Laterally transferred genomic islands in Xanthomonadales related to
Transcrição
Laterally transferred genomic islands in Xanthomonadales related to
RESEARCH LETTER Laterally transferred genomic islands in Xanthomonadales related to pathogenicity and primary metabolism Wanessa C. Lima1, Apuã C.M. Paquola1, Alessandro M. Varani2, Marie-Anne Van Sluys2 & Carlos F.M. Menck1 1 Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil; and 2Department of Botany, Institute of Biosciences, University of São Paulo, São Paulo, SP, Brazil Correspondence: Carlos F. M. Menck, Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Lineu Prestes, 1374, ICB II, São Paulo, SP, Brazil. Tel.: 155 11 3091 7357; fax: 155 11 3091 7354; e-mail: [email protected] Received 10 September 2007; accepted 7 January 2008. First published online February 2008. DOI:10.1111/j.1574-6968.2008.01083.x Editor: Ross Fitzgerald Keywords lateral gene transfer; horizontal gene transfer; Xanthomonas ; genomic islands; categorization. Abstract Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes. Introduction Evolutionary molecular biology started in the 1960s, when Zuckerkandl & Pauling (1965) noticed that nucleic acid and protein sequences are sources of information to infer organism evolution. Since then, biologists have tried to reconstruct the evolutionary history of species, based on sequence similarity of highly conserved genes such as 16S rRNA and recA (Woese & Fox, 1977; Eisen, 1995). Nevertheless, there are several examples of genes that do not follow the classical topology of these phylogenetic trees, and this has been attributed to the existence of lateral gene transfer (LGT) events between species (Syvanen, 1994; Gogarten et al., 1999), contrasting with the vertical process of inheritance whereby traits are transmitted from parents to offspring (Eisen, 2000). Because of the increase in available genomic data, it is now easier to evaluate the extent by which organisms increase their genetic diversity through the acquisition of genes by LGT. Nevertheless, identifying LGT within a given bacterial genome is not a trivial task. The approaches are FEMS Microbiol Lett 281 (2008) 87–97 based on the analysis of DNA composition, distinguishing foreign from indigenous DNA (including GC content, GC skew, dinucleotide relative abundance and codon usage bias); analysis of unusual phyletic patterns (as unexpected ranking of similarities or unusual gene content); unexpected phylogenetic tree topology; and the presence of mobile genetic elements (Eisen, 1998; Ragan, 2001a). The ability to perform comparative analyses on several genomes at the same time, in order to predict LGT, has gained precision and is currently becoming the method of choice for identifying LGT. Currently, several papers assessing the amount of laterally transferred genes in completely sequenced genomes are available, using different methodologies. The data indicate the overall contribution of LGT events in shaping microbial genomes, in proportions ranging from virtually none to more than 25% of genes as resultant from transfers (depending on the organism, its taxonomy position and its life style) (Ochman et al., 2000; Merkl, 2004; Beiko et al., 2005; Shi et al., 2005). Xanthomonadales are gram-negative Gammaproteobacteria including important plant pathogen species (Vauterin et al., 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 88 W.C. Lima et al. 1995; Van Sluys et al., 2002), although phylogenetic reconstruction of conserved genes (as 16S rRNA gene and recA) branches them very close to the root of the Beta- and Gammaproteobacteria subdivision (Martins-Pinheiro et al., 2004; Lima et al., 2005). Some studies even state that the extensive transfers among Xanthomonadales and the three major proteobacterial clades (alpha, beta and gamma) make it difficult to place them in one of the former groups (Comas et al., 2006). In Xanthomonas campestris pv. campestris (XCC) and Xanthomonas axonopodis pv. citri (XAC), estimates of the number of laterally transferred genes vary between 5% and 20%, depending on the methodology used (Garcia-Vallve et al., 2003; Merkl, 2004; Nakamura et al., 2004). The identification, at a genomic scale, of gene clusters or gene islands laterally transferred was carried out in a few cases, in general relating them to pathogenicity or virulence traits. Nakamura et al. (2004) identified four PAIs (pathogenicity islands) in XAC and three in XCC, all of them containing virulence genes or gene regulators. Mantri & Williams (2004), searching for islands delivered by sitespecific integrases, identified four islands in each genome containing transposases, tRNAs, virulence or phage-related genes, hypothetical genes, peptidases and regulators. More recently, the genomic repertoire of mobile genetic elements from six Xanthomonadales species was described, and several of these elements are located at one of the borders of a genomic island (Monteiro-Vitorello et al., 2005). Finally, Chen (2006) identified four islands in XAC and one in XCC, based on the cumulative GC profile method. In a previous work, we identified 25% of Xanthomonas genes as potentially acquired through LGT, by unusual ranking of sequence similarity and clustering analysis (Lima et al., 2005). In this work, further analysis distinguished potentially late and early events of gene transfer in Xanthomonas genome diversification, where recently acquired islands are bordered by mobile genetic elements, and old islands have lost most of the LGT identifiers except for the atypical phylogenetic pattern. Functional categorization of these clustered genes reveals a bias towards biological roles, including pathogenicity and virulence traits, transport, energy metabolism and regulatory functions. The data reveal a group of potentially transferred genes that are not usually detected by nucleotide composition methods, and indicate that LGT may also contribute to build prokaryotic genomes by replacing genes normally inherited by vertical processes. were the GC content bias, the dinucleotide content bias and the codon usage bias, as described, respectively, in GarciaVallve et al. (2000), Karlin (2001) and Hsiao et al. (2003). Genes were considered extraneous in terms of (1) the GC content if their GC content deviated by 4 1.5s (SD) from the mean value of their genome; (2) the dinucleotide frequency if the gene deviated 4 2.0s away from the genome mean; and (3) the codon usage when the gene had a Mahalanobis distance of 4 2s from the mean genome value. Genes were defined as possessing an odd nucleotide composition when displaying at least one of these three parameters deviant from the genome average. Analysis of tetranucleotide usage variance was performed by the implementation of the OLIGOWORDS program (Reva & Tümmler, 2005; Klockgether et al., 2007). Parameters of distance (D) and oligonucleotide usage variance (OUV) were calculated using a sliding window of 8 kbp and a step size of 2 kbp (both calculations were normalized by monoand dinucleotide frequencies). Materials and methods Functional categorization Nucleotide composition analyses Each island was inspected for the unusual nucleotide composition of their genes. The three parameters analyzed 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Phylogenetic analyses Automatic, large-scale phylogenetic reconstruction was undertaken through the implementation of a modified PYPHY algorithm (Sicheritz-Ponten & Andersson, 2001). This software performs a BLAST similarity search and keyword search on the Swiss-Prot and TrEMBL databases, and the sequences matching predefined criteria are aligned with CLUSTAL software. Distance-based phylogenetic trees (using the NEIGHBOR-JOINING program implemented in the PHYLIP package) are then generated for all genes in both genomes, whenever the number of homologs was at least four proteins. Following this first phylogenetic screening, manual and detailed phylogenetic reconstructions were made to all genes belonging to some gene cluster of interest, including the trees shown in this study. In these cases, protein sequences were aligned with the CLUSTALX program (Thompson et al., 1997), and regions of the alignments that were ambiguous, hypervariable or containing gaps were excluded from subsequent analysis (GENEDOC program; Nicholas et al., 1997). Distancebased phylogenetic trees were generated from these alignments using the neighbor-joining algorithm (NEIGHBORJOINING program from PHYLIP package; Felsenstein, 1989). Bootstrap assessment of tree topology (one thousand replicates) was performed with the SEQBOOT program (PHYLIP). The functional, biological role of each gene within the islands was assigned through the Xanthomonas genome project homepage (da Silva et al., 2002) and the Comprehensive Microbial Resource in TIGR (Peterson et al., 2001). FEMS Microbiol Lett 281 (2008) 87–97 89 LGT islands in Xanthomonas Table 1. Analysis of unusual nucleotide composition of Xanthomonas islands Number of genes % of atypical genes Number of mobile genetic elements Island XAC XCC XAC XCC XAC XCC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A1 A2 A3 A4 A5 C1 C2 C3 C4 C5 70 20 24 19 28 18 43 39 34 17 19 13 22 18 10 30 13 13 18 77 17 23 34 19 16 30 25 24 16 108 25 17 36 32 14 – – – – – 42 21 23 17 51 24 30 33 39 20 22 13 13 19 10 31 13 14 18 32 23 27 27 24 15 11 30 22 16 94 – – – – – 57 50 19 59 36 81 60 29 56 64 50 33 44 44 35 21 38 55 56 20 37 15 23 44 77 47 56 44 32 37 60 40 37 19 48 96 29 53 97 50 – – – – – 62 43 43 82 69 92 40 39 59 40 18 15 46 63 20 39 15 29 61 78 57 37 37 46 60 45 30 41 56 59 – – – – – 95 58 95 75 81 3 3 0 0 1 2 2 0 0 0 0 0 0 0 0 5 0 1 2 25 0 0 2 2 0 7 1 0 0 12 14 0 2 4 0 – – – – – 0 2 0 0 4 2 0 0 1 0 1 0 0 0 0 5 0 1 1 6 0 2 0 0 0 0 2 0 0 13 – – – – – 19 23 9 29 14 The percentages consider the number of genes within each island displaying one or more of the following