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J Mol Evol (2008) 66:266–275 DOI 10.1007/s00239-008-9082-8 Replacement of the Arginine Biosynthesis Operon in Xanthomonadales by Lateral Gene Transfer Wanessa C. Lima Æ Carlos F. M. Menck Received: 24 March 2007 / Accepted: 25 January 2008 / Published online: 28 February 2008 Ó Springer Science+Business Media, LLC 2008 Abstract The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the c-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only W. C. Lima C. F. M. Menck Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil C. F. M. Menck (&) Av. Prof. Lineu Prestes 1374, CEP 05508-900 São Paulo, SP, Brazil e-mail: [email protected] 123 under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution. Keywords Lateral gene transfer Horizontal gene transfer Xanthomonas Xanthomonadales Arginine biosynthesis Selfish operon Introduction Lateral gene transfer (LGT) has been considered a major driver of genome evolution in prokaryotes, since it can dramatically alter the biochemical repertoire of host organisms and potentially create structural or functional novelties, thus allowing for the exploitation of new environments and survival under strong selection regimes (Ochman et al. 2000). However, the successful mobilization of complex metabolic traits requires the physical clustering of genes, and as a result, lateral inheritance will select for gene clusters and operons (Lawrence and Roth 1996). LGT can bring not only entirely new sequence families into the genome, but also sequences that are homologous to existing genes, which may lead to the replacement of autochthonous sequences with the acquired copies (Ochman 2001). The potential for acquiring and replacing an existing gene generally decreases with the phylogenetic distance between the donor and the recipient lineages, due to sequence checking barriers, such as mismatch repair (Vulic et al. 1999). However, this is still a matter of debate, as growing evidence suggests that LGT may occur at vast phylogenetic distances, changing the metabolic character of bacterial species. J Mol Evol (2008) 66:266–275 267 The extent of potential LGT in two phytopathogenic bacteria from the genus Xanthomonas, X. axonopodis pv. citri and X. campestris pv. campestris, was previously assessed, based on atypical similarity ranking, unusual nucleotide composition, and atypical phylogenetic reconstruction (Lima et al. 2005, 2008). Several genomic islands were detected by this approach, including clusters of genes related to essential metabolic functions. In particular, the genomic island bearing the genes responsible for arginine biosynthesis showed striking features; these are investigated further in this work. Arginine biosynthesis is notable for its complexity and variability at the genetic level and for its connection with several other pathways, such as pyrimidine and polyamine biosynthesis, besides diverse degradative pathways. Moreover, the pattern of arginine synthesis is not unique, since two completely different enzymes may catalyze the formation of a key intermediate, ornithine (Fig. 1) (Xu et al. 2000). The initial steps in the arginine biosynthetic pathway from glutamate proceed via N-acetylated intermediates. Two alternative ways have evolved to split off the acetyl group from N-acetylornithine: in the linear pathway, which is mostly found in the Enterobacteriaceae, acetylornithine deacetylase (EC 3.5.1.16; encoded by argE) catalyzes the hydrolysis of N-acetylornithine into the arginine precursor ornithine and acetate. Other prokaryotes and the eukaryotic microbes use an energetically more economic pathway which recycles the acetyl group onto glutamate, yielding acetylglutamate. This reaction is catalyzed by ornithine acetyltransferase (OAT; EC 2.3.1.35; encoded by argJ). In these organisms the first step in the pathway fulfills an anaplerotic role: once N-acetylornithine Fig. 1 General scheme of the arginine biosynthesis pathway. The linear pathway proceeds from argA to argH. Left: The reactions catalyzed by the bifunctional enzyme ArgJ. Right: The different roles played by the proteins encoded by argE’ and argF’ in Xanthomonadales. In each text box are shown the enzyme name and synonyms, the EC number, and the coding gene 123 268 is synthesized, the action of N-acetylglutamate synthase (NAGS; EC 2.3.1.1; encoded by