Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, Japan
Correspondence: E-mail: tgojobor{at}genes.nig.ac.jp.
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Abstract |
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Key Words: vitamin B6 metabolic network gene gain gene loss complete genome
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Introduction |
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Many studies of VB6 metabolism have been conducted in Escherichia coli (White and Dempsey 1970; Lam and Winkler 1990; Zhao et al. 1995; Yang, Zhao, and Winkler 1996; Man, Zhao, and Winkler 1996) and fungi such as Cercospora nicotianae, Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae (Ehrenshaft et al. 1999; Osmani, May, and Osmani 1999; Bean et al. 2001; Ehrenshaft and Daub 2001). For PLP biosynthesis, three pathways have been characterized, and a total of 10 genes are involved in the pathways in bacteria and fungi (fig. 1). In the case of E. coli, both de novo and salvage pathways have been identified. These two pathways include enzymes encoded by eight genes in total (Mittenhuber 2001) and share only one gene, pdxH. In the de novo pathway, the pyridine ring of VB6 is generated from D-erythrose-4-phosphate (E4P) and glyceraldehyde-3-phosphate. On the other hand, in the salvage pathway, PLP is synthesized without pyridine ring generation. A corresponding de novo pathway has not been discovered in fungi or plants. Instead, fungi were found to have another biosynthetic pathway, the fungal type pathway. This pathway has two genes, SNZ and SNO, whose functions are currently unknown. Therefore, species that synthesize PLP have been reported to have at least one of the three PLP biosynthetic pathways.
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To study the evolutionary process of VB6 metabolism, we investigated the 10 genes involved in the three pathways for PLP biosynthesis. We were particularly interested in learning when the individual genes were gained or lost during evolution. Therefore, we focused on the gain and loss of these 10 genes in 122 species in the three domains of life, namely eubacteria, archaebacteria, and eukaryotes (Woese, Kandler, and Wheelis 1990). We estimated the pertinent gene set of the common ancestor of the 122 species on the basis of their genealogical relationships. Next, we identified the gain and loss events for the genes by comparing the gene sets between the ancestral and extant species. On the basis of the results obtained, we report the evolutionary features of the formation and dysfunction processes of VB6 metabolism from the view of the gains and losses of the genes.
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Materials and Methods |
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Phylogenetic Tree
To estimate the gene sets involved with the PLP biosynthetic pathways of ancestors of the 122 species, we basically employed the eukaryotic lineages of Baldauf et al. (2000) and the eubacterial and archaebacterial lineages of Nelson et al. (2000). For missing species in the eubacterial and archaebacterial lineages, such as proteobacteria, firmicutes, actinobacteria, chlamydia, spirochete, cyanobacteria, euryarchaeota, and crenarchaeota, we constructed their phylogenetic trees for the same gene (16s rRNA) as that used by Nelson et al. (2000). To do this, we applied their 16s rRNA sequences to the ClustalW program with 1,000 bootstrap trials (Thompson, Higgins, and Gibson 1994). We excluded the positions with gaps and corrected for multiple substitutions.
Estimation of the Gene Set of the PLP Biosynthetic Pathways in the Ancestor
We assumed that a single gene was acquired only once during the evolution of the 122 species examined in this model and ignored horizontal gene transfer and parallel evolution. The method for estimating the set of genes in the ancestor was as follows. As shown in figure 2, we used the following two states to represent whether a species had a particular gene (Gene-A): "1" was used when the species had at least one homologous gene to Gene-A, and "0" was used when the species had no homologous genes to Gene-A.
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The Order of the Losses Among the Genes
Using the results for the estimation of the gene losses, we investigated the order of the losses among the 10 genes. If the loss of a gene occurred randomly during the evolution of the PLP biosynthetic pathways, skewness of the early loss of either one of the two genes may not be observed (null hypothesis). Therefore, we considered that the frequency of the order of gene loss between two genes followed the binominal distribution. For all pairs among the 10 genes, we examined which gene was lost first during the evolution of the 122 species and statistically tested the frequency of the order of gene loss on the basis of the binominal distribution. If the losses of two genes occurred on the same branch in the phylogenetic tree, we did not count them, because we did not know which gene was lost first.
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Results |
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Moreover, we found that gene sets containing all seven genes for the de novo pathway (gapA, dxs, pdxA, pdxB, pdxF, pdxH, and pdxJ) were only found in 18 eubacteria. These bacterial species were all -proteobacteria, indicating that the de novo pathway may only function in
-proteobacteria.
