An Unorthodox Bacteriophytochrome from Rhodobacter sphaeroides Involved in Turnover of the Second Messenger c-di-GMP

Marina Tarutina,Dmitri A Ryjenkov,Mark Gomelsky

doi:10.1074/jbc.m604819200

Abstract

Bacteriophytochromes are bacterial photoreceptors that sense red/far red light using the biliverdin chromophore. Most bacteriophytochromes work as photoactivated protein kinases. The Rhodobacter sphaeroides bacteriophytochrome BphG1 is unconventional in that it has GGDEF and EAL output domains, which are involved, respectively, in synthesis (diguanylate cyclase) and degradation (phosphodiesterase) of the bacterial second messenger c-di-GMP. The GGDEF-EAL proteins studied to date displayed either diguanylate cyclase or phosphodiesterase activity but not both. To elucidate the function of BphG1, the holoprotein was purified from an Escherichia coli overexpression system designed to produce biliverdin. The holoprotein contained covalently bound biliverdin and interconverted between the red (dark) and far red (light-activated) forms. BphG1 had c-di-GMP-specific phosphodiesterase activity. Unexpectedly for a photochromic protein, this activity was essentially light-independent. BphG1 expressed in E. coli was found to undergo partial cleavage into two species. The smaller species was identified as the EAL domain of BphG1. It possessed c-di-GMP phosphodiesterase activity. Surprisingly, the larger species lacking EAL possessed diguanylate cyclase activity, which was dependent on biliverdin and strongly activated by light. BphG1 therefore is the first phytochrome with a non-kinase photoactivated enzymatic activity. This shows that the photosensory modules of phytochromes can transmit light signals to various outputs. BphG1 is potentially the first "bifunctional" enzyme capable of both c-di-GMP synthesis and hydrolysis. A model for the regulation of the "opposite" activities of BphG1 is presented.

Highlights

We have demonstrated that R. sphaeroides can synthesize c-di-GMP (23); the functions of c-di-GMP in this organism are yet to be identified
We found that BphG1, which is different from the majority of Bphs in that it contains the GGDEF-EAL output module, responds to light in a manner similar to that of the Bphs containing kinase outputs
Our data on the PAS-GAF-PHY-GGDEF fragment of BphG1 suggest that light-induced conformational changes are efficiently transduced to the GGDEF domain to result in activation of the diguanylate cyclase (DGC) activity

Summary

The abbreviations used are

Pr, red-absorbing form of a phytochrome; Pfr, far red-absorbing form of a phytochrome; Bph, bacteriophytochrome; BV, biliverdin; c-di-GMP, cyclic dimeric GMP; DGC, diguanylate cyclase; PDE, phosphodiesterase; HPLC, high-pressure liquid chromatography; MALDITOF, matrix-assisted laser desorption ionization time-of-flight. The GGDEF domains possess diguanylate cyclase (DGC) activity; i.e. they synthesize c-di-GMP from two GTP molecules (23–25). C-di-GMP is degraded into linear dimeric GMP by the EAL domains that possess phosphodiesterase (PDE) activity (24, 27–29). The BphG proteins contain GGDEF and EAL domains arranged in tandem (GGDEF-EAL). These proteins could possess either DGC or PDE activity or both. Prior to this study, no proteins containing both activities have been identified. It was unclear whether the “opposite” activities, i.e. c-di-GMP synthesis and c-di-GMP hydrolysis, could coexist in a single protein, and if they could, how they would be regulated. We found that (i) similar to conventional Bphs, these proteins undergo reversible photoconversion between the Pr and Pfr forms, i.e. they are photochromic; (ii) they possess two enzymatic activities; only one activity is light-dependent; and (iii) they may employ an apparently unique switch between the opposite enzymatic activities

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION