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

Feng Jiang,Yiping Wang,Zhexian Tian

doi:10.1007/s11434-012-5360-3

Abstract

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.

Highlights

CAMP receptor protein (CRP) plays profound roles in many bacteria as a global regulator
In order to investigate the role of P. putida cAMP receptor protein (CRP) as a transcription factor, plasmid expressing CRPP. putida was introduced into the crp cya double mutant E. coli strain BD7000
Substantial lac promoter activity was observed in the strain expressing CRPP. putida even under the condition without any exogenous ligand was supplied in the medium

Summary

Introduction

CAMP receptor protein (CRP) plays profound roles in many bacteria as a global regulator. Coli) is probably the best-characterized protein in the CRP-FNR family It regulates the transcription of more than 200 genes in E. coli [5]. Coli undergoes certain conformational changes, which in turn facilitate recognition of specific DNA target site When it is the sole activator, DNA target site bound CRPE. In E. coli, as the allosteric ligand of CRP protein, cAMP is synthesized by the adenylate cyclase encoded by cyaA. Intracellular cAMP concentration is tightly controlled and fluctuates significantly in response to quality change of carbon source This control is achieved by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Coli-cAMP complex participates mostly in the regulation of genes which involved in the transport and metabolism of carbon and nitrogen source [5]. CRPE. coli can bind cGMP, no evidences show that cGMP-binding could induce CRPE. coli’s conformational change required for DNA binding and transcriptional regulation [12]

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Conclusion