Two independent, light‐sensing two‐component systems in a filamentous cyanobacterium

Abstract

Two ORFs, cphA and cphB, encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp. PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp. PCC7601. While CphA carries a cysteine within a highly conserved amino-acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine. Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two-component signal transduction. In Calothrix, all four genes are expressed under white light irradiation conditions, albeit in low amounts. For heterologous expression and convenient purification, the cloned genes were furnished with His-tag encoding sequences at their 3¢ end and expressed in Escherichia coli. The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the P r and P fr forms (red and far-red absorbing forms, respectively) of the plant phytochromes and Cph1. A red shift in the absorption maxima of the CphB/PCB complex (k max ¼ 685 and 735 nm for P r and P fr , respectively) is indicative for a noncovalent incorporation of the chromophore (k max of P r , P fr of CphA: 663, 700 nm). A CphB mutant generated at the chromophore-binding position (Leu246 fi Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB. The kinetics of the light-induced P fr formation of the CphA-PCB chromoprotein, though similar to that of its ortholog from Synechocystis, showed differences in the kinetics of the P fr formation. The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator. In contrast, the light-induced kinetics of the CphB-PCB complex was markedly different, clearly due to the noncovalently bound chromophore.Keywords: Calothrix sp. strain PCC 7601; flash photolysis; heterologous expression; phytochrome-like apoproteins CphA and CphB; response regulators RcpA and RcpB.A precise qualitative and quantitative measurement of the surrounding light is essential to photosynthetic organisms in order to either adapt to the environmental light conditions or, in the case of motility, to proceed towards a favorable habitat. Higher plants have developed the phytochromes, a chromoprotein family, which absorb light in the longwavelength region and regulate numerous photomorphogenetic processes [1][2][3]. In cyanobacteria, the presence and molecular structure of comparable sensory system(s) have long been debated. It has been suggested that the photosynthetic apparatus itself or blue-light-and/or other noncharacterized light-absorbing photoreceptors might fulfil these functions [4][5][6][7].The complete s...

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

“…Com- pared with PCB adducts of CysA-phytochromes such as Synechocystis Cph1, PCB-Agp1 (Fig. 3b) has a red-shifted absorbance maximum of 685 nm (see Table 3, which is published as supporting information on the PNAS web site, www.pnas.org) such as the non-CysA phytochromes Deinococcus BphP (3) and Calothrix CphB (18). Compared with PCB-Agp1, BV-Agp1 is further red-shifted to 701 nm ( Fig.…”

“…Dark-reversion has long been known for plant phytochromes (13,14,16) but is missing in cyanobacterial PCB-Cph1 and PCB-CphA (18). It is likely that the ancestors of phytochromes were photoreceptors with only one thermostable form, and that the lifetime of the designated Pfr intermediate was later increased.…”

“…In general, the phytochrome chromophore is covalently bound. However, covalent binding seems not to be required for full spectral activity, as shown for CphB, for example (18). Covalent associations of biliproteins are usually analyzed by SDS͞PAGE and Zn 2ϩ -induced fluorescence (33).…”

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

“…Sample Generation-Recombinant CphA and CphB from the cyanobacterium Tolypothrix PCC7601 and their cognate response regulators RcpA and RcpB were heterologously expressed as previously described (10,12,14). The HK domain of CphA was generated by introducing a start codon at positions 1375-1377 of cphA (originally coding for Glu-459) and subsequent cloning of this new open reading frame into pET28a between restriction sites for NcoI and XhoI.…”

“…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.).…”