Hindawi Publishing Corporation International Journal of Evolutionary Biology Volume 2012, Article ID 745931, 6 pages doi:10.1155/2012/745931

Research Article Evolution of Lysine Biosynthesis in the Phylum Deinococcus-Thermus

Hiromi Nishida1 and Makoto Nishiyama2

1 Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 2 Biotechnology Research Center, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Correspondence should be addressed to Hiromi Nishida, [email protected]

Received 28 January 2012; Accepted 17 February 2012

Academic Editor: Kenro Oshima

Copyright © 2012 H. Nishida and M. 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.

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 species acquired genes from various other bacteria to survive different kinds of environmental stresses, whereas Thermus The Deinococcus-Thermus phylum constitutes one of the species have acquired genes from thermophilic bacteria to major bacterial evolutionary lineages [1, 2]. At present, the adapt to high-temperature environments [11]. genome sequence data of 6 genera (13 organisms) belonging The distribution of lysine biosynthetic genes in the to this phylum are available in the Kyoto Encyclopedia of Deinococcus-Thermus phylum has not been clearly described. Genes and Genomes (KEGG) database [3]. In this study, we compared the distribution of the genes Two pathways for lysine biosynthesis have been de- for lysine biosynthesis between 13 organisms (D. deserti, α scribed, namely, the -aminoadipate (AAA) pathway and the D. geothermalis, D. maricopensis, D. proteolyticus, D. radio- diaminopimelate (DAP) pathway [5]. The AAA pathway has durans, Marinithermus hydrothermalis, Meiothermus ruber, ff two di erent types [6]. In T. thermophilus,ageneclusterwas M. silvanus, Oceanithermus profundus, T. scotoductus, T. found for lysine biosynthesis not through the DAP pathway thermophilus HB8, T. thermophilus HB27, and Truepera but through the AAA pathway [6–8]. Although Deinococcus radiovictrix). radiodurans has genes homologous to the T. thermophilus lysine biosynthetic genes, these genes are scattered on the 2. Methods genome [9]. In addition, the D. radiodurans aspartate kinase that catalyzes the phosphorylation of l-aspartate (the first We analyzed the distribution of each of the following reaction in the DAP pathway) is structurally and phylogenet- 10 enzymes related to lysine biosynthesis through the ically very different from that of T. thermophilus [10]. Recent AAA pathway in the Deinococcus-Thermus phylum: α- studies have shown that the genome signatures of these 2 bac- aminoadipate aminotransferase, homoisocitrate dehydro- teria are different [4], supporting the theory that Deinococcus genase, LysW-γ-l-lysine aminotransferase, LysW-γ-l-lysine 2 International Journal of Evolutionary Biology ∗ ∗ ∗ ∗ ∗ ∗ ∗ 1358 0673 1257 1779 13980 1238 1388 c01850 Dgeo TTC1550 Trad Deide TTHA1914 Ocepr TSC Marky Deima ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 1355 0672 1780 2728 0442 1156 1610 DR 1389 13470 c01880 TTC1547 Trad Dgeo Mesil Mrub TTHA1911 Ocepr TSC Marky Deima Deide ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 1353 0671 1781 2727 0441 1154 1614 DR 1390 13460 c01890 phylum. TTC1546 Trad Dgeo Mesil Mrub TTHA1910 Ocepr TSC Marky Deima Deide ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 1350 0668 1784 2724 0438 1151 2194 DR 1392 13430 c01920 TTC1543 Trad Mesil Mrub TTHA1907 Ocepr TSC Marky Deima ∗ ∗ ∗ Deinococcus-Thermus ∗ ∗ ∗ ∗ ∗ ∗ 1349 0667 0685 Dgeo 1788 10350 Deide 2723 0436 0963 DR 1395 c01930 TTC1542 Trad Mesil Mrub TTHA1904 Ocepr TSC Marky Deima ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 1346 0666 0678 Dgeo 1796 10430 Deide 2721 0435 1420 DR 1399 c01940 Trad Ocepr Marky -aminoadipate pathway in the ∗ α ∗ ∗ 2593 Deima 0027 Mrub 0663 0347 Mesil 1391 Dgeo 1798 17960 Deide c3520 TSC 1413 DR 1404 Trad Ocepr Marky ∗ ∗ ∗ 2454 Deima 0348 Mesil 0665 1416 Dgeo 16910 Deide 1797 0794 DR 1401 c03550 TSC 1545 Deima 1533 Marky 1387 Ocepr 2738 Mrub 1337 Mesil 1458 Dgeo 2841 Trad 09240 Deide 1674 DR c20650 TSC Marky 1: Genes for lysine biosynthesis through the 0046 Deima 0871 Mrub 0213 2567 Mesil 2084 Dgeo Table c05810 TSC TTC0043 TTC1012 TTC1393 TTC1396 TTC1541 TTHA0411 TTHA1378 TTHA1755 TTHA1757 TTHA1903 HB27 HB8 -aminoadipate kinase. -aminoadipyl-6-phosphate reductase. α α -lysine aminotransferase. -lysine hydrolase. - - l l l l - - - - γ γ γ γ -aminoadipate aminotransferase. -aminoadipate-LysW ligase LysX. α α More than 3 genes are clustered. OrganismThermus thermophilus Thermus thermophilus Thermus scotoductusMeiothermus ruberMeiothermus silvanusOceanithermus profundus TSC EnzymeMarinithermus 1hydrothermalis Mrub Mesil EnzymeDeinococcus 2 radiodurans Enzyme 3 Ocepr Deinococcus Enzyme proteolyticus 4 Deipr Enzyme 5 DR Enzyme 6 Enzyme 7 Enzyme 8 Enzyme 9 Enzyme 10 Truepera radiovictrix Trad Deinococcus deserti Deide Deinococcus geothermalis Dgeo Deinococcus maricopensis Deima Enzyme 2, Homoisocitrate dehydrogenase. Enzyme 3, LysW- Enzyme 1, ∗ Enzyme 4, LysW- Enzyme 5, LysW- Enzyme 6, LysW- Enzyme 8, LysU. Enzyme 9, LysT. Enzyme 10, Homocitrate synthase. Enzyme 7, International Journal of Evolutionary Biology 3

