Dynamique des Membranes Végétales, Complexes Protéines-Pigments, CNRS UMR 8543, Ecole Normale Supérieure, 46 rue dUlm 75230 Paris Cedex 05, France1
Author for correspondence: Jean Houmard. Tel: +33 144323519. Fax: +33 144323935. e-mail: jhoumard{at}biologie.ens.fr
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ABSTRACT |
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Keywords: CRP, cyclic-nucleotide gated channel
Abbreviations: cNMP, cyclic nucleotide monophosphate; CNG, cyclic-nucleotide- gated; CRP, cAMP receptor protein
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INTRODUCTION |
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Intracellular levels of cAMP and cGMP depend on the relative rates at which they are synthesized by the adenylyl- (EC 4 . 6 . 1 . 1) or guanylyl-cyclases (EC 4 . 6 . 1 . 2), degraded by the cAMP- (EC 3 . 1 . 4 . 17) or cGMP- (EC 3 . 1 . 4 . 35) phosphodiesterases, and eventually excreted. The association of cAMP and cGMP with protein receptors in turn determines the physiological response(s). At present, only the cAMP and cGMP biosynthetic enzymes have been identified and characterized in cyanobacteria (Kasahara & Ohmori, 1999 ; Katayama & Ohmori, 1997
; Ochoa de Alda et al., 2000
; Terauchi & Ohmori, 1999
).
Cyclic-nucleotide phosphodiesterases play a pivotal role in cAMP and cGMP signal transduction by regulating the intracellular levels of cyclic nucleotides. The two known classes of phosphodiesterases comprise the seven families of eukaryotic cyclic-nucleotide phosphodiesterases (Beavo, 1995 ) and the proteobacterial cAMP-phosphodiesterases (Macfadyen et al., 1998
). Although cAMP-phosphodiesterase activities have been described in cyanobacteria (Sakamoto et al., 1991
), no homologue of the proteobacterial enzymes has been recognized in the genome of Synechocystis PCC 6803 (Macfadyen et al., 1998
). Five hypothetical coding regions in the Synechocystis PCC 6803 genome contain, however, a phosphohydrolase domain, named HD, characteristic of the N-terminal half region of the eukaryotic cAMP- phosphodiesterase catalytic centres (Aravind & Koonin, 1998
). Mutational analysis of the HD domain of a cGMP-specific phosphodiesterase showed that the more conserved residues within HD domains are involved in catalysis (Turko et al., 1998
).
In vertebrates, cAMP and cGMP couple visual and olfactory signals to electrical excitation and Ca2+ signalling by modulating cyclic-nucleotide-gated (CNG) channels (Zagotta & Siegelbaum, 1996 ). Both nucleotides can also activate protein kinases, which in turn regulate enzymes and proteins involved in intermediary metabolism as well as in transcription (Daniel et al., 1998
). In Escherichia coli, the real occurrence of cGMP [<10-9 mol·(mg protein)-1 ] is debated (Herdman & Elmorjani, 1988
; Vogler & Lengeler, 1987
). In this bacterium, cAMP functions as a cofactor of the cAMP-receptor protein (CRP) rather than as an activator of a protein kinase. The cAMP-receptor protein CRP, also known as CAP (Catabolite gene Activator Protein), is an allosteric DNA- binding protein that modulates the transcription of several genes (Gralla & Collado-Vides, 1996
; Kolb et al., 1993
). At present, all the cAMP-dependent responses in bacteria appear to be mediated through the binding of cAMP to its receptor, which in turn regulates directly or indirectly genes involved in pH regulation, sugar metabolism and taxis (Botsford & Harman, 1992
).
The allosteric effect promoted by cyclic nucleotides on CRP, kinases and the aforementioned channels is exerted through a similar cyclic nucleotide monophosphate (cNMP)-binding domain (Zagotta & Siegelbaum, 1996 ). The homodimeric three-dimensional structure of the E. coli cAMP-receptor protein (CRP-Ec ) has been determined (Weber & Steitz, 1987
). The structural data combined with site-directed mutagenesis studies showed the importance of the residues that are conserved among the cyclic- nucleotide-binding domains of CRPs, kinases and cyclic-nucleotide-gated channels (Varnum et al., 1995
; Woodford et al. , 1989
; Zagotta & Siegelbaum, 1996
). All the well-characterized cyclic-nucleotide-binding domains can accommodate both cGMP and cAMP, but the degree of activation that they confer upon the output domain depends on the bound nucleotide. Both cAMP and cGMP bind to the E. coli cAMP-receptor protein but cGMP does not activate transcription (Ebright et al., 1985
). The olfactory cyclic- nucleotide-gated channel is fully activated by cGMP and cAMP. In contrast, for the photoreceptor channels, cAMP acts as a partial agonist, producing only a fraction of the current induced by cGMP (Zagotta & Siegelbaum, 1996
).