argA) becomes superfluous (Cunin et al. 1986; Glansdorff 1987). In Xanthomonas, biosynthesis proceeds via the linear pathway, but with notable modifications (Fig. 1). While in the canonical biosynthetic pathway, acetylornithine deacetylase catalyzes the conversion of N-acetylornithine to ornithine, which is then converted to citrulline by ornithine transcarbamylase (OTCase; encoded by argF), in Xanthomonas, the argF’-encoded enzyme shows no catalytic activity toward ornithine (Shi et al 2005a). Instead, a novel N-acetylornithine transcarbamylase (AOTCase) catalyzes the carbamylation of N-acetylornithine rather than ornithine. In the same way, a novel N-acetylcitrulline deacetylase (encoded by argE’) is able to catalyze the deacetylation of N-acetylcitrulline to produce citrulline (Shi et al 2005b). Consequently, a new pathway has been proposed for Xanthomonas, inverting the order of transcarbamylation and deacetylation (Morizono et al. 2006). Moreover, recently the argB found in the Xanthomonas genus was proposed to encode a fusion protein with both NAGS and NAG kinase activities (NAGS/K) (Shi et al. 2006; Qu et al. 2007). Working with the genomic data of Xanthomonadales, we investigated the evolution of the genes involved in arginine biosynthesis, which are mostly found as a cluster forming a potential operon (referred hereafter as arg operon). Not only do these bacteria show a distinct pathway, as described above, but also most of the genes in the arg operon have atypical phylogenies, branching close to homologues from otherwise unrelated organisms. The data provide strong evidence for a foreign origin of the whole operon coding for this pathway. This supports an important role for LGT that may directly affect primary metabolism through gene replacement from distantly related organisms. J Mol Evol (2008) 66:266–275 BRENDA enzyme database (Schomburg et al. 2004; http://www.brenda.uni-koeln.de), and the Genome Properties server (Haft et al. 2005; http://cmr.tigr.org). The most relevant genomic sequences for this study were as follows: Xanthomonas axonopodis pv. citri 306 (XAC; GenBank accession no. AE008923), Xanthomonas campestris pv. campestris ATCC33913 (XCC; GenBank accession no. AE008922), Xanthomonas campestris 8004 (XC_; GenBank accession no. CP000050), Xanthomonas campestris pv. vesicatoria str. 85–10 (XCV; GenBank accession no. AM039952), Xanthomonas oryzae KACC10331 (XOO; GenBank accession no. AE013598), Xanthomonas oryzae pv. oryzicola BLS256 (XOM; GenBank accession no. NZ_AAQN00000000), Xanthomonas campestris pv. armoraciae 756C (XCA; no GenBank accession number associated; sequenced by TIGR institute), Xylella fastidiosa 9a5c (XF; GenBank accession no. AE003849), and Xylella fastidiosa Temecula1 (PD; GenBank accession no. AE009442). Phylogenetic Analyses Materials and Methods Protein sequences were aligned with the CLUSTALX program (Thompson et al. 1997), and regions of the alignments that were ambiguous, were hypervariable, or contained gaps were excluded from subsequent analysis (GENEDOC program [Nicholas et al. 1997]). Distancebase phylogenetic trees were generated using a neighborjoining algorithm (Neighbor-Joining program from the PHYLIP package [Felsenstein 1989]). Bootstrap assessment of tree topology (1000 replicates) was performed with the SEQBOOT program (PHYLIP). Maximum likelihood trees were made using TREE-PUZZLE (Schmidt et al. 2002), and parameters used were 100,000 puzzling quartets, gamma-distributed rates over eight categories and the c-parameter estimated from the data set, and an automatically selected model of substitution. Trees were visualized using the TREEVIEW program (Page 1996). Identification of the Arginine Biosynthetic Genomic Island in Xanthomonadales Results The pipeline for detection of laterally transferred islands in Xanthomonas genomes was previously described (Lima et al. 2005). Sequence similarity searches were performed using the BLASTP program as implemented in the NCBI server (http://www.ncbi.nlm.nih.gov/), Expasy Proteomics Server (Gasteiger et al. 2003; http://us.expasy.org/), and CMR-TIGR (Peterson et al. 2001; http://cmr.tigr.org/). The database used for these searches was completely set up from fully sequenced genomes by December 2006. Metabolic pathways were analyzed through the KEGG web service (Kanehisa et al. 2006; http://www.genome.jp/kegg/), the 