GBB, Department of Biochemistry, University of Groningen, Nijenborgh, The Netherlands
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Abstract |
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Introduction |
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The completion of sequencing of the genomes of an expanding number of organisms, ranging from bacteria to human, has resulted in a better insight into the role of horizontal gene transfer and gene loss during evolution (Koonin et al. 1997
; Aravind et al. 1998
; Andersson and Andersson 1999
; Nelson et al. 1999
; Aravind et al. 2000
). Among prokaryotes, horizontal gene transfer as well as gene loss seem to have occurred frequently and are expected to be major contributors to the diversity among prokaryotes (Ochman and Moran 2001
). For multicellular eukaryotes, the chance of a horizontal gene transfer event seems low because this transfer should occur in the germ line to be transferred to a future generation. For these organisms, gene duplication and the combination of protein domainencoding sequences seems to be the main path to obtain new genes. From an analysis of the recently published human genome, 113 genes were identified that were proposed to be most likely derived from bacteria by horizontal gene transfer to the vertebrate lineage (Lander et al. 2001
) because orthologs are present in several bacteria but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cereviciae. However, a more comprehensive analysis suggests that a significant portion of the cases can be explained by gene loss (Roelofs and Van Haastert 2001c
; Salzberg et al. 2001
; Stanhope et al. 2001
). One of these genes is a soluble adenylyl cyclase (sAC) (Buck et al. 1999
) that belongs to the class-III ACs (Danchin 1993
). These ACs form an interesting phylogenetic family of genes because in eukaryotes it consists of two main groups, a very large group of prevailing cyclases present in nearly all eukaryotes encoding membrane-bound ACs and GCs and soluble GCs and a very small group of genes related to sAC present in only a few bacteria, Dictyostelium, and several vertebrates. In the present study, we investigate the origin of the small family of genes related to sAC.
The three-dimensional structure of mammalian AC (Tesmer et al. 1997
; Zhang et al. 1997
) reveals that the catalytic core is formed by two cyclase domains that are associated in an antiparallel manner. This dimer can be formed by two identical as well as two different domains. For the heterodimer, these domains can be derived from one polypeptide or two different polypeptides. In metazoa, most AC enzymes consist of 12 transmembrane segments and two cyclase domains, C1 and C2; these enzymes are regulated by G-proteins (Hanoune and Defer 2001
). Two forms of GC enzymes are present; a soluble enzyme composed of two different subunits and a membrane-bound enzyme with one transmembrane segment and one catalytic domain that functions as the homodimer (Wedel and Garbers 2001
). sAC is present in human and rat and contains two cyclase domains and a
1,000 amino acid long C-terminal region (Buck et al. 1999
). The enzyme is regulated by bicarbonate and involved in sperm maturation (Chen et al. 2000
). The cyclase domains of sAC share the highest degree of identity with the cyclase domains of type-III bacterial cyclases and far less identity with the cyclase domains of other vertebrate adenylyl or guanylyl cyclases. Recently we identified a putative ortholog of sAC in the eukaryotic microorganism Dictyostelium (Roelofs et al. 2001b
), which is, however, a guanylyl cyclase (sGC). It shares significant sequence identity with the corresponding cyclase domains and the
1,000 amino acid C-terminal region of mammalian sAC, indicating common ancestry. To trace the possible evolutionary origin of human sAC and Dictyostelium sGC, we have analyzed the protein and nucleotide databases for genes that share significant identity with segments of human sAC and Dictyostelium sGC. The results suggest that in bacteria a protein segment of
1,000 amino acids was fused with a cyclase domain. In bacteria, the 1,000 amino acid segment was shortened, whereas in the eukaryotic lineage a second cyclase domain was added. The gene was retained in the eukaryotic lineage up to human and rat but was apparently lost independently in several other lineages.
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Materials and Methods |
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The SMART program (Schultz et al. 1998, 2000
) was used to analyze the domain structure of the retrieved sequences. Multiple sequence alignments were constructed using the CLUSTAL W program (Thompson, Higgins, and Gibson 1994
), followed by manual optimization. In DdsGC, three short (
18 amino acids) and three long (39114 amino acids) stretches of repetitive sequence within the sCKH region were deleted; repetitive sequences are often found in Dictyostelium proteins, even within very conserved domains. Distance matrices were constructed from the alignments with the PROTDIST program of the PHYLIP package, which uses the Dayhoff's PAM 001 matrix for the calculation of evolutionary distances (PHYLIP 3.5, J. Felsenstein 1993 [Felsenstein 1996
]). Phylogenetic trees were generated by using the FITCH program of the PHYLIP package, with 250 bootstrap replications to assess the reliability of the nodes. Tree topologies obtained by FITCH were confirmed using Neighbor-Joining and Protein Parsimony from the same PHYLIP package.
