Identification and Characterization of a Novel cAMP Receptor Protein in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

Three open reading frames of Synechocystis sp. PCC 6803 encoding a domain homologous with the cAMP binding domain of bacterial cAMP receptor protein were analyzed. These three open reading frames, sll1371, sll1924, and slr0593, which were named sycrp1, sycrp2, and sypk, respectively, were expressed in Escherichia coli as His-tagged or glutathione S-transferase fusion proteins and purified, and their biochemical properties were investigated. The results obtained for equilibrium dialysis measurements using these recombinant proteins suggest that SYCRP1 and SYPK show a binding affinity for cAMP while SYCRP2 does not. The dissociation constant of His-tagged SYCRP1 for cAMP is approximately 3 M. A cross-linking experiment using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide revealed that His-tagged SYCRP1 forms a homodimer, and the presence or absence of cAMP does not affect the formation of the homodimer. The amino acid sequence reveals that SYCRP1 has a domain similar to the DNA binding domain of bacterial cAMP receptor protein in the COOH-terminal region. Consistent with this, His-tagged SYCRP1 forms a complex with DNA that contains the consensus sequence for E. coli cAMP receptor protein in the presence of cAMP. These results strongly suggest that SYCRP1 is a novel cAMP receptor protein.

SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross‐talk and sheds light on regulation by effector molecules

“…The virtually complete lack of binding of CRP to its cognate promoter fragment in the absence of cAMP, and the high affinity of CRP for cAMP both in the presence (our results) and in the absence [8] of the promoter, strongly suggests that the site for cAMP on CRP is always accessible, and that its occupation by cAMP triggers a conformational change that promotes the binding of CRP to its DNA box. A different model for 2OG activation of NtcA is suggested by the present results.…”

“…This question is pertinent, since some cyanobacteria have CRP protein-encoding genes [7], as in Synechocystis sp. PCC6803 (from now on termed Synechocystis), where a CRP protein called Synechocystis CRP (SYCRP1) is expressed from the homonymous gene and transcriptionally regulates expression of a number of genes such as cccS in a cAMP-dependent manner [6,8,9]. Even in cyanobacteria in which there is no gene encoding CRP (such as Synechococcus elongatus), screening methods [7] detect several CRP boxes having an uncertain function, which could be the targets of NtcA regulation.…”

“…While CRP is activated by cAMP [5,8,9], NtcA is activated by 2-oxoglutatare (2OG) [10,11], an indicator of the cellular nitrogen level [12], and it is coactivated by a small (10 kDa) monomeric protein called PII-interacting protein X (PipX) [13,14] (Fig. 1).…”

Biochemical Properties of a cAMP Phosphodiesterase in the Cyanobacterium Anabaena sp. strain PCC 7120

“…The cells were then disrupted by sonication using an Insonator model 201M (Kubota Co., Tokyo, Japan), the resulting crude extract was centrifuged at 20,000´g for 15 min, and the supernatant was further centrifuged at 245,000´g for 60 min. According to methods reported previously 21) , the supernatant was loaded onto a HiTrap Chelating column (Amersham Biosciences, NJ) connected to a FPLC system that had been equilibrated with buffer A, and then eluted using a step gradient of 50 and 200 mM imidazole in buffer A. The 200 mM imidazole fractions were collected and concentrated by Ultrafree-15 (Millipore, MA).…”

Adenylyl Cyclase Activity of Cya1 from the Cyanobacterium Synechocystis sp. Strain PCC 6803 Is Inhibited by Bicarbonate

“…strain PCC 6803, cAMP has also been demonstrated to be a second messenger in the transduction of a light signal (reviewed in reference 9). In particular, transfer of the cells from the dark to blue light results in an increase in the level of cellular cAMP, which is bound to the cAMP receptor protein SYCRP1 involved in the biogenesis of pili (7,(12)(13)(14). This mechanism is thought to allow cells to adjust their motility in response to environmental changes in light conditions via cAMP levels.…”