atypical nucleotide composition features: GC content, codon usage and dinucleotide frequency. Results Detection of unusual features in laterally transferred islands Xanthomonas genomes comprise an elevated number of genes with the highest similarity to genes from phylogenetiFEMS Microbiol Lett 281 (2008) 87–97 cally distant organisms, and in most cases the genes are grouped into islands instead of simply being scattered throughout the genome. In a previous report, we identified 40 islands whose best matches (through BLAST searches) were to distantly related organisms (non-Gammaproteobacteria) in both XAC and XCC, including 30 islands shared by both genomes (islands 01–30, in Table 1) and five exclusive to either one (identified in Table 1 as A1–A5 when belonging to XAC, or C1–C5 when belonging to XCC) (Lima et al., 2005; the genomic coordinates for all islands are provided as supplementary material). Genomes, publically available, from other Xanthomonas species were also assessed, and, as expected, almost all islands are also present, with the exception of those classified as exclusive. XAC-exclusive islands were not found in the other six Xanthomonas genomes, while XCC-exclusive islands were found in other X. campestris strains (data not shown). In this study, genes within the islands were further investigated with respect to nucleotide composition and phylogenetic position. A gene is considered to have unusual nucleotide composition if it differs significantly from the genome average in at least one of the following characteristics: GC content, codon usage and dinucleotide frequency. As shown in Table 1, the islands have different proportions of atypical genes, ranging from 15% to 97%. The exclusive islands were among the islands with the highest proportion of atypical genes, and the presence of an elevated number of mobile genetic elements within these islands strengthens the hypothesis of recent events of lateral transmission originating from such exclusive islands. However, it is worth noting that several islands potentially originated by LGT events show low levels of deviation in nucleotidic parameters. In such cases, the concomitant findings of atypical phylogenetic reconstruction and atypical similarity ranking of genes, and in most cases the association with mobile genetic elements (MGEs), support their origin via lateral gene transfer events. These islands may be the result of an early LGT event and, due to the amelioration process, the nucleotide composition became similar to the host’s genome. A second approach to assess the deviation on the nucleotide composition was also employed, and involved the analysis of the tetranucleotide usage variance in each island, compared with the median values of the whole host chromosome (Reva & Tümmler, 2005; Klockgether et al., 2007). It is important to note that, differently from the approaches presented before, the tetranucleotide parameters (denoted here by OUV) were calculated based on the entire island (using a sliding window of 2 Kbp) and not individual genes. In general, the islands with higher levels of genes with atypical nucleotide composition also present tetranucleotide signatures distinct from that of the host chromosome and those with less atypical genes present OUV values in 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 90 W.C. Lima et al. Fig. 1. Tetranucleotide usage variance of two more-deviant (a) and two less-deviant (b) islands in XAC and XCC genomes. The horizontal lines correspond to the limits of the 95% confidence interval of OUV in a randomly generated sequence with the same length and tetranucleotide content as the host genome. accordance with the genome average. In Fig. 1, this analysis is illustrated for one island more deviant and one less deviant of each genome (and in the Supplementary Fig. S1, the variance of tetranucleotide frequency is shown for the three other islands, in each genome, with the highest frequencies of genes with atypical nucleotide composition). Clearly, the results indicate that the islands denoted as more deviant present regions with strong heterogeneity of tetranucleotide frequencies, but for most of the islands these values are consistently different from the median OUV values for the two Xanthomonas genomes. These data are in agreement with a potential recent acquisition of these islands by LGT. To support the best-BLAST-match analysis, first used to detect the laterally transferred islands, a large-scale phylogenetic analysis was conducted in both XAC and XCC genomes. As expected, to the genes where it was possible to generate a tree (at least four homologs found in GenBank), most of them displayed an atypical phylogenetic behavior (data not shown). A gene is considered to have atypical phylogenetic reconstruction if it branches outside the Beta/ Gammaproteobacteria group. Both groups (beta and gamma) were considered as typical branching to Xanthomonas genomes by the close