Characterization of ligand response properties of the CRP protein from Pseudomonas putida

cAMP receptor protein(CRP) plays profound roles in many bacteria as a global regulator.In Escherichia coli,CRP E.coli modulates the expression of many operons involved in carbon catabolism,in response to the fluctuation of intracellular cAMP level caused by carbon catabolism.A crp homologue gene has been identified in the genome of Pseudomonas putida,however,little is known about its cellular function.In this work,we investigated ligand response properties of this CRP protein(CRP P.putida).The results showed that in the presence of exogenous cAMP or cGMP,CRP P.putida can activate the lac promoter in E.coli cya crp mutant.In vitro isothermal titration calorimetry(ITC) assays indicated that CRP P.putida could bind cAMP as well as cGMP.Its affinity to cAMP is much higher than CRP E.coli.Sequence alignment of the CRP proteins suggested that the Thr132 of CRP P.putida(analogous to Ser128 of CRP E.coli) could be the key determinant for all ligand responsive properties observed above.When Thr132 of CRP P.putida is mutated to Serine,two phenomena were observed:(i) its affinity to cAMP or cGMP was reduced to a level similar to CRP E.coli ;(ii) its transcriptional activation activity on E.coli lac promoter was diminished.The potential physiological implications of these ligand binding properties are discussed.

cAMP receptor protein （CRP） plays profound roles in many bacteria as a global regulator. In Escherichia coli, CRPe. coli modulates the expression of many operons involved in carbon catabolism, in response to the fluctuation of intracellular cAMP level caused by carbon catabolism. A crp homologue gene has been identified in the genome of Pseudomonas putida, however, little is known about its cellular function. In this work, we investigated ligand response properties of this CRP protein （CRPp. purida）. The results showed that in the presence of exogenous cAMP or cGMP, CRPp. purida can activate the lac promoter in E. coli cya crp mutant. In vitro isothermal titration calorimetry （ITC） assays indicated that CRPp. putiac, could bind cAMP as well as cGMP. Its affinity to cAMP is much higher than CRPE.ctoni. Sequence alignment of the CRP proteins suggested that the Thr132 of CRPp. purida （analogous to Ser128 of CRPL.. coli） could be the key determinant for all ligand responsive properties observed above. When Thr132 of CRPp.puti,la is mutated to Serine, two phenomena were observed： （i） its affinity to cAMP or cGMP was reduced to a level similar to CRPE coli （ii） its transcriptional activation activity on E. coli lac promoter was diminished. The potential physio- logical implications of these ligand binding properties are discussed.