The protein GAF domains (encountered in cGMP-specific phosphodiesterases, Adenylyl cyclases and Formate hydrogen lyases) which may allosterically regulate catalytic activities via ligand binding represent another type of cyclic nucleotide receptor (Aravind & Ponting, 1997 ). GAF domains participate in the architecture of many signalling proteins (phytochromes, ethylene receptors and members of two-component regulatory systems). The GAF domain of cGMP-stimulated phosphodiesterases binds cGMP. Structurefunction studies of this GAF domain performed by alanine mutagenesis have identified a motif [N(K/R)XnD] necessary for full cGMP-binding activity (McAllister-Lucas et al., 1995
; Turko et al., 1996
).
Apart from the adenylyl cyclase Cya1 (Slr1991), which has just been shown to be involved in the regulation of cell motility in Synechocystis PCC 6803 (Terauchi & Ohmori, 1999 ), cGMP and cAMP signalling pathways in cyanobacteria remain sketchy. Here we report on the identification of putative components of the cGMP and cAMP cyanobacterial signalling pathways by making use of: (i) the complete genome sequence of Synechocystis PCC 6803 (Terauchi & Ohmori, 1999
); (ii) recently available programs and databases (Table 1
); and (iii) the data obtained by sequencefunction studies for cAMP/cGMP-dependent proteins. Our study leads in particular to the presumptive identification of a prokaryotic cyclic-nucleotide-gated channel protein. Hypotheses are presented that could serve as good starting-points for functional genomic studies.
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RESULTS AND DISCUSSION |
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Cyclic nucleotide receptor proteins
Protein domain analysis of the Synechocystis genome, using the SMART program, indicated that single or multiple GAF domains are present in 28 ORFs of the Synechocystis genome (12 of which also containing a PAS domain). SMART server provides a CLUSTAL W alignment of GAF domains that permits comparison of the Synechocystis GAF domains with those of some eukaryotic phosphodiesterases. The GAF domain of ORF Slr2104 contains all the determinants (Asn258, Lys259, Asp267) for cGMP-binding. That some of these residues are missing in other GAF domains of Synechocystis may indicate: (i) that these sites do not participate in cGMP binding; (ii) that the substituted residues can functionally replace those found in GAF domains of eukaryotic phosphodiesterases; or (iii) that this GAF domain serves some function other than cGMP binding.
Based on protein domain sequence analyses (SMART), 12 ORFs of the Synechocystis genome could have cNMP-binding domains (Fig. 2 ). All of them contain a highly conserved stretch of approximately 120 amino acids that is homologous to the cNMP- binding domains of other proteins, including the cAMP- and cGMP- dependent protein kinases, the cyclic-nucleotide-gated channels and the cAMP receptor protein (CRP). In five of these ORFs the cNMP-binding domain is joined to putative transmembrane helices (Slr0593, Slr1529, Slr1575, Slr0510 and Sll1180), whereas in another five it is attached to a C-terminal domain which contains helixturnhelix (HTH) DNA-binding motifs (Sll1371, Sll1924, Sll1169, Slr0449 and Slr1423).
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In general, ligand discrimination of cNMP-binding domains is achieved by the residues analogous to Ser83 and Thr127 of CRP-Ec (Altenhofen et al., 1991 ; Lee et al. , 1994
; Shabb et al., 1990
; Varnum et al. , 1995
). The Ser83 residue (or a conservative replacement of it such as Thr) allows the binding of both cGMP and cAMP. Since in CRP-Syn, Slr1575, Slr0593 and Slr0842 the position analogous to Ser83 of CRP-Ec is occupied by either a Ser or a Thr residue, this position probably does not allow discrimination between cAMP and cGMP. In CRP-Ec, residues Thr127 and Ser128 determine nucleotide selectivity and participate in the intersubunit communication. Amino acid substitutions that introduce a hydrophobic amino acid side chain at positions 127 or 128 decrease CRP-Ec discrimination between cAMP and cGMP (Lee et al., 1994
). Mutation T127L results in an activation of transcription upon the binding of cGMP instead of cAMP (Moore et al., 1996
). According to these data, CRP-Syn, Slr1575 and Slr0593 would be activated by cGMP, since the positions equivalent to Thr127 in CRP-Ec are occupied by either Leu or Ala residues (Fig. 2
). However, reconstruction of the putative cNMP- binding domain using the CRP-Ec structure as a template (Fig. 3
) showed that: (i) the substitution of Ser128 by Asn (in CRP-Syn and Slr0842) preserved the interactions with positions N-6 and N-7 of the adenine; and (ii) the loss of specificity that would result from the substitution of Thr127 by Leu or Ala (as in CRP-Syn and Slr0593) could be overcome by the substitution of Ser62 by an Asn, which could create a new hydrogen bond with the N1 of the adenine (Fig. 3
). Thus we propose that the cNMP-binding domains of CRP-Syn, Slr0593 and Slr0842 would be activated preferentially by cAMP (Fig. 3
). This deductive method cannot be used to infer the specificity of Slr1575 because the aforementioned critical positions are occupied by non-polar amino acids.
