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Molecular Phylogenetics and Evolution xxx (2009) xxx–xxx

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev

Evolutionary placement of Xanthomonadales based on conserved proteinsignature sequences

Ania M. Cutiño-Jiménez a, Marinalva Martins-Pinheiro b, Wanessa C. Lima b,1, Alexander Martín-Tornet c,Osleidys G. Morales a, Carlos F.M. Menck b,*

a Department of Biology, Facultad de Ciencias Naturales, Universidad de Oriente, Ave. Patricio Lumumba s/n., Santiago de Cuba, CP 90 500, Cubab Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Ave. Prof. Lineu Prestes, 1374, São Paulo, SP, Brazilc Department of Informatics, Centro de Ingeniería Genética y Biotecnología (CIGB), Ave. 31 e/158 y 190, Playa, P.O. Box 6162, Habana 10600, Cuba

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 May 2009Revised 11 September 2009Accepted 21 September 2009Available online xxxx

Keywords:XanthomonadalesXanthomonasXylellaDNA repairINDELs

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2009.09.026

* Corresponding author. Address: Ave. Prof. LineuBiomedical Sciences, ICB II/USP, Cidade UniversitáriaBrazil. Fax: +55 11 3091 7357.

E-mail address: [email protected] (C.F.M. Menck)1 Present address: Department Cell Physiology an

Geneva, R. Michel Servet 1, 1211, Geneva, CH, Switzerl

Please cite this article in press as: Cutiño-Jiméquences. Mol. Phylogenet. Evol. (2009), doi:10.1

Xanthomonadales comprises one of the largest phytopathogenic bacterial groups, and is currently clas-sified within the gamma-proteobacteria. However, the phylogenetic placement of this group is not clearlyresolved, and the results of different studies contradict one another. In this work, the evolutionary posi-tion of Xanthomonadales was determined by analyzing the presence of shared insertions and deletions(INDELs) in highly conserved proteins. Several distinctive insertions found in most of the members ofthe gamma-proteobacteria are absent in Xanthomonadales and groups such as Legionelalles, Chromati-ales, Methylococcales, Thiotrichales and Cardiobacteriales. These INDELs were most likely introducedafter the branching of Xanthomonadales from most of the gamma-proteobacteria and provide evidencefor the phylogenetic placement of the early gamma-proteobacteria. Moreover, other proteins containinsertions exclusive to the Xanthomonadales order, confirming that this is a monophyletic group and pro-vide important specific genetic markers. Thus, the data presented clearly support the Xanthomonadalesgroup as an independent subdivision, and constitute one of the deepest branching lineage within thegamma-proteobacteria clade.

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1. Introduction

With the advent of the genomic era, new molecular approachesto identify evolutionary relationships among organisms have cometo light, and since then extensive work has been done in bacterialtaxonomic classification, mainly focusing in the use of conservedprotein sequences to reconstruct evolutionary histories. However,the use of single gene or protein sequences to reconstruct organismevolution has been contested (Korbel et al., 2002; Ochman, 2001),mainly due to inherent methodological problems during phyloge-netic reconstruction and analysis or to systematic errors from thesequences. Moreover, phylogenetic trees based on rRNA or proteinsequences are unable to determine how certain divisions withinBacteria are related to each other and how they have evolved froma common ancestor (Gupta and Griffiths, 2002). New sequence-based approaches for assessing evolutionary relationships have re-

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Prestes, 1374, Institute of, São Paulo, CEP 05508-900,

.d Metabolism, University ofand.

nez, A.M., et al. Evolutionary p016/j.ympev.2009.09.026

duced some of the biases in earlier methodology that can lead tothe misinterpretation of phylogenetic results. Detection of signa-ture sequences in the primary structure of a protein is a reliableand intuitive method for examining evolutionary relationships.Specific changes in