Distribution of the Genes in the Three Domains of Life
Based on the gene sets of the 122 species, we compared the gene sets among the three domains of life (fig. 3). SNZ, SNO, and pdxF were observed in all the domains, indicating that they existed before the divergence into the three domains. This result also indicates that the fungal type pathway composed of SNZ and SNO was formed before the divergence into the three domains. The other four genes in the de novo and salvage pathways (gapA, dxs, pdxH, and pdxK) were discovered in eukaryotes and eubacteria, indicating that part of these pathways was present in eukaryotes and eubacteria. Based on our assumption that a single gene was acquired only once during the evolution of the 122 species examined, we interpret this result to indicate that the salvage pathway composed of pdxH and pdxK was formed before the divergence into the three domains. We also note that pdxA, pdxB, and pdxJ were only observed in eubacteria. When we focused on the eubacteria, we found that both pdxA and pdxJ were observed not only in proteobacteria but also firmicutes, cyanobacteria, chlorobi, and aquificae, whereas pdxB only existed in -proteobacteria. Therefore, we consider that both pdxA and pdxJ were generated in the eubacterial lineage after the divergence from the other two domains and that pdxB was generated in the
-proteobacterial lineage after the divergence from the other lineages. These three genes are, therefore, considered to have contributed to the formation of the de novo pathway in the eubacterial lineage. In particular, pdxB may be the most important gene for the completion of this pathway because it was only generated in
-proteobacteria.
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Next, we examined the order of the losses for the 10 genes. When we compared the order of the losses between two genes, a significant bias was observed in nine particular combinations of genes: pdxH and dxs, pdxJ and pdxH, pdxH and pdxF, pdxA and dxs, pdxJ and pdxA, pdxJ and dxs, pdxJ and pdxK, pdxJ and SNO, and pdxK and pdxF (appendix B of Supplemental Material online). In every pair of these nine combinations, we observed that the loss of the latter gene occurred more frequently after the loss of the former gene. These biases were statistically significant against the binominal distribution (P < 0.05). From this observation, we deduced the patterns of losses of five genes, as shown in figure 6. The loss of pdxJ caused the loss of at least one of pdxA, pdxH, and pdxK. These four genes encode enzymes whose reactions are connected through pyridoxine 5'-phosphate (PNP). Moreover, the losses of these four genes caused the loss of at least one of pdxF and dxs. These two genes encode enzymes whose reactions are connected to pdxA and pdxJ through 4-phosphohydroxy-L-threonine (4PHT) and 1-deoxyxylulose 5-phosphate (DXP), respectively. If we designate the two genes encoding enzymes that catalyze two consecutive reactions the neighbor genes, we can, thus, conclude that the loss of one gene accelerates the loss of the neighbor gene.
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Discussion |
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We also revealed three aspects to the evolution of the PLP biosynthetic pathways by estimating the gain and loss of the 10 genes. The first aspect is related to the evolutionary order of the generation of the three PLP biosynthetic pathways. From the distribution of the 10 genes in the 122 species examined, we found that the fungal type and salvage pathways were probably older than the de novo pathway on the basis of the following two results. First, pdxK and pdxH of the salvage pathway exist in eubacteria and eukaryotes, and SNO and SNZ of the fungal type pathway are found in all the three domains, namely, eubacteria, archaebacteria, and eukaryotes. Therefore, we consider that the fungal type and salvage pathways both existed before the separation of the three domains of life. Second, pdxB of the de novo pathway only exists in -proteobacteria, indicating that it was generated in
-proteobacteria after the divergence of the three domains of life.
Applying the second result mentioned above to the existing model for explaining the evolution of the metabolic networks, the patchwork model and de novo invention (Jensen 1976; Schmidt et al. 2003), we propose that the process for the formation of the de novo pathway in -proteobacteria was as follows (fig. 7). Originally, the common ancestor of the 97 eubacterial species studied had part of the de novo pathway involving five genes (dxs, pdxA, pdxF, pdxH, and pdxJ). Because gapA functioned not only in the PLP biosynthetic pathways but also in glycolysis (Seta et al. 1997), we think that this gene also existed in the common ancestor. As shown in figure 7, when pdxB was generated in the lineage of
-proteobacteria, the reaction catalyzed by the product of pdxB was connected to the two metabolic reactions that were separately catalyzed by the products of gapA and pdxF. As a result, the de novo pathway was completed by the presence of the seven genes (dxs, gapA, pdxA, pdxB, pdxF, pdxH, and pdxJ) in
-proteobacteria. We have, therefore, reached the same conclusion as Mittenhuber (2001) but via a different process. Mittenhuber postulated that the de novo pathway was largely restricted to
-proteobacteria on the basis of the functions of pdxA and pdxJ and the requirement of VB6 in the de novo pathway.