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Slr1575 is a putative cyclic-nucleotide-gated channel (CNG).
ORF Slr1575 possesses structural elements clearly related to voltage-gated and cyclic-nucleotide-gated channels (Fig. 4 ). In such channels, the major determinants in channel gating have been mapped in the cytoplasmic linker (C-linker) that connects the last transmembrane segments of the channel and the cNMP-binding domain (Zong et al., 1998
). The alignment of the cyclic-nucleotide-gated bovine cone photoreceptor channel (CNG3) with Slr1575 (Fig. 4a
) showed that two (I439 and D481) of the three amino acids in the C- linker that determine the high specificity of the cone photoreceptor channel for cGMP were conserved, the Asp494 of CNG3 being replaced by a Gln residue. This alignment strongly suggests that Slr1575 would be activated preferentially by cGMP.
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The subunit composition of cyclic-nucleotide-gated channels, heteromeric or homomeric, modulates both cyclic nucleotide sensitivity (Bradley et al., 1994 ) and cation permeation (Dzeja et al., 1999
). Although Slr0510 does not contain the determinants for cyclic nucleotide binding (Fig. 1
), this hypothetical protein is homologous to Slr1575 in both sequence (33% identity and 51% similarity) and domain architecture (Fig. 2
). By analogy with eukaryotic cyclic-nucleotide- gated channels, the specificity for cations and cyclic nucleotides of Slr1575 homomeric channels might be modulated by heteromerization with Slr0510.
Slr0593 is a putative cyclic-nucleotide-modulated permease.
In both Cyanobase and Genequiz databases, Slr0593 is annotated as a cAMP protein kinase regulatory chain because the cNMP-binding domains of these proteins share a high degree of homology (30% identity and 48% similarity). Our sequence analysis indicates, however, that of the five domains that define such proteins (Taylor et al., 1990 ), two were not conserved, namely the dimer-interaction site and the peptide-inhibitory site. Two programs aimed at predicting transmembrane helices and protein topology (PHDhtm and HMMTOP) predict eight transmembrane helices for Slr0593, the cNMP-binding domain being located on the cytosolic side, between the third and fourth helices (Fig. 2
). The three C-terminal membrane helices of Slr0593 show 27% identity (43% similarity) with three membrane helices of the amino acid permease ROCC of Bacillus subtilis (PID g730600) (Glaser et al., 1993
). The five C- terminal membrane helices of Slr0593 show 26% identity (42% similarity) with the phenylacetic acid permease PhaJ of Pseudomonas putida (PID g3253206) (Olivera et al., 1998
). Although the specificity of the permease cannot be inferred from sequence comparison, our results indicate that Slr0593 is likely to be a permease, the activity of which would be modulated by cyclic nucleotides.
Slr0842 is a putative signal transducer.
In ORF Slr0842, the cNMP-binding domain is joined to a DUF2 domain (Fig. 2). This domain was first recognized in the N-terminal region of Acetobacter xylinum enzymes that control the turnover of c-di-GMP:diguanylate cyclase and phosphodiesterase A (Tal et al., 1998
). It is possible that DUF2 domains mediate one of these activities. Using a program for detecting characteristic architecture domains (SMART), we have found DUF2 domains in 13 ORFs of Synechocystis PCC 6803 (Slr0842, Sll0267, Slr1588, Slr1102, Sll0821, Slr1103, Slr0359, Slr1104, Sll1895, Slr1305, Slr1593, Slr2077 and Slr1692). They may be preceded by input sensory signalling domains like response regulators, GAF domains, FHA ( Forkhead-associated domain) domains and/or PAS domains. Hence, DUF2 domains can probably be regulated by different signals. We conclude that ORF Slr0842 might be a cyclic- nucleotide-modulated signal transducer of a pathway yet to be determined.
The cAMP receptor protein (CRP-Syn, sll1371) is a transcription factor.