relationship they present in phylogenetic trees (Figure S2 shows a phylogenetic tree for the 16S 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c rRNA gene, depicting the position of the Xanthomonadales group inside the Bacteria clade). Functional categorization of genes within islands The classification of genes within the islands into functional categories was assessed, and the proportion of category distribution is presented in Fig. 2, compared with the average found in the entire genome. As expected for laterally transferred genes, the functional categories that have higher proportions of genes within the islands are those related to pathogenicity and virulence as well as MGEs. The distribution of functional categories was also assessed discriminating islands with the highest as well as the lowest levels of deviation in nucleotide composition (the seven more-or-less deviants in each genome). As shown in Fig. 3a, islands with highly divergent genes (potentially recent LGT events) display a clear predominance of genes related to MGE, besides pathogenicity and virulence. In fact, for these islands the other functional categories are barely represented, except as regards cell structure. On the contrary, islands with lower levels of atypical nucleotide composition, although also carrying genes related to FEMS Microbiol Lett 281 (2008) 87–97 91 LGT islands in Xanthomonas (a) Frequency (%) * * * (b) * Frequency (%) * * * Fig. 2. Distribution of functional categories within islands (light bars) compared with genes in the whole genome (dark bars), in XAC (a) and XCC (b). Categories were defined by the original description (http://cancer.lbi.ic.unicamp.br/ xanthomonas/]). pathogenicity and virulence, have an increased number of genes related to intermediary metabolism and cellular processes (Fig. 3b). It is worth noting the presence of entire clusters of genes related to metabolic pathways, among islands with a low percentage of genes with discrepant nucleotide composition (Table 2). Notable examples are islands involved in the metabolism of NAD, arginine and cysteine and in energetic metabolism, including tricarboxylic acid cycle (Lima & Menck, 2008). Phylogenetic reconstructions for such genes confirm these islands as the result of transfer from distantly related organisms, considering closely related organisms from the Beta/Gammaproteobacteria group (Fig. S2). In some of these important metabolic pathways, we found genes branching close to homologs of the Eukarya and Archaea clades, as well as other Bacteria groups distantly related, such as Actinobacteria, Firmicutes and Bacteroidetes, instead of branching with closely related homologs from proteobacteria. These results support the origin of such genes from LGT events, despite the fact that these genes are related to primary metabolic functions in the cell. One clear example of this ‘core metabolism gene transfer’ refers to the island bearing the genes related to cystein FEMS Microbiol Lett 281 (2008) 87–97 biosynthesis. Five genes (cysNC, cysD, cysJ, cysI and cysH), responsible for the assimilation and activation of inorganic sulfate to organic sulfide, are clustered, and phylogenetic reconstructions branch them with non-Gammaproteobacteria groups. This is illustrated by Fig. 4, as CysI, which encodes the beta subunit of NADPH sulfite reductase, groups with Firmicutes and Fungi, in a separate branch from the other Proteobacteria. Concerning recently acquired islands, seven are related to virulence and pathogenicity, which may provide selective traits advantageous to these plant pathogens (Table 2). These islands carry genes related to type II, III and IV secretion systems, xanthan gum production and host cell wall degradation, and some of them have been described previously (Van Sluys et al., 2002; Lima et al., 2005). The majority are associated to MGEs (phages, transposons or tRNA), and have discrepant nucleotide composition (Table 1). Phylogenetic trees of these genes also reveal an atypical pattern. For example, the island carrying the xanthan gum operon (composed of 14 genes) has strongest similarity to Bacillus species, although the genes from this gram-positive bacterium were not annotated as being involved in xanthan 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 92 W.C. Lima et al. XAC (a) XCC * * * * * * * (b) * * * * XAC XCC ** * * ** * * ** Fig. 3. Distribution of functional categorization of island genes according to atypical nucleotide composition, showing the highest (a) and the lowest (b) levels of deviation from the nucleotide composition. Genes within islands (light bars) were compared with genes in the whole genome (dark bars), in XAC and XCC. For columns marked with one asterisk, the difference in relation to the total genome is statistically significant (P o 0.01, determined for each pair with a w2 test). In the columns marked with two asterisks, a P o 0.05 was considered. The following islands were