For the calculation of the divergence time (Nei, Xu, and Glazko 2001
), linearized forms of the trees, as presented in figures 2 and 3 , were used for the C1 and C2 cyclase domains and the first and second half of the sCKH region; bacterial sequences were AsCYAA for cyclase domains and AsKin for sCKH regions. The evolutionary distance between human and bacterial orthologs of a protein should be between the origin of eubacteria, about 3,500 Myr, and endosymbiotic acquisition of organelles, about 2,000 Myr (Doolittle et al. 1996
; Feng, Cho, and Doolittle 1997
). The absolute divergence time of the bacteria-eukaryote separation is the subject of substantial debate because fossil-based records are missing. Therefore, we use a relative time for the origin of eukaryotes. The divergence times of Dictyostelium and rat from the human sAC were calculated as described (Nei, Xu, and Glazko 2001
) by using a relative distance of 1.00 from bacteria to human.
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Sequences used are: RnACV, NP072122; RnACII, P26769; RnGCE, P51840; RnGC2, NP076446; HssAC1, NP060887; RnsAC1, NP067716; DdsGC, AF361947; AsCYAA, BAA13997; MlsAC, CAA19149; SmsAC, S60684; MlosAC1, AP003001; MlosAC2, AP002995; AsKin, AF230361; BrKin, AF222754; SpKin1, CAA20836; SpKin2, CAB11683; CaKin, AAC39451; ReKin, BAA34261; ScLux1, CAB37582; ScLux2, CAB93733; ReLux, AAD28307. Abbreviations of species used in sequence names are: Hs, Homo sapiens; Rn, Rattus norvegicus; Dd, Dictyostelium discoideum; As, Anabaena sp.; Ml, Mycobacterium leprae; Sm, Sinorhizobium meliloti; Mlo, Mesorhizobium loti; Sp, Schizosaccharomyces pombe; Ca, Candida albicans; Sc, Streptomyces coelicolor; Br, Bradyrhizobium sp.; and Re, Rhodococcus erythropolis.
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Results |
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The amino acid sequences of the C-terminal region of HssAC1, DdsGC, and MlsAC were used to search for genes that could provide information on the origin of this C-terminal region. Two bacterial genes, AsKin and BrKin, that show significant sequence identity over the entire length of the cyclase C-terminal region were identified (fig. 1 ). These bacterial proteins are complex kinases with an N-terminal serine-threonine-tyrosine kinase and a C-terminal extension with a GAF domain, a histidine kinase, and a H-ATPase. We therefore addressed the C-terminal region as soluble Cyclase-Kinase Homology (sCKH) region. Further database searches with the sCKH region of HssAC and AsKin uncovered several bacterial sequences with an N-terminal kinase and a part of the sCKH region (e.g., ReKin; data not shown) and complex yeast kinases with a similar topology as AsKin, except for an additional C-terminal histidine kinase receiver (Rec) domain (fig. 1 ). Finally, a group of bacterial transcription factors containing the sCKH region and a C-terminal Lux-domain were identified. Subsequent database searches with complete or partial sequences of the Lux-containing proteins or the yeast kinases did not reveal new sequences in bacteria or eukaryotes that share significant sequence identity with soluble cyclases.
The sCKH Region
On the basis of sequence alignment, the sCKH region can be divided into four tentative segments AD (fig. 1
). When no indications for a specific domain identity can be given, we prefer to use the neutral terms "region" and "segment" for longer and shorter sequences, respectively. The first segment, A, of the sCKH region is about 260 amino acids in length and clearly encodes an AAA ATPase domain with a P-loop motif found in many ATP and GTP-binding proteins. This domain is observed in all proteins with the sCKH region. The second segment, B, also present in all the proteins, is about 250 amino acids in size but has no domain identity in databases. The third segment, C, is the least conserved, even in relative closely related proteins such as HssAC1 and RnsAC1. The C and D segments of the sCKH region, about 400 and 150 amino acids, respectively, are present in a subset of the proteins. MlsAC and MlosAC1 only comprise segments A and B, whereas SmsAC and MlosAC2 also comprise segment D, but lack most of segment C. The yeast kinases and the bacterial Lux-transcription factors do have amino acid sequence corresponding to the length of segments C and D, but these show little sequence identity with the sCKH region of sAC1 (with the exception of segment D of SpKin2).