Table 2: Genes for lysine biosynthesis through the diaminopimelate pathway in the Deinococcus-Thermus phylum.

Aspartate- ll- Aspartate Dihydrodipicolinate Dihydrodipicolinate Diaminopimelate Organism semialdehyde diaminopimelate kinase synthase reductase decarboxylase dehydrogenase aminotransferase 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 Marky 1492 Marky 1381 Marky 1261 hydrothermalis Deinococcus radiodurans DR 1365 DR 2008 DR 1758 Deinococcus geothermalis Dgeo 1127 Dgeo 1782 Dgeo 0790 Deide 1p00310, Deide 12830, Deinococcus deserti Deide 11430 Deide 15740 Deide 3p00120, Deide 21880 Deide 3p01100 Deinococcus maricopensis Deima 1822 Deima 2680 Deima 2660 Deipr 0627, Deinococcus proteolyticus Deipr 0941 Deipr 0985 Deipr 1377∗ Deipr 1378∗ Deipr 1376∗ Deipr 1375∗ Truepera radiovictrix Trad 0977 Trad 0289 Trad 1893 Trad 0134 ∗ More than 3 genes are clustered.

33 Spirochaeta thermophila STHERM c00170 16 Denitrovibrio acetiphilus Dacet 0798 Spirochaeta caldaria DSM 7334 Spica 0951 35 100 Treponema primitia TREPR 1186 Calditerrivibrio nitroreducens Calni 0514 35 89 Flexistipes sinusarabici Flexsi 1574 97 Methanohalophilus mahii Mmah 0892 Methanococcoides burtonii Mbur 0628 98 100 Methanosarcina barkeri Mbar A1641 100 Methanosarcina mazei MM 1885 100 Methanosarcina acetivorans MA0726 Selenomonas sputigena Selsp 2045 56 Syntrophobotulus glycolicus Sgly 1852 61 Desulfitobacterium hafniense DCB-2 Dhaf 4876 100 Desulfitobacterium hafniense Y51 DSY4977 Spirochaeta smaragdinae Spirs 1460 100 Deinococcus proteolyticus Deipr 1375 Kytococcus sedentarius Ksed 00760 100 Spirochaeta sp. Buddy SpiBuddy 1124 100 Spirochaeta coccoides Spico 0777

0.1 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. 4 International Journal of Evolutionary Biology