considered as less deviant in XAC (03, 11, 15, 17, 18, 29, A2) and in XCC (11, 12, 15, 17, 18, 27) and as more deviant in XAC (01, 02, 05, 20, 26, A1, A4) and XCC (04, 06, 20, C1, C3, C4, C5). Table 2. Islands identified in XAC and XCC related to pathogenicity and central metabolic pathways Island Function and/or prominent genes Comments 5 8 13 16 18 19 20 Type III secretory system, effector proteins and host cell wall degradation genes (b-galactosidases) Type II secretory system (operon 1) and host cell wall degradation genes (b-galactosidases) NAD metabolism Energetic metabolism (TCA cycle) Arginine biosynthesis Xanthan gum production genes Type IV secretory system, fimbriae production and phage genes tRNA-flanked, presence of transposase 22 23 24 25 30 Host cell wall degradation genes (glucosidases, mannosidases, b-galactosidase, rhamnosidase) Cysteine metabolism Host cell wall degradation genes (esterase, amylase, rhamnogalacturonase and cellulases) Type II secretory system (operon 2) and proteases Host cell wall degradation genes (xylosidases, glucosidases, xylanases, b-galactosidase) production. Trees for two genes belonging to this system (gumF presented in Fig. 5a and gumH in Fig. 5b) reveal that both genes appear only in distantly related groups, such as Archaea, Bacteroidetes and gram-positive bacteria (Actino2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Presence of several tRNAs tRNA-flanked tRNA-flanked, presence of integrase tRNA-flanked, presence of transposase and integrase Presence of transposase Presence of transposase Presence of transposase tRNA-flanked, presence of transposase bacteria and Firmicutes). However, in organisms other than Xanthomonas these homologous genes are responsible for the assembly of some other polysaccharide, because xanthan gum is exclusive of this genus. FEMS Microbiol Lett 281 (2008) 87–97 93 LGT islands in Xanthomonas Fungi Firmicutes Xanthomonadales 10 Proteobacteria Fig. 4. Distance tree of cysI gene computed by the neighbor-joining method. Numbers within the tree correspond to the bootstrap assessment (based on 1000 replicates). Values below 50% are not shown. Discussion In this work, we present a concerted analysis of genomic islands potentially originating through LGT events. The initial and fast approach, the detection of clusters of genes showing atypical ranking of similarity, is now complemented by analyses of unusual nucleotide composition and atypical phylogenetic branching. Although the use of several methods may fail to identify a common set of genes, as previously observed in Escherichia coli (Ragan, 2001b), the finding of two or more parameters indicating LGT in genes within the islands supports the explanation of an alien origin for those clustered genes. As expected, the phylogenetic reconstruction of genes within islands reinforced their potential foreign origin, while the analysis of atypical nucleotide composition offered some hints into the time of introgression for some of the FEMS Microbiol Lett 281 (2008) 87–97 islands. Some authors consider codon bias and base composition as poor indicators for defining gene transfer events; however, this method has been widely used in the search for lateral transfer in prokaryotes, with the advantage of yielding information through the analysis of single genomes (Lawrence & Ochman, 1998). In this work, atypical nucleotide composition and tetranucleotide usage variance were used as additional criteria to validate cluster of genes previously detected as a result of LGT. The high percentage of genes featuring atypical nucleotide composition, with consistent tetranucleotide distinct signatures, indicates that a given island is a recently acquired island. In fact, many of the islands with this feature were found as exclusive for one of the two Xanthomonas genomes analyzed, and carry many MGEs, confirming that they may be the result of recent transfer events. In some cases, these islands contain phagerelated sequences, an indication of phage transduction. 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 94 W.C. Lima et al. (a) Xanthomonas oryzae gumG Xanthomonas axonopodis gumG Xanthomonadales Xanthomonas campestris gumG 100 Xanthomonas campestris gumF Xylella fastidiosa gumF Xylella fastidiosa gumG Xanthomonas axonopodis gumF 99 100 100 Staphylococcus saprophyticus 99 57 Xanthomonas oryzae gumF 96 55 100 100 Staphylococcus aureus Polaromonas sp. Lactobacillus plantarum Staphylococcus epidermidis Clostridium perfringens Oceanobacillus iheyensis Bacteroides fragilis 76 100 Clostridium acetobutylicum 100 Firmicutes and Bacteroidetes Bacteroides thetaiotaomicron 79 Methanosarcina acetivorans Bacteroides fragilis _100 Methanosarcina mazei Bacillus clausii Bacillus licheniformis (b) Methanosarcina mazei Methanosarcina acetivorans Methanospirillum hungatei Mycobacterium bovis Archaea Haloarcula marismortui 99 Mycobacterium tuberculosis Pyrococcus abyssi Frankia sp. Archaeoglobus fulgidus 100 98 55 Mycobacterium paratuberculosis 100 Methanosarcina mazei 68 100 100 62 100 100 Streptomyces coelicolor 100 Methanosarcina barkeri 99 Streptomyces avermitilis 999 Brucella melitensis Actinobacteria Brucella suis Xylella fastidiosa Acetobacter xylinus 918 Xanthomonas campestris Xanthomonadales _100 Xanthomonas axonopodis Xanthomonas oryzae Fig. 5. Distance tree of gumF (a) and gumH (b) genes computed by the neighbor-joining method. Numbers within the tree correspond to the bootstrap assessment (based on 1000 replicates). Values below 50% are not shown. 