Phylogeny of the sCKH Region
To deduce the evolutionary origin of human sAC, we first analyzed the phylogeny of the sCKH regions from the different groups of proteins and then compared the observed phylogenetic relations with those deduced from the cyclase and kinase domains. Because only the first half of the sCKH region is well conserved among all proteins, this segment was used to construct a phylogenetic tree (fig. 2
). Bootstrap analyses suggest four sister groups: bacterial cyclases, eukaryotic cyclases, transcription factors, and kinases. Within these sister groups branching is strongly supported (bootstrap values above 85); the positions of the four sister groups relative to each other is less well resolved (bootstrap above 50). In the group of sCKH regions from bacterial cyclases, MlosAC1 groups with MlsAC, and MlosAC2 with SmsAC, which is in agreement with the classification based upon the overall topology of these four proteins (fig. 1
). The sCKH regions from eukaryotic cyclases form a monophyletic group, with human and rat sAC1 in the crown; human sAC2 apparently arose before the division of human and rat and the Dictyostelium gene from a branching halfway between vertebrates and the presumed bacterial origin. The third sister group of sCKH regions is formed by the Lux-containing transcription factors, whereas the fourth sister group consists of all kinases, with the yeast kinases in the crown and the bacterial kinases together closer to the root. In this analysis, the cyclase and kinase domains were not included; nevertheless, the phylogeny of the first half of the sCKH region places all sequences in accordance with the domains found outside the sCKH region.
The phylogeny of the N-terminal kinase domain and the C-terminal GAF/H-ATPase region were analyzed separately by bootstrap analysis to investigate whether the evolutionary relations seen in the sCKH region are also present in other domains of these proteins. The phylogeny of the STY-kinase domains of the yeast and bacterial proteins is weakly resolved (some bootstrap values are below 40; data not shown). In contrast, the GAF/H-ATPase regions are well resolved (all bootstap values above 98), revealing coevolution of sCKH region and the GAF/H-ATPase domains (data not shown). This strongly suggests that the sCKH region was combined in bacteria with the N-terminal STY kinase domain and the C-terminal GAF, H kinase, and H-ATPase domains, and that the Rec domain was added to this sequence in the yeast lineage.
Phylogeny of the Cyclase Domain
The phylogeny of the cyclase domains (fig. 3
) suggests the presence of four sister groups: one group of bacterial sAC enzymes and three eukaryotic cyclase groups. Other bacterial cyclases (only AsCYAA is included in the fig. 3
) are positioned close to the region of the tree where the four sister groups come together. The three groups of eukaryotic cyclases are characterized by the C1 domains of sAC (including the C1 of DdsGC), the C2 domains of sAC (including the C2 of DdsGC), and all other vertebrate cyclases; this third group contains many members (all 12 transmembrane AC and all animal guanylyl cyclases) and will be referred to in this article as the prevailing eukaryotic cyclases. Bootstrap analysis demonstrates that the three eukaryotic sister groups are well resolved. The positioning of the bacterial sAC cyclase domains into one group is not well supported, which is also the case for the positioning of this group relative to the eukaryotic cyclases. In some analysis, members of the group of bacterial sAC associate with the C1 domains of eukaryotic soluble cyclases and in other experiments with the C2 domains. Within the group of bacterial cyclases, the cyclase domains of MlosAC1 and MlsAc are always positioned close together and that of MlosAC2 with SmsAC, as was observed for the sCKH regions.
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Discussion |
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Deducing the origin of a complex protein such as sAC with sequence information from only a few organisms is difficult but might be possible when information on amino acid sequence comparison and domain composition are combined. Proteins of the class of sAC enzymes contain one or two cyclase domains and a long C-terminal sCKH region that is found in a limited set of other proteins. The phylogeny of the sCKH region points to four sister groups: bacterial cyclases, eukaryotic soluble cyclases, transcription factors, and complex kinases. For all proteins, the phylogeny of the sCKH region is convergent with the phylogeny of the other domains. This suggests that an sCKH region was fused with other domains early during evolution, leading to the four groups of proteins, as we recognize them presently (figs. 1 and 2 ).
The coevolution of the sCKH region and cyclase domains strongly suggests that the sCKH region and at least one cyclase domain were combined in bacteria. From this ancestor sAC enzyme, the four presently known bacterial sAC enzymes were likely derived by deletion of the second half of the sCKH region in MlsAC and MlosAC1 and by the deletion of an internal segment of sCKH in SmsAC and MlosAC2 (fig. 1 ). In eukaryotic sAC enzymes, a second cyclase domain has been added. This occurred very early during evolution, possibly in bacteria or early eukaryotes, because no common phylogenetic trait can be identified for the soluble cyclase C1 and C2 domains in eukaryotes. In the group of prevailing eukaryotic ACs, which also has two cyclase domains, the C1 and C2 domains appear to have diverged much later during eukaryotic evolution (see fig. 3 ).