100 Anabaena variabilis Ava 2354 59 Anabaena sp. PCC7120 alr5103 81 Cyanothece sp. PCC 7425 Cyan7425 4424 89 Acaryochloris marina AM1 1880 87 Synechococcus elongatus PCC7942 Synpcc7942 0853 100 Synechococcus elongatus PCC6301 syc0687 c 72 Synechococcus sp. CC9605 Syncc9605 0311 Opitutus terrae Oter 4620 Pelobacter carbinolicus Pcar 2423 100 Geobacter sp. M21 GM21 4142 100 Geobacter bemidjiensis Gbem 4052 94 Geobacter metallireducens Gmet 0213 100 Geobacter uraniumreducens Gura 0238 74 Geobacter sp. FRC-32 Geob 1134 50 Geobacter lovleyi Glov 3040 Spirochaeta smaragdinae Spirs 1460 100 Deinococcus proteolyticus Deipr 1376 100 Kytococcus sedentarius Ksed 0070 58 Spirochaeta coccoides Spico 0778 99 Spirochaeta sp. Buddy SpiBuddy 1123

0.1 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.

100 Desulfovibrio vulgaris DP4 Dvul 1296 99 Desulfovibrio vulgaris Hildenborough DVU1868 67 Desulfovibrio vulgaris Miyazaki F DvMF 0562 72 Desulfovibrio desulfuricans G20 Dde 1797 100 Desulfomicrobium baculatum Dbac 0647 Desulfohalobium retbaense Dret 1874 50 76 Desulfovibrio salexigens Desal 1588 67 Candidatus Nitrospira defluvii NIDE0489 70 Desulfurivibrio alkaliphilus DaAHT2 2217 Deferribacter desulfuricans SSM1 DEFDS 0215 94 Calditerrivibrio nitroreducens Calni 1811 Spirochaeta thermophila STHERM c08670 35 Uncultured Termite bacterium phylotype Rs-D17 TGRD 641 50 Aquifex aeolicus aq 1143 100 Hydrogenobacter thermophilus HTH 1231 Spirochaeta smaragdinae Spirs 1462 100 Spirochaeta sp. Buddy SpiBuddy 1122 55 Spirochaeta coccoides Spico 0779 59 Deinococcus proteolyticus Deipr 1377 100 Kytococcus sedentarius Ksed 00780

0.1 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. International Journal of Evolutionary Biology 5

88 Pedobacter heparinus Phep 3529 100 Pedobacter saltans Pedsa 0258 Muricauda ruestringensis Murru 0253 49 93 Leadbetterella byssophila Lbys 0935 Waddlia chondrophila wcw 0766 24 75 Parachlamydia acanthamoebae PUV 02330 51 Candidatus Protochlamydia amoebophila pc0687 Spirochaeta caldaria DSM 7334 Spica 0948 48 97 Treponema azotonutricium TREAZ 1304 82 Treponema primitia TREPR 2975 Treponema succinifaciens Tresu 2067 36 Chlorobaculum parvum NCIB 8327 Cpar 0353 Spirochaeta thermophila STHERM c08680 100 Rhodothermus marinus DSM 4252 Rmar 1657 97 Rhodothermus marinus SG0.5JP17-172 Rhom172 1129 Salinibacter ruber SRM 01279 52 Spirochaeta coccoides Spico 0780 50 Deinococcus proteolyticus Deipr 1378 37 Spirochaeta sp. Buddy SpiBuddy 1121 45 Kytococcus sedentarius Ksed 00790