123 Identification of the Genomic Island Coding for Arginine Biosynthesis in Xanthomonadales In previous studies, potential laterally transferred genomic islands were identified in the genome of both Xanthomonas axonopodis pv. citri (XAC) and Xanthomonas campestris pv. campestris (XCC) (Lima et al. 2005). Several of these islands contained gene clusters related to primary metabolism, including biosynthesis of cysteine, NAD metabolism, the TCA cycle, and energetic metabolism. One of the most interesting islands contained the genes J Mol Evol (2008) 66:266–275 269 responsible for arginine metabolism, and this potential arg operon was further investigated in this work. The presence of such an arg operon was also found in seven other Xanthomonadales genomes, with minor modifications (Fig. 2). In the Xanthomonas genus, the island comprises almost 15 kbp and contains 13 to 15 genes, depending on the genome considered, 8 of which may be related to the arginine metabolism pathway, and the region is flanked by a Pro-tRNA in all organisms. All genes coding for the eight steps of the arginine pathway, from glutamate to arginine, are present within the island (Table 1), except for the argD gene, which is found elsewhere in the genome. Although NAGS and NAGK represent a fusion gene (argB), one putative acetyltransferase GNAT-family gene (designated gnat) was detected, and it might perform an analogue glutamate synthase function. Two other genes, proA and proB, are not directly related to arginine metabolism but share glutamate as the initial substrate. These genes are close to each other, being transcribed in the same direction, thus suggesting that they form an operon. The other five genes present inside this island are not clearly correlated among themselves (Table 1). The aehX Fig. 2 Graphical representation of the arginine biosynthesis genomic island in Xanthomonadales. Genes are represented by arrows according to their transcription direction; the arrow length is not proportional to the gene length. Gene names are shown above the arrows. In dark gray, genes related to the arginine pathway; the black arrow indicates a Pro-tRNA; within the arrows, the corresponding gene number (as defined during the initial sequencing project). XAC, Xanthomonas axonopodis pv. citri 306; XCC, Xanthomonas campestris pv. campestris ATCC33913; XC_, Xanthomonas campestris 8004; XOO, Xanthomonas oryzae KACC10331; XOM, Xanthomonas oryzae pv. oryzicola BLS256; XCA, Xanthomonas campestris pv. armoraciae 756C; XCV, Xanthomonas campestris pv. vesicatoria str. 85–10; XF, Xylella fastidiosa 9a5c; PD, Xylella fastidiosa Temecula1 Table 1 Genes within the arg genomic island, in X. axonopodis pv. citri (XAC) and X. campestris pv. campestris (XCC) XAC XCC Gene name Gene function TIGR-associated role 2340 2236 cynX MFS transporter Transport and binding 2341 2237 aehX a-Amino acid ester hydrolase Cellular processes 2342 2238 proA c-Glutamyl phosphate reductase Amino acid biosynthesis 2343 2239 proB Glutamate 5-kinase Amino acid biosynthesis 2344 2240 – Hypothetical protein Hypothetical – 2241 – Hypothetical protein Hypothetical 2345 2242 argH Argininosuccinate lyase Amino acid biosynthesis 2346 2243 argC Glutamyl-phosphate reductase Amino acid biosynthesis 2347 2244 Gnat1 Acetyltransferase GNAT family Amino acid biosynthesis 2348 2349 2245 2246 argB argE Acetylglutamate kinase N-Acetylcitrulline deacetylase Amino acid biosynthesis Amino acid biosynthesis 2350 – Gnat2 Acetyltransferase GNAT family Amino acid biosynthesis 2351 2247 argG Argininosuccinate synthase Amino acid biosynthesis – 2248 ohr Organic hydroperoxide resistance protein Cellular processes 2352 2249 argF N-Acetylornithine transcarbamylase Amino acid biosynthesis 123 270 J Mol Evol (2008) 66:266–275 gene encodes for an a-amino acid ester hydrolase, responsible for the hydrolysis and synthesis of esters and acids with an a-amino group, and is economically important for b-lactam antibiotics synthesis in a more efficient way (Barends et al. 2003). The ohr gene encodes for an organic hydroperoxide resistance protein, involved in the detoxification of organic hydroperoxides generated by the host plant during microbial infection. This gene may play an important role in the oxidative stress response in host-pathogen interactions (Mongkolsuk et al. 1998). The cynX gene belongs to a family of MFS