C, F) of M. sativa. (G) spdA-lacZ expression was monitored ex planta in S. meliloti 1021 strain after addition of 5 mM 3′, 5′cAMP or water as a negative control. smc02178-lacZ was used as a control.

Mentions: We probed expression of a translational spdA-lacZ fusion (pGD2179, See Additional file 2) that contained the intergenic region between smc02178 and spdA (Figure 1A) as well as the first 12 codons of spdA. The spdA-lacZ fusion did not detectably express ex planta and instead expressed in Medicago sativa nodules with the same pattern as smc02178[3]i.e. expression in young nodule primordia and in zones II and III of mature nodules (Figure 2A-F). However, spdA expression in planta was independent of clr, and ex planta expression could not be induced by exogenous 3′, 5′cAMP, in contrast to smc02178 expression (Figure 2G). None of the environmental conditions or compounds which we have tested was able to stimulate spdA expression ex planta, including 3′, 5′cGMP, 2′, 3′cAMP, 5′AMP, nodule extracts, root exudates or several growth and stress conditions (See Additional file 3).


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)

SpdA is expressed in planta, independently of clr. Expression of a spdA-lacZ reporter gene fusion in S. meliloti 1021 [A-C] and clr mutant [D-F], in infection threads (A, D), young nodules (7 dpi) (B, E) and mature nodules (14 dpi) (C, F) of M. sativa. (G) spdA-lacZ expression was monitored ex planta in S. meliloti 1021 strain after addition of 5 mM 3′, 5′cAMP or water as a negative control. smc02178-lacZ was used as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: SpdA is expressed in planta, independently of clr. Expression of a spdA-lacZ reporter gene fusion in S. meliloti 1021 [A-C] and clr mutant [D-F], in infection threads (A, D), young nodules (7 dpi) (B, E) and mature nodules (14 dpi) (C, F) of M. sativa. (G) spdA-lacZ expression was monitored ex planta in S. meliloti 1021 strain after addition of 5 mM 3′, 5′cAMP or water as a negative control. smc02178-lacZ was used as a control.
Mentions: We probed expression of a translational spdA-lacZ fusion (pGD2179, See Additional file 2) that contained the intergenic region between smc02178 and spdA (Figure 1A) as well as the first 12 codons of spdA. The spdA-lacZ fusion did not detectably express ex planta and instead expressed in Medicago sativa nodules with the same pattern as smc02178[3]i.e. expression in young nodule primordia and in zones II and III of mature nodules (Figure 2A-F). However, spdA expression in planta was independent of clr, and ex planta expression could not be induced by exogenous 3′, 5′cAMP, in contrast to smc02178 expression (Figure 2G). None of the environmental conditions or compounds which we have tested was able to stimulate spdA expression ex planta, including 3′, 5′cGMP, 2′, 3′cAMP, 5′AMP, nodule extracts, root exudates or several growth and stress conditions (See Additional file 3).

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