The transition of the bacterial ancestor sAC to the eukaryotic lineage could be either by generation-to-generation gene transfer (vertical) or by species-to-species gene transfer (horizontal), with important implications for each hypothesis. Figure 4 presents our current knowledge on the phylogeny of eukaryotic species (dotted lines) combined with phylogeny of sAC enzymes (thick lines). The normal vertical gene transfer of sAC implies that the gene was present during all the lineage deviations up to human and thus has been lost independently in several lineages after their separation from the lineage leading to human. On the other hand, horizontal gene transfer should explain how Dictyostelium and the vertebrate lineage received the gene. One explanation would be two events of horizontal gene transfer from bacteria to Dictyostelium and to vertebrates, respectively. Another explanation would be normal evolution to the Dictyostelium lineage (which implies loss in plants), loss in the fungi-animal clade, and horizontal gene transfer from Dictyostelium or bacteria to the vertebrate lineage.
The hypothesis that genes have entered the eukaryotic cell by horizontal gene transfer in the period of the earliest eukaryotes is widely accepted (Doolittle 1998; Gray 1999
). Two arguments are used for proposing a more recent transfer of a gene from bacteria to vertebrates (Wolf, Kondrashov, and Koonin 2000
; Lander et al. 2001
; Ponting 2001
). First, the amino acid sequence encoded by a eukaryotic candidate gene shows considerably higher sequence similarities with bacterial proteins than with proteins in closer related organisms. A second argument is a limited presence of the gene in eukaryotes and a widespread presence of the bacterial counterpart over different bacterial lineages.
The following lines of evidence suggest the vertical gene transfer of human sAC. First, sAC1, sAC2, and DdsGC show a long monophyletic origin. The phylogenetic distance of human sAC to bacteria is even longer than the distance of the large group of prevailing human cyclases to bacterial cyclases. Thus, there are no indications of a recent or even ancient horizontal transfer of the gene. Second, the estimated relative divergence times of Dictyostelium sGC and rat sAC on a relative bacteria-human time scale are not significantly different from the proposed divergence times of the species, as deduced from the phylogeny of many proteins (table 1
[Baldauf et al. 2000
; Nei, Xu, and Glazko 2001
]). This strongly suggests a normal evolutionary trait of sAC from bacteria via eukaryotic microorganisms to vertebrates, which implies the loss of the gene in multiple lineages. The family of sAC genes is an example of a dozen cases in which Dictyostelium forms the only phylogenetic connection identified so far between bacteria and vertebrates (Roelofs and Van Haastert 2001c
).
The difference in evolutionary success between the subclasses of cyclases is intriguing. Although the sAC branch is almost completely lost in many species, the group of prevailing eukaryotic cyclases is extensively used in several organisms. Vertebrates have nine subtypes of the 12 transmembrane ACs (Hanoune and Defer 2001
). Also membrane GCs and soluble GCs have expanded in animals. In C. elegans the membrane GC family contains even more than 29 members (Yu et al. 1997
). Possibly the genes of the sAC group have been fixed only in organisms where it received an important function, such as chemotaxis in Dictyostelium and sperm maturation in vertebrates. The imbalance in the number of sequences present in the sAC group compared with the prevailing eukaryotic cyclases makes it tempting to refer to sAC as closely related to bacterial ACs (Buck et al. 1999
; Hanoune and Defer 2001
; Roelofs et al. 2001a, 2001b
). When performing blast searches using sAC cyclase domains as input (e.g., C1 domain of HssAC1), the first bacterial sequence appears at position 6, whereas for a cyclase domain of the prevailing group (e.g., RnGCE), the first bacterial sequence does not come before position 260. However, the expectation values obtained are 6 x 10-7 and 5 x 10-15 for HssAC1 and RnGCE, respectively, suggesting that the evolutionary distances toward bacterial cyclases is slightly shorter for RnGCE than for HssAC1, which was also observed in the phylogenetic analysis (fig. 3
). The phylogenetic analysis of the family of cyclase domains in eukaryotes suggests that both the small group of sACs and the large group of prevailing cyclase are derived from the cyclase domain in bacteria. Whereas the prevailing cyclases became very successful during evolution, the soluble cyclases have become fixed in only a few eukaryotes and bacteria but have not become ubiquitous among them.
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Acknowledgements |
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Footnotes |
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Abbreviations: AC, adenylyl cyclase; GC, guanylyl cyclase; sCKH region, soluble cyclase-kinase homology region.
Keywords: Dictyostelium
evolution
guanylyl cyclase
gene loss
adenylyl cyclase
Address for correspondence and reprints: Peter J. M. Van Haastert, GBB, Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: p.j.m.van.haastert{at}chem.rug.nl
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