Evolution of Lysine Biosynthesis in the Phylum Deinococcus-Thermus

Thermus thermophilus biosynthesizes lysine through the α-aminoadipate (AAA) pathway: this observation was the first discovery of lysine biosynthesis through the AAA pathway in archaea and bacteria. Genes homologous to the T. thermophilus lysine biosynthetic genes are widely distributed in bacteria of the Deinococcus-Thermus phylum. Our phylogenetic analyses strongly suggest that a common ancestor of the Deinococcus-Thermus phylum had the ancestral genes for bacterial lysine biosynthesis through the AAA pathway. In addition, our findings suggest that the ancestor lacked genes for lysine biosynthesis through the diaminopimelate (DAP) pathway. Interestingly, Deinococcus proteolyticus does not have the genes for lysine biosynthesis through the AAA pathway but does have the genes for lysine biosynthesis through the DAP pathway. Phylogenetic analyses of D. proteolyticus lysine biosynthetic genes showed that the key gene cluster for the DAP pathway was transferred horizontally from a phylogenetically distant organism.


Introduction
The Deinococcus-Thermus phylum constitutes one of the major bacterial evolutionary lineages [1,2]. At present, the genome sequence data of 6 genera (13 organisms) belonging to this phylum are available in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [3].
Two pathways for lysine biosynthesis have been described, namely, the α-aminoadipate (AAA) pathway and the diaminopimelate (DAP) pathway [5]. The AAA pathway has two different types [6]. In T. thermophilus, a gene cluster was found for lysine biosynthesis not through the DAP pathway but through the AAA pathway [6][7][8]. Although Deinococcus radiodurans has genes homologous to the T. thermophilus lysine biosynthetic genes, these genes are scattered on the genome [9]. In addition, the D. radiodurans aspartate kinase that catalyzes the phosphorylation of l-aspartate (the first reaction in the DAP pathway) is structurally and phylogenetically very different from that of T. thermophilus [10]. Recent studies have shown that the genome signatures of these 2 bacteria are different [4], supporting the theory that Deinococcus species acquired genes from various other bacteria to survive different kinds of environmental stresses, whereas Thermus species have acquired genes from thermophilic bacteria to adapt to high-temperature environments [11].
The distribution of lysine biosynthetic genes in the Deinococcus-Thermus phylum has not been clearly described. In this study, we compared the distribution of the genes for lysine biosynthesis between 13 organisms (D.

Methods
We analyzed the distribution of each of the following 10 enzymes related to lysine biosynthesis through the AAA pathway in the Deinococcus-Thermus phylum: αaminoadipate aminotransferase, homoisocitrate dehydrogenase, LysW-γ-l-lysine aminotransferase, LysW-γ-l-lysine   Figure 4: Phylogenetic relationship between Deinococcus proteolyticus dihydrodipicolinate reductase and related proteins. Multiple alignment was obtained using the top 20 amino acid sequences of the BLASTp search result for D. proteolyticus dihydrodipicolinate reductase (Deipro 1378), as based on the KEGG database. The maximum-likelihood tree was constructed using MEGA software version 5 [12]. The WAG model was used as the amino acid substitution model. The nearest neighbor interchange was used for the maximum-likelihood heuristic method. The γ-distributed rate was considered, and the number of discrete γ categories was 3. Bootstrap analysis was performed with 100 replicates. Red indicates D. proteolyticus.
hydrolase, LysW-γ-l-α-aminoadipate kinase, LysW-γ-l-αaminoadipyl-6-phosphate reductase, α-aminoadipate-LysW ligase LysX, LysU, LysT, and homocitrate synthase. In addition, we analyzed the distribution of each of the following 6 enzymes related to lysine biosynthesis through the DAP pathway: aspartate kinase, aspartate-semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, ll-diaminopimelate aminotransferase, and diaminopimelate decarboxylase. Homologous genes were selected on the basis of BLASTp search results by using each T. thermophilus enzyme for lysine biosynthesis through the AAA pathway and each D. proteolyticus enzyme for lysine biosynthesis through the DAP pathway. Multiple alignments were obtained using 20 amino acid sequences, with the highest to the 20th highest score by the BLASTp result. Maximum-likelihood trees were constructed using MEGA software version 5 [12]. The WAG model [13] was used as the amino acid substitution model. The nearest neighbor interchange was used for the maximum-likelihood heuristic method. The γ-distributed rate was considered, and the number of discrete γ categories was 3. Bootstrap analysis was performed with 100 replicates.

Results and Discussion
Genes homologous to the T. thermophilus genes for lysine biosynthesis through the AAA pathway were found to be widely distributed in bacteria belonging to the Deinococcus-Thermus phylum, except for D. proteolyticus (Table 1). Among the 13 organisms examined, Marinithermus, Oceanithermus, and Truepera have the largest gene cluster, containing 8 lysine biosynthetic genes (Table 1). In each phylogenetic analysis of the 10 enzymes, lysine biosynthetic genes of the Deinococcus-Thermus phylum were found to have a common ancestor (See in Supplementary Material Figures S1−S10 available online at doi:10.1155/2012/745931). We hypothesize that a common ancestor of the Deinococcus-Thermus phylum biosynthesized lysine through the AAA pathway.
In contrast, the distribution of genes for lysine biosynthesis through the DAP pathway was found to be limited in the Deinococcus-Thermus phylum (Table 2). Thus, lldiaminopimelate aminotransferase and dihydrodipicolinate reductase were identified in no bacteria other than D. proteolyticus (Table 2). This observation supports our hypothesis that a common ancestor of the Deinococcus-Thermus phylum biosynthesized lysine not through the DAP pathway, but through the AAA pathway.
Interestingly, D. proteolyticus was found to have the genes for lysine biosynthesis through the DAP pathway ( Table 2). D. proteolyticus has 2 diaminopimelate decarboxylases, namely, Deipro 0627 and Deipro 1375 (Table 2), which are structurally different from each other. Because Deipro 1375 forms a gene cluster with other genes for lysine biosynthesis through the DAP pathway, we used Deipro 1375 as a query sequence in the BLASTp search. Each phylogenetic tree based on diaminopimelate decarboxylase (Figure 1), ll-diaminopimelate aminotransferase (Figure 2), dihydrodipicolinate synthase (Figure 3), and dihydrodipicolinate reductase (Figure 4) showed that the D. proteolyticus enzyme is closely related to that of the genera Kytococcus (a member of Actinobacteria) and Spirochaeta (a member of Spirochaetes) (Figures 1−4). The 3 phyla Actinobacteria, Deinococcus-Thermus, and Spirochaetes do not form a monophyletic lineage in the phylogenetic tree, as based on genomewide comparative studies [14]. In addition, the 4 genes encoding diaminopimelate decarboxylase, lldiaminopimelate aminotransferase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase are clustered in each genus (Figures 1−4). Thus, these findings strongly suggested that a DNA fragment including the 4 D. proteolyticus genes was horizontally transferred from a phylogenetically distant organism. This horizontal transfer event may have induced the loss of the genes for lysine biosynthesis through the AAA pathway in D. proteolyticus.