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The second aspect is related to the losses of SNO and SNZ in animal lineages. Among animals, these two genes have only been discovered in the marine sponge S. domuncula (Seack et al. 2001). Therefore, it is plausible that the losses of SNZ and SNO occurred only once in the Eumetazoan lineage after its divergence from the Poriferal lineage. We found that the two genes were present in the complete genome sequence of C. intestinalis in the present study. This species is more closely related to mammals than D. melanogaster and C. elegans, neither of which have the two genes (fig. 5). Therefore, we consider that SNZ and SNO existed in animals after the divergence between invertebrates and vertebrates and that their losses occurred independently at least three times in the animal lineage, as shown in figure 5. We reject the possibility of horizontal gene transfer from the bacterial lineage to C. intestinalis not only by the homology of the two genes but also by the order and orientation of the two genes in the genome (fig. 4).
The third aspect is related to the evolutionary order of the gene loss. The losses of five genes occurred in the order shown in figure 6 during the evolution of the PLP biosynthetic pathways. Historically, five models have been proposed to explain the formation of metabolic pathways: the retrograde model, the patchwork model, de novo invention, specialization of a multifunctional enzyme, and pathway duplication (Horowitz 1945; Jensen 1976; Schmidt et al. 2003). However, these models only consider the gene gain. Because there are other reports that gene losses have often occurred in the metabolic pathways in bacterial lineages (Tatusov et al. 1996; Shigenobu et al. 2000), it is not sufficient to only consider the gain of genes for the evolution of metabolic pathways in bacterial lineages.
Therefore, we propose a new model based on our results that explains the evolution of metabolic pathways by gene loss. Once the loss of a gene has occurred in a metabolic pathway, the neighboring gene is more easily lost than other genes in the pathway. This can be explained by functional constraints. The breakdown of a metabolic pathway by gene loss will decrease the functional constraints on the other genes of the pathway. Our model suggests that the functional constraint on the proximal genes to the lost gene decreases more extensively than that on the distant genes. Of course, it is possible that the functional constraint is affected by other pathways. For example, if a gene is also involved in another metabolic pathway, as in the case of gapA, its functional constraint may not be changed.
Our approach to estimating the gain and loss of genes is affected by at least two points. The first point is the frequency of the gain and loss of genes. In this study, we considered that gene gain occurred only once, even though gene loss could have occurred more than once in the evolution of the 122 species examined. As a result, we concluded that there were seven genes in the common ancestor of the 122 species examined and that a total of 132 gene losses took place during the evolution of the 122 species. However, when we performed an estimation based on the parsimony method, there were only three genes in the common ancestor and the number of gene losses was underestimated because of overestimation of the gene gain (data not shown). These results indicate that the prediction of the gene set in the ancestor and the gain and loss of genes are clearly affected by the initial assumption.
However, we can emphasize the low possibility of gene gain for the following reasons. The cause of gene gain is mainly horizontal gene transfer or parallel evolution. Therefore, if there is a difference in the gene sets among closely related species, the number of gene losses is expected to be larger than that of gene gains. If horizontal gene transfer and parallel evolution occurred, then gene loss would decrease and gene gain would increase. When we have evidence for horizontal gene transfer and parallel evolution of the 10 genes in this study, it will be possible to estimate the times of the gain and loss of the genes more accurately. In fact, we examined the probability of horizontal gene transfer for the 10 genes between eubacteria and archaebacteria using the method of Nakamura et al. (2004) and concluded that the probability was negligible.
The second point is that our results are affected by the topology of the phylogenetic tree. If the topology is changed, the estimation of the evolutionary times of the gain and loss of genes are changed accordingly. As a result, it is possible to miscount the total numbers of gains and losses of the genes. However, our results showed that the sets of genes were different among the 122 species examined (appendix A of Supplemental Material online). Because the gain and loss of genes cause the differentiation of the sets of genes in the species, our conclusion that the evolutionary process of VB6 metabolism has been quite dynamic regarding the events of gain and loss of genes, under some constraints, is not altered, even when the topology of the phylogenetic tree changes.
Studies using comparative analysis have often shown differences in gene sets involved in metabolic pathways among species (Huynen, Dandekar, and Bork 1999). By estimating the gain and loss of genes, we are able to learn not only the differences in a metabolic pathway among the species examined but also in which lineage the change in the metabolic pathway occurred during evolution. This means that we will be able to understand the evolutionary processes of the metabolic networks by evaluating the gains and losses of genes. In some metabolic pathways, dysfunctions in particular lineages have been reported (Smirnoff 2001; Meganathan 2001). By applying our approach to these metabolic pathways, we will be able to elucidate the dysfunctions in these pathways by the gain and loss of genes. It is also possible to further extend our approach to other metabolic networks in the KEGG (Kanehisa et al. 2002) and EcoCyc (Karp et al. 2002) databases, to more clearly understand the evolutionary processes of the metabolic pathways they contain.
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Acknowledgements |
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Footnotes |
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References |
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