Phosphorylation of proteins in the light‐dependent signalling pathway of a filamentous cyanobacterium

Abstract

The genome of the filamentous cyanobacterium Calothrix sp. PCC7601 contains two genes, cphA and cphB, encoding proteins with similarity to plant phytochromes and bacterial histidine kinases. In vitro, CphA and CphB readily attach a tetrapyrrole chromophore to develop spectrally active holoproteins that are photointerconvertible between a red light‐absorbing and a far‐red light‐absorbing form. Together with the putative response regulators, RcpA and RcpB, the putative histidine kinases, CphA and CphB, are suggested to constitute two two‐component systems of light‐dependent signal transduction. In this report, we demonstrate the kinase activity of both CphA and CphB. In vitro experiments carried out on the purified proteins show that CphA and CphB are autophosphorylated in the presence of ATP and that phospho‐CphA is capable of efficient phosphotransfer to RcpA as is phospho‐CphB towards RcpB. The autophosphorylation and the phosphorelay are dependent on light. Both activities are reduced under red light vs. far‐red light irradiation. No phosphoryl transfer occurred between phospho‐CphA and RcpB or between phospho‐CphB and RcpA. The response regulators RcpA and RcpB can receive a phosphoryl moiety also from the small phospho‐donor acetyl phosphate. The stability of the phosphorylated regulators is not affected by CphA and CphB or light.

A Non-hydrolyzable ATP Derivative Generates a Stable Complex in a Light-inducible Two-component System

“…CphB, instead, binds biliverdin covalently in its N-terminally located PAS domain, as shown previously by us by homologous expression in Tolypothrix, affinity purification of the recombinant protein, and spectroscopic and kinase activity analysis (12). Irrespective of the differences in their chromophore binding properties, both phytochromes exhibit light-regulated HK activity and a highly specific phosphate transfer upon incubation with their cognate response regulators, RcpA and RcpB (11). Interestingly, both phytochromes showed a high kinase activity and transphosphorylation to the response regulators in their dark-adapted (Pr) states.…”

“…Light induction of HKs has been reported for several systems, amongst which the first identified cyanobacterial phytochrome, Cph1 (3), for bacteriophytochromes (7,8), and even for blue light-sensitive photoreceptors, where a flavin chromophore-binding domain activates an HK just in the same way as in the red light-sensing phytochromes (9). Recently, we have demonstrated light-inducible kinase activity for two cyanobacterial phytochromes, CphA and CphB from Tolypothrix PCC7601, and the transfer of the phosphate group from the histidine in the HK domain to an added response regulator (10,11). These two proteins show a very similar overall architecture (as deduced from sequence comparison, and secondary and tertiary structure prediction); however, they * This work was supported in part by the Helmholtz-Society (VIBS VH-VI-157, to M. E. and W. G.) and the International Max Planck Research School Chemical Biology (to Y. J. K.).…”

Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site

“…2b), Agp1 and Agp2 carry a valin and isoleucin instead. The Cys also is lacking some cyanobacterial phytochromes (21) and all known proteobacterial orthologs (11,31).…”

“…With Agp1, phosphorylation was stronger in the Pr form (Fig. 4); thus, it matches with the cyanobacterial orthologs (19)(20)(21) and not with Pseudomonas BphP (11). Quite interestingly, Apo-Agp1 incorporated more phosphate than Holo-Agp1.…”

“…His-kinase activity is light-modulated; cyanobacterial phytochromes are more active in the Pr form (19)(20)(21), whereas phytochrome BphP from the proteobacterium Pseudomonas aeruginosa is more active in the Pfr form (11). In general, His kinases transphosphorylate particular response regulators (22); this mechanism also has been shown for bacterial phytochromes (11,19,21).…”