transporters, specifically involved in the active transport of cyanate and detoxification of environmental cyanate (Sung and Fuchs 1988). The gene order of the genomic island is conserved among all Xanthomonas genomes (Fig. 2). Few differences involve the hypothetical genes flanking the proA and proB genes, the ohr gene, and a second copy of the putative acetyltransferase GNAT-family gene (gnat2). In Xylella, however, only genes related to arginine metabolism plus proAB genes are present. Analysis of nucleotide composition revealed no atypical features. The average GC content of XAC and XCC genomes is 65%, and the average GC content on the arg island is 65% for XAC and 66% for XCC. Other parameters, such as dinucleotide frequency and codon usage, are also similar to the genome average. The same is observed for the other genomes (data not shown). The similarity of nucleotide composition of this island with the host genome is in fact expected, as it was probably acquired by an ancestral xanthomonad, and with time, it may have evolved and ameliorated (Lawrence and Ochman 1997). Table 2 Percentage identity of proteins from the arg genomic island to their first-match organism 123 Arginine Biosynthesis Genes Display an Atypical Similarity Profile Similarity searches performed for each gene belonging to the arg genomic island revealed an atypical pattern of similarity ranking, i.e., to organisms phylogenetically distant from the Xanthomonadales group (Table 2). Xanthomonadales belong to the c-proteobacteria group, although phylogenetic reconstructions with several proteins and methodologies place them as a deep branch of the b-/cproteobacteria group, along with Pseudomonadales (Martins-Pinheiro et al. 2004; Comas et al. 2006). Of all the genes within the island, only three (argE and the two acetyltransferases) presented typical best-match species in a similarity search (i.e., species belonging to bor c-proteobacteria). The other five arg genes and proB showed atypical best-match organisms, mainly Eukarya and Bacteroidetes species. This is strong evidence of the potentially foreign origin of such genes, reinforced by the high levels of sequence similarity between the Xanthomonas genes and the sequences found as best match (between 50% and 70% identity) (Table 2). As a matter of comparison, sequence identity between Xanthomonas and E. coli genes, a closely related c-proteobacteria, ranges from 25% to 45% for genes of the arginine pathway (Table 2). Phylogenetic Analysis of the arg Genes Reveals an Atypical Branching Pattern To further explore the results obtained through similarity searches, phylogenetic reconstructions were carried out XAC XCC Gene name First-match organism Taxonomic group % identity to 1st-match organism % identity to E. coli homologue 2340 2236 cynX Nocardia farcinica Actinobacteria 63 2341 2237 aehX Zymomonas mobilis a-Proteobacteria 74 2342 2238 proA Geobacter metalli-reducens d-Proteobacteria 70 44 2343 2239 proB Salinibacter ruber Bacteroidetes 45 44 2344 2240 – Nocardia farcinica Actinobacteria 63 – 2241 – Bacillus halodurans Firmicutes 62 2345 2346 2242 2243 argH argC Flavobacteria bacterium Aspergillus fumigatus Bacteroidetes Eukarya 57 66 2347 2244 Gnat1 Ralstonia solanacearum b-Proteobacteria 66 2348 2245 argB Aspergillus fumigatus Eukarya 62 27 2349 2246 argE Marinobacter aquaeolei c-Proteobacteria 40 26 2350 – Gnat2 Burkholderia ambifaria b-Proteobacteria 67 2351 2247 argG Salinibacter ruber Bacteroidetes 56 – 2248 ohr Agrobacterium tumefaciens a-Proteobacteria 72 2352 2249 argF Salinibacter ruber Bacteroidetes 55 32 29 26 31 J Mol Evol (2008) 66:266–275 271 Table 3 Phylogenetic position of Xanthomonas genes and support assessment XAC XCC Gene name Nearest neighbor NJ bootstrap (%) ML puzzlings (%) 2340 2236 cynX Actinobacteria 100 97 2341 2237 aehX a-Proteobacteria 100 98 2342 2238 proA d-Proteobacteria 93 85 \50 \50 68 66 2343 2239 proB c-Proteobacteria 2344 2240 – Actinobacteria – 2241 – Actinobacteria and Firmicutes 2345 2242 argH Bacteroidetes and Eukarya 2346 2243 argC Eukarya 2347 2244 Gnat1 Actinobacteria and a-proteobacteria 100 99 \50 \50 100 87 80 74 2348 2245 argB Eukarya 100 84 2349 2350 2246 – argE Gnat2 a- and b-proteobacteria b-/c-Proteobacteria and Eukarya 100 89 56 90 2351 2247 argG Bacteroidetes and Archaea – 2248 ohr Proteobacteria and Actinobacteria 2352 2249 argF