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c FEMS Microbiol Lett 281 (2008) 87–97 95 LGT islands in Xanthomonas These results confirm previous observations where MGES were associated to genomic islands that distinguish XAC and XCC genomes, defined by a different approach (MonteiroVitorello et al., 2005). On the other hand, islands carrying most of the genes with nucleotide content similar to the rest of the genome may still result from LGT events. These genes may result from an ancient event, but their nucleotide composition may have evolved to a composition similar to that of the host genome, or, in other words, may have ameliorated (Lawrence & Ochman, 1997). However, these islands may also have been recently acquired, albeit from genomes with a nucleotide composition similar to Xanthomonas. For these islands, the concomitant atypical phylogenetic reconstruction supports their origin via LGT, which would not be detected simply by genome composition analyses, certifying the methods of best-match screenings for the search of LGT events. Functional analyses of genes found in islands corroborate previous observations in whole-sequenced genomes (Merkl, 2004; Nakamura et al., 2004; Lima et al., 2005). One interesting feature is certainly the high number of genes related to virulence and pathogenicity that accounts for a metabolic role in the life style of the phytopathogens. Another relevant consideration is the under-representation of informational genes (those related to macromolecule metabolism) in these islands. However, the most striking finding is the strong bias of genes playing roles in the core metabolism of bacteria (intermediate and cellular processes categories), especially in islands that carry genes with low nucleotide composition discrepancy compared with the whole genome. The presence of gene clusters, such as those related to amino acid biosynthesis (arginine and cysteine), NAD and energetic metabolism, points to the potential relevance of LGT contributing to primary functions in bacterial genomes, and supports the selfish-operon model (Lawrence & Roth, 1996). According to this model, transfer of clustered genes confers a selectable function, facilitating its maintenance in the recipient genome. Transfer of single genes that would provide only part of a metabolic function would most likely be lost during evolution. This seems to be the case for the clusters reported in this work, as the genes analyzed present a clear phylogenetic proximity to organisms unrelated to Xanthomonadales. Transfer of a single gene within the integron/gene cassette system has been reported, including for the Xanthomonas species (Gillings et al., 2005), but this was not detected by our strategy. Although the characteristics of a genome (such as nucleotide composition features) usually place a threshold in the search for atypical genes, the evolutionary traits of a gene (similarity and phylogeny) are more direct to identify potential LGT events, making it possible to identify gene FEMS Microbiol Lett 281 (2008) 87–97 clusters normally not detected by the traditional compositional approaches. Islands from potentially recent LGT events carry genes that enlarge the adaptive responses of bacteria in the environment, and are maintained, as they probably facilitate the pathogenic life of these bacteria in plants. The findings of this work are in agreement with observations from literature that recently transferred genes are under fast and relaxed evolution, and many of them may be lost quickly from the genome, when compared with ancient genes (Hao & Golding, 2006). On the other hand, some of the other islands seem to provide genes related to core metabolic pathways, replacing and maintaining functions that are normally acquired by vertical inheritance. These data support the mosaic structure of Xanthomonas genomes and the dynamic exchange of genetic information among bacteria. Acknowledgements This work was supported by FAPESP (São Paulo, SP, Brazil) and CNPq (Brası́lia, DF, Brazil). W.C.L. has a fellowship from FAPESP, and A.C.M.P. and A.M.V have fellowships from CAPES (Brası́lia, DF, Brazil). C.F.M.M. is a Fellow of the John Simon Guggenheim Memorial Foundation (New York, USA). References Beiko RG, Harlow TJ & Ragan MA (2005) Highways of gene sharing in prokaryotesn. 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