The homology of CRP-Syn with the E. coli cAMP receptor protein (CRP-Ec) extends to the helixturnhelix DNA-binding domain (Fig. 5 ). The crystal structure of the CRP-DNA complex enabled the identification of the residues of CRP-Ec that interact with the DNA (Schultz et al., 1991
), and the compilation of several CRP-DNA binding sites led to the identification of a 22 bp palindromic consensus site of sequence AAATGTGATCT*AGA TCACATTT (the most conserved bases are underlined and the base pairs that interact with the protein are doubly underlined) (Berg & von Hippel, 1988
). Interactions of CRP-Ec with the consensus DNA-binding sequence involve specific base contacts with Arg180, Glu181 and Arg185, the determinants of specificity (Kolb et al., 1993
). These and other less selective contacts involved in the specific recognition of the consensus DNA binding site by E. coli cAMP receptor protein are conserved in the Synechocystis PCC 6803 protein (Fig. 5a
). This suggests that CRP-Syn could recognize the same DNA sequence as CRP-Ec. Upstream of the CRP- Syn coding region we found DNA sequences showing a high degree of similarity with the promoter region of the E. coli crp, including the two CRP binding sites (Fig. 5b
). These identities reinforce the hypothesis that CRP-Syn binds to the same DNA sequence as CRP-Ec, and suggest that crp is autoregulated as it is in E. coli (Hanamura & Aiba, 1991
). While this paper was under review, Yoshimura et al. (2000)
reported that sll1371 (referred to as CRP- Syn in this article) not only binds cAMP but also, when complexed with cAMP, can alter the electrophoretic mobility of an oligonucleotide that contains the E. coli CRP binding site. Slr0593 (referred to as a cyclic nucleotide permease in this article) also bound cAMP while sll1924 could not. These data are in agreement with the hypotheses proposed in this article.
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Concluding remarks
After standard search for identification by homology, about half of the putative ORFs of every fully sequenced genome are annotated as hypothetical proteins. More thorough analyses, coupled with the physiological and structurefunction data that are available, make it possible to refine the assignments and/or propose working hypotheses (Pallen, 1999 ). Using this approach in conjunction with targeted gene inactivation, we have recently shown that Synechocystis Cya2, a protein previously identified as an adenylyl cyclase (Kaneko et al., 1996
), is responsible for cGMP synthesis in this organism, thus identifying the first prokaryotic guanylyl cyclase (Ochoa de Alda et al., 2000
). Although phosphodiesterase activities have been measured in cyanobacteria (Herdman & Elmorjani, 1988
), including Synechocystis PCC 6803, no obvious homologues of proteobacterial enzymes have been previously found; we tentatively propose Sll1624 and Slr2100 as being cyanobacterial phosphodiesterases.
Among 40 ORFs showing a high overall homology with cyclic nucleotide receptors, we identified 5 that contain the determinants required for ligand binding. One important conclusion derived from the presence of cGMP and cAMP at similar levels in Synechocystis PCC 6803 (Herdman & Elmorjani, 1988 ), and from the presence of the aforementioned cNMP-binding do-mains, is that one cyclic nucleotide could function as an inhibitor or as an agonist of the activity induced by the other, both of them being able to bind to the cNMP-binding domain but only one of the two inducing full activation.
Our sequence analysis study of the genome of Synechocystis PCC 6803 revealed the previously unrecognized presence of eukaryotic- type cGMP/cAMP signalling components in a prokaryote, such as the cyclic-nucleotide-gated channels and the cGMP-binding GAF domain. The presence of the genes encoding eukaryotic-type cGMP/cAMP signalling components that are detected, together with that of a putative CRP modulon, suggests that the complexity of the cAMP/cGMP signalling pathways in cyanobacteria is greater than in other bacteria, in agreement with the striking morphological and physiological diversity found in the cyanobacteria. This complexity could be even higher in some filamentous cyanobacteria able to carry out cellular differentiation, because Anabaena PCC 7120 for example has five different nucleotide cyclases (Katayama & Ohmori, 1997 ).
Targeted inactivation of the ORFs that we have characterized would be a first approach to validate our hypotheses. In parallel, site- directed mutagenesis of the hypothetical residues essential for the function of the identified domains would give more information about the signal transduction pathways in which they participate. Substitution of the gene encoding slr2100 by a mutated gene, for example, should remove the phosphodiesterase activity and affect the phosphorelay(s) in which cyclic nucleotides are involved.
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ACKNOWLEDGEMENTS |
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Received 7 February 2000;
revised 14 August 2000;
accepted 30 August 2000.