0.1

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-α- to be widely distributed in bacteria belonging to the aminoadipyl-6-phosphate reductase, α-aminoadipate-LysW Deinococcus-Thermus phylum, except for D. proteolyticus ligase LysX, LysU, LysT, and homocitrate synthase. In (Table 1). Among the 13 organisms examined, Marinither- addition, we analyzed the distribution of each of the mus, Oceanithermus,andTruepera have the largest gene following 6 enzymes related to lysine biosynthesis through cluster, containing 8 lysine biosynthetic genes (Table 1). the DAP pathway: aspartate kinase, aspartate-semialdehyde In each phylogenetic analysis of the 10 enzymes, lysine dehydrogenase, dihydrodipicolinate synthase, dihydrodipi- biosynthetic genes of the Deinococcus-Thermus phylum colinate reductase, ll-diaminopimelate aminotransferase, were found to have a common ancestor (See in Sup- and diaminopimelate decarboxylase. plementary Material Figures S1−S10 available online at Homologous genes were selected on the basis of BLASTp doi:10.1155/2012/745931). We hypothesize that a common search results by using each T. thermophilus enzyme for ancestor of the Deinococcus-Thermus phylum biosynthesized lysine biosynthesis through the AAA pathway and each lysine through the AAA pathway. D. proteolyticus enzyme for lysine biosynthesis through the In contrast, the distribution of genes for lysine biosyn- DAP pathway. Multiple alignments were obtained using 20 thesis through the DAP pathway was found to be limited amino acid sequences, with the highest to the 20th highest in the Deinococcus-Thermus phylum (Table 2). Thus, ll- score by the BLASTp result. Maximum-likelihood trees diaminopimelate aminotransferase and dihydrodipicolinate were constructed using MEGA software version 5 [12]. The reductase were identified in no bacteria other than D. prote- WAG model [13] was used as the amino acid substitution olyticus (Table 2). This observation supports our hypothesis model. The nearest neighbor interchange was used for the that a common ancestor of the Deinococcus-Thermus phylum maximum-likelihood heuristic method. The γ-distributed biosynthesized lysine not through the DAP pathway, but rate was considered, and the number of discrete γ categories through the AAA pathway. was 3. Bootstrap analysis was performed with 100 replicates. Interestingly, D. proteolyticus was found to have the genes for lysine biosynthesis through the DAP pathway 3. Results and Discussion (Table 2). D. proteolyticus has 2 diaminopimelate decar- boxylases, namely, Deipro 0627 and Deipro 1375 (Table 2), Genes homologous to the T. thermophilus genes for which are structurally different from each other. Because lysine biosynthesis through the AAA pathway were found Deipro 1375 forms a gene cluster with other genes for lysine 6 International Journal of Evolutionary Biology biosynthesis through the DAP pathway, we used Deipro Bioscience, Biotechnology and Biochemistry, vol. 71, no. 4, pp. 1375 as a query sequence in the BLASTp search. Each 1015–1020, 2007. phylogenetic tree based on diaminopimelate decarboxylase [11] M. V. Omelchenko, Y. I. Wolf, E. K. Gaidamakova et al., “Com- (Figure 1), ll-diaminopimelate aminotransferase (Figure 2), parative genomics of Thermus thermophilus and Deinococcus dihydrodipicolinate synthase (Figure 3), and dihydrodipi- radiodurans: divergent routes of adaptation to thermophily colinate reductase (Figure 4) showed that the D. prote- and radiation resistance,” BMC Evolutionary Biology, vol. 5, article 57, 2005. olyticus enzyme is closely related to that of the genera [12]K.Tamura,D.Peterson,N.Peterson,G.Stecher,M.Nei, Kytococcus (a member of Actinobacteria) and Spirochaeta and S. Kumar, “MEGA5: molecular evolutionary genetics − (a member of Spirochaetes) (Figures 1 4). The 3 phyla analysis using maximum likelihood, evolutionary distance, Actinobacteria, Deinococcus-Thermus, and Spirochaetes do and maximum parsimony methods,” Molecular Biology and not form a monophyletic lineage in the phylogenetic tree, as Evolution, vol. 28, no. 10, pp. 2731–2739, 2011. based on genomewide comparative studies [14]. In addition, [13] S. Whelan and N. Goldman, “A general empirical model the 4 genes encoding diaminopimelate decarboxylase, ll- of protein evolution derived from multiple protein families diaminopimelate aminotransferase, dihydrodipicolinate syn- using a maximum-likelihood approach,” Molecular Biology thase, and dihydrodipicolinate reductase are clustered in each and Evolution, vol. 18, no. 5, pp. 691–699, 2001. genus (Figures 1−4). Thus, these findings strongly suggested [14] H. Nishida, T. Beppu, and K. Ueda, “Whole-genome compari- that a DNA fragment including the 4 D. proteolyticus genes son clarifies close phylogenetic relationships between the phyla was horizontally transferred from a phylogenetically distant Dictyoglomi and Thermotogae,” Genomics,vol.98,no.5,pp. 370–375, 2011. organism. This horizontal transfer event may have induced the loss of the genes for lysine biosynthesis through the AAA pathway in D. proteolyticus.

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