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.

1. 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 [6-8]. 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. deserti, D. geothermalis, D. maricopensis, D. proteolyticus, D. radiodurans, Marinithermus hydrothermalis, Meiothermus ruber, M. silvanus, Oceanithermus profundus, T. scotoductus, T. thermophilus HB8, T. thermophilus HB27, and Truepera radiovictrix).

2. 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: α-aminoadipate aminotransferase, homoisocitrate dehydrogenase, LysW-γ-l-lysine aminotransferase, LysW-γ-l-lysine hydrolase, LysW-γ-l-α-aminoadipate kinase, LysW-γ-l-α-aminoadipyl-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.

3. 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.

Table 1

Genes for lysine biosynthesis through the α-aminoadipate pathway in the Deinococcus-Thermus phylum.

Organism	Enzyme 1	Enzyme 2	Enzyme 3	Enzyme 4	Enzyme 5	Enzyme 6	Enzyme 7	Enzyme 8	Enzyme 9	Enzyme 10
Thermus thermophilus HB27	TTC0043	TTC1012	TTC1393	TTC1396	TTC1541*	TTC1542*	TTC1543*	TTC1546*	TTC1547*	TTC1550*
Thermus thermophilus HB8	TTHA0411	TTHA1378	TTHA1755	TTHA1757	TTHA1903*	TTHA1904*	TTHA1907*	TTHA1910*	TTHA1911*	TTHA1914*
Thermus scotoductus	TSC_c05810	TSC_c20650	TSC_c03550	TSC_c3520	TSC_c01940*	TSC_c01930*	TSC_c01920*	TSC_c01890*	TSC_c01880*	TSC_c01850*
Meiothermus ruber	Mrub_0871	Mrub_2738		Mrub_0027	Mrub_2721*	Mrub_2723*	Mrub_2724*	Mrub_2727*	Mrub_2728*
Meiothermus silvanus	Mesil_2567	Mesil_1337	Mesil_0348	Mesil_0347	Mesil_0435*	Mesil_0436*	Mesil_0438*	Mesil_0441*	Mesil_0442*
Oceanithermus profundus		Ocepr_1387	Ocepr_1797*	Ocepr_1798*	Ocepr_1796*	Ocepr_1788*	Ocepr_1784*	Ocepr_1781*	Ocepr_1780*	Ocepr_1779*
Marinithermus hydrothermalis		Marky_1533	Marky_0665*	Marky_0663*	Marky_0666*	Marky_0667*	Marky_0668*	Marky_0671*	Marky_0672*	Marky_0673*
Deinococcus radiodurans		DR_1674	DR_0794	DR_1413	DR_1420	DR_0963	DR_2194	DR_1614	DR_1610	DR_1238
Deinococcus geothermalis	Dgeo_2084	Dgeo_1458	Dgeo_1416	Dgeo_1391	Dgeo_0678	Dgeo_0685	Dgeo_1151*	Dgeo_1154*	Dgeo_1156*	Dgeo_1257
Deinococcus deserti		Deide_09240	Deide_16910	Deide_17960	Deide_10430	Deide_10350	Deide_13430*	Deide_13460*	Deide_13470*	Deide_13980
Deinococcus maricopensis	Deima_0046	Deima_1545	Deima_2454	Deima_2593	Deima_1346*	Deima_1349*	Deima_1350*	Deima_1353*	Deima_1355*	Deima_1358*
Deinococcus proteolyticus	Deipr_0213
Truepera radiovictrix		Trad_2841	Trad_1401*	Trad_1404*	Trad_1399*	Trad_1395*	Trad_1392*	Trad_1390*	Trad_1389*	Trad_1388*

Enzyme 1, α-aminoadipate aminotransferase.

Enzyme 2, Homoisocitrate dehydrogenase.

Enzyme 3, LysW-γ-l-lysine aminotransferase.

Enzyme 4, LysW-γ-l-lysine hydrolase.

Enzyme 5, LysW-γ-l-α-aminoadipate kinase.

Enzyme 6, LysW-γ-l-α-aminoadipyl-6-phosphate reductase.

Enzyme 7, α-aminoadipate-LysW ligase LysX.

Enzyme 8, LysU.

Enzyme 9, LysT.

Enzyme 10, Homocitrate synthase.

*More than 3 genes are clustered.

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, ll-diaminopimelate 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.

Table 2

Genes for lysine biosynthesis through the diaminopimelate pathway in the Deinococcus-Thermus phylum.

Organism	Aspartate kinase	Aspartate-semialdehyde dehydrogenase	Dihydrodipicolinate synthase	Dihydrodipicolinate reductase	ll-diaminopimelate aminotransferase	Diaminopimelate decarboxylase
Thermus thermophilus HB27	TTC0166	TTC0177	TTC0591
Thermus thermophilus HB8	TTHA0534	TTHA0545	TTHA0957
Thermus scotoductus	TSC_c07050	TSC_c08140	TSC_c10420			TSC_c10870
Meiothermus ruber	Mrub_0976	Mrub_1641	Mrub_1335			Mrub_0798
Meiothermus silvanus	Mesil_1711	Mesil_2173	Mesil_2308			Mesil_0318
Oceanithermus profundus	Ocepr_1316	Ocepr_1018				Ocepr_2076
Marinithermus hydrothermalis	Marky_1492	Marky_1381	Marky_1261
Deinococcus radiodurans	DR_1365	DR_2008				DR_1758
Deinococcus geothermalis	Dgeo_1127	Dgeo_1782				Dgeo_0790
Deinococcus deserti	Deide_11430	Deide_15740	Deide_1p00310, Deide_3p00120, Deide_3p01100			Deide_12830, Deide_21880
Deinococcus maricopensis	Deima_1822	Deima_2680				Deima_2660
Deinococcus proteolyticus	Deipr_0941	Deipr_0985	Deipr_1377*	Deipr_1378*	Deipr_1376*	Deipr_0627, Deipr_1375*
Truepera radiovictrix	Trad_0977	Trad_0289	Trad_1893			Trad_0134

*More than 3 genes are clustered.

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, ll-diaminopimelate 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.

Figure 1

Phylogenetic relationship between Deinococcus proteolyticus diaminopimelate decarboxylase and related proteins. Multiple alignment was obtained using the top 20 amino acid sequences of the BLASTp search result for D. proteolyticus diaminopimelate decarboxylase (Deipro 1375), as based on the Kyoto Encyclopedia of Genes and Genomes (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.

Figure 2

Phylogenetic relationship between Deinococcus proteolyticus ll-diaminopimelate aminotransferase and related proteins. Multiple alignment was obtained using the top 20 amino acid sequences of the BLASTp search result for D. proteolyticus ll-diaminopimelate aminotransferase (Deipro 1376), 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.

Figure 3

Phylogenetic relationship between Deinococcus proteolyticus dihydrodipicolinate synthase and related proteins. Multiple alignment was obtained using the top 20 amino acid sequences of the BLASTp search result for D. proteolyticus dihydrodipicolinate synthase (Deipro 1377), 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.

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.

Phylogenetic relationships among enzymes related to lysine biosynthesis through the AAA pathway.

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