# Evolution of Lysine Biosynthesis in the PhylumDeinococcus-Thermus

Authors: Hiromi Nishida; Makoto Nishiyama
Journal: International Journal of Evolutionary Biology
 (2012)

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## 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.


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## Body

## 1. Introduction

TheDeinococcus-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, theα-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 theDeinococcus-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 theDeinococcus-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 eachT. 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 theT. 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 theα-aminoadipate pathway in the Deinococcus-Thermus phylum.
OrganismEnzyme 1Enzyme 2Enzyme 3Enzyme 4Enzyme 5Enzyme 6Enzyme 7Enzyme 8Enzyme 9Enzyme 10Thermus thermophilus HB27TTC0043TTC1012TTC1393TTC1396TTC1541*TTC1542*TTC1543*TTC1546*TTC1547*TTC1550*Thermus thermophilus HB8TTHA0411TTHA1378TTHA1755TTHA1757TTHA1903*TTHA1904*TTHA1907*TTHA1910*TTHA1911*TTHA1914*Thermus scotoductusTSC_c05810TSC_c20650TSC_c03550TSC_c3520TSC_c01940*TSC_c01930*TSC_c01920*TSC_c01890*TSC_c01880*TSC_c01850*Meiothermus ruberMrub_0871Mrub_2738Mrub_0027Mrub_2721*Mrub_2723*Mrub_2724*Mrub_2727*Mrub_2728*Meiothermus silvanusMesil_2567Mesil_1337Mesil_0348Mesil_0347Mesil_0435*Mesil_0436*Mesil_0438*Mesil_0441*Mesil_0442*Oceanithermus profundusOcepr_1387Ocepr_1797*Ocepr_1798*Ocepr_1796*Ocepr_1788*Ocepr_1784*Ocepr_1781*Ocepr_1780*Ocepr_1779*Marinithermus hydrothermalisMarky_1533Marky_0665*Marky_0663*Marky_0666*Marky_0667*Marky_0668*Marky_0671*Marky_0672*Marky_0673*Deinococcus radioduransDR_1674DR_0794DR_1413DR_1420DR_0963DR_2194DR_1614DR_1610DR_1238Deinococcus geothermalisDgeo_2084Dgeo_1458Dgeo_1416Dgeo_1391Dgeo_0678Dgeo_0685Dgeo_1151*Dgeo_1154*Dgeo_1156*Dgeo_1257Deinococcus desertiDeide_09240Deide_16910Deide_17960Deide_10430Deide_10350Deide_13430*Deide_13460*Deide_13470*Deide_13980Deinococcus maricopensisDeima_0046Deima_1545Deima_2454Deima_2593Deima_1346*Deima_1349*Deima_1350*Deima_1353*Deima_1355*Deima_1358*Deinococcus proteolyticusDeipr_0213Truepera radiovictrixTrad_2841Trad_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 theDeinococcus-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 theDeinococcus-Thermus phylum.
OrganismAspartate kinaseAspartate-semialdehyde dehydrogenaseDihydrodipicolinate synthaseDihydrodipicolinate reductasell-diaminopimelate aminotransferaseDiaminopimelate decarboxylaseThermus thermophilus HB27TTC0166TTC0177TTC0591Thermus thermophilus HB8TTHA0534TTHA0545TTHA0957Thermus scotoductusTSC_c07050TSC_c08140TSC_c10420TSC_c10870Meiothermus ruberMrub_0976Mrub_1641Mrub_1335Mrub_0798Meiothermus silvanusMesil_1711Mesil_2173Mesil_2308Mesil_0318Oceanithermus profundusOcepr_1316Ocepr_1018Ocepr_2076Marinithermus hydrothermalisMarky_1492Marky_1381Marky_1261Deinococcus radioduransDR_1365DR_2008DR_1758Deinococcus geothermalisDgeo_1127Dgeo_1782Dgeo_0790Deinococcus desertiDeide_11430Deide_15740Deide_1p00310, Deide_3p00120, Deide_3p01100Deide_12830, Deide_21880Deinococcus maricopensisDeima_1822Deima_2680Deima_2660Deinococcus proteolyticusDeipr_0941Deipr_0985Deipr_1377*Deipr_1378*Deipr_1376*Deipr_0627, Deipr_1375*Truepera radiovictrixTrad_0977Trad_0289Trad_1893Trad_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 betweenDeinococcus 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 betweenDeinococcus proteolyticusll-diaminopimelate aminotransferase and related proteins. Multiple alignment was obtained using the top 20 amino acid sequences of the BLASTp search result for D. proteolyticusll-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 betweenDeinococcus 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 betweenDeinococcus 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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*Source: 745931-2012-05-08.xml*