Bacteroidetes and Eukarya 90 60 100 99 90 76 Note: NJ, neighbor joining; ML, maximum likelihood for each gene belonging to the arg genomic island, using both minimum distance and maximum likelihood methodologies (Table 3). As expected, the phylogenies were congruent with the BLAST results, and they are supported by high bootstrap and quartet puzzling values. In only three cases was the nearest neighbor in the tree different from the best-match organism: the proB, argE, and gnat1 genes. Five arg genes (argB, argC, argF, argG, and argH) branched with Eukarya or Archaea species with high support (except for argH, with \50%) (Table 3). To illustrate the most prominent examples, Figs. 3–5 present the phylogenetic trees for the argB, argC, and argG genes. The argB and argC genes have as ‘‘nearest neighbor’’ several species of Fungi, and this group is supported by high bootstrap values (100%) (Figs. 3 and 4). It is important to note that the phylogenetic reconstruction of the argB gene was carried out only with the domain encoding NAGK activity. Phylogenetic trees generated with the NAGS domain (corresponding to argA) place the Xanthomonadales along with fungi and mammalian orthologues (data not shown). On the other hand, the argG gene branches with Bacteroidetes and Archaea species (90% bootstrap support) (Fig. 5). It is interesting to note that genes coding for the arginine pathway are widespread throughout all domains, appearing in Eukarya, Archaea, and Bacteria, and in several bacterial groups (such as proteobacteria, high-GC gram positives, and cyanobacteria). In spite of this, the Xanthomonas genes present a clearly atypical branching pattern, which significant evidence for lateral acquisition of the operon. Discussion The evolution of metabolic pathways is a subject of much current interest, and the current availability of several completely sequenced genomes makes this question easier to address. In this work, we provide evidence of the acquisition of a primary metabolic pathway through LGT in the Xanthomonadales group, with the data pointing to a foreign origin for the arginine biosynthesis operon. It is well known that the acquisition of genes from other species is by far the most rapid evolutionary process, frequently occurring without loss of existing functions, and is central to the evolution of bacteria, increasing fitness in certain environmental niches. However, how does it occur for genes related to central functions, such as amino acid metabolic pathways? It has been argued that core genes are unlikely to be successfully transferred, since recipient taxa would already bear functional orthologues which would have experienced long-term coevolution with the rest of the cellular machinery. In contrast, it is proposed that those under weak or transient selection (such as nonessential catabolic processes, new operons, or those providing new niche adaptive changes) are likely to be successfully transferred and retained (Lawrence 1999a). However, we found that LGT is not limited to genes associated with niche adaptation, virulence, or pathogenicity, but rather it also occurs with genes related to primary metabolism (Lima et al. 2008). Omelchenko et al. (2003), on analyzing 41 complete genomes, found several instances of ‘‘neighborhood genes’’ that probably originated via LGT, with different evolutionary scenarios, involving either the transfer of whole 123 272 J Mol Evol (2008) 66:266–275 Fig. 3 Distance tree of argB gene computed by the neighbor-joining method. Numbers within the tree correspond to bootstrap assessment (based on 1000 replicates). Values \50% are not shown operons or a patchy inheritance (generating operon mosaicism). Among their findings, the displacement of tryptophan and arginine biosynthesis genes (argB and argG) is related. In another, more recent work, Qu et al. (2007) also observed the relationship among Xanthomonadales fusion ArgB protein and mammalian homologues. These results are in agreement with those presented here, and indicate that LGT of entire operons is the most likely explanation for most of the findings of colocalized alien genes in a genome, which is generally consistent with the selfish operon model. This model proposes that a gene cluster was initially beneficial to the genes themselves, not to their host organism; since individual genes cannot confer a selectable function alone, the clustering is essential for successful lateral transfer. In this way, the selfish operon 123 allows phenotypic information to be transferred among genomes, because all genes conferring a selectable function may be introduced simultaneously (Lawrence 1999b). The parasitic lifestyle of Xanthomonas might contribute to the explanation of how resident copies could be replaced by xenologous ones. The probable scenario invokes loss of the resident pathway and its replacement by laterally introduced genes. Due to the parasitic lifestyle of all Xanthomonadales species, we might guess that the ancestor of the group displayed a similar behavior. Thus, loss of the original genes responsible for arginine production could occur if the lack of this amino acid was fulfilled by the host. Finally, the restriction of this amino acid could lead to selection of bacteria that have acquired the arg operon from a eukaryotic donor, or eventually from Bacteroidetes J Mol Evol (2008) 66:266–275 273 Fig. 4 Distance tree of the argC gene computed by the neighbor-joining method. Numbers within the tree correspond to bootstrap assessment (based on 1000 replicates). Values \50% are not shown species, as we cannot discriminate which of the bacteria received this operon first. One possibility for restriction of arginine in the medium could occur as a result of an eventual free-living lifestyle of the bacterium (as also occurs in Xanthomonas). Selection as a result of adaptation to a restrictive environment favors the uptake of genes suitable to the host organism, thus making LGT a powerful driving force. Although common features of genomic islands, such as nucleotide composition bias and the presence of integrases flanking the island, are not found, the presence of a tRNA and discordance among the phylogenetic trees and similarity searches of arg genes corroborate the foreign origin of the entire island. The absence of any significant difference in nucleotide parameters (such as GC content, codon usage, and dinucleotide frequency) may be explained in either of two ways: the incoming operon could have had a nucleotide compositional pattern similar to the average recipient genome pattern or perhaps enough time has elapsed since acquisition of the operon in such a way that mutational pressure has ameliorated the within genes. Neither explanation contradicts the hypothesis of a foreign origin for the operon. The aberrant branching pattern shown for arg genes branching with Archaea, Eukarya, and the distantly related Bacteroidetes may be most parsimoniously explained as a result of LGT events, though we cannot exclude the possibility of hidden paralogies (caused by independent gene duplications and losses in different groups). However, a common trend in all ‘‘paralogy scenarios’’ involves multiple copies of the orthologous and paralogous genes, spread among the different taxonomical groups within the phylogeny, with random gaps reflecting differential losses. And this is not the case in all the phylogenies analyzed 123 274 J Mol Evol (2008) 66:266–275 Fig. 5 Distance tree of the argG gene computed by the neighbor-joining method. Numbers within the tree correspond to the bootstrap assessment (based on 1000 replicates). Values \50% are not shown here. Just one copy of each gene is present, and the high bootstrap values support the sister-grouping seen. Moreover, although the arginine pathway is universally conserved among prokaryotes, Xanthomonadales genes are grouping with distantly related organisms, indicating the replacement of native by foreign copies. In conclusion, this work presents data indicating the possibility of gene replacement in primary metabolism by LGT events. Although several features often associated with laterally acquired DNA are lacking, the presence of a tRNA integration site, the phylogenetic discordance, and the genome context information all support the foreign origin of the arg island in Xanthomonadales. Although this type of gene substitution is rare in nature, it may play an important role in the evolution of certain organisms. The sequencing and analysis of more genomes, especially bacterial ones, may facilitate the scrutiny of such events, 123 thus providing more data for studies of genome evolution in general and of arginine metabolism in particular. 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