International Journal of Evolutionary BiologyVolume 2012 (2012), Article ID 745931, 6 pagesdoi:10.1155/2012/745931

Research Article

Evolution of Lysine Biosynthesis in the Phylum Deinococcus-Thermus

Hiromi Nishida¹ and Makoto Nishiyama²

¹Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan²Biotechnology Research Center, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received 28 January 2012; Accepted 17 February 2012

Academic Editor: Kenro Oshima

Copyright © 2012 Hiromi Nishida and Makoto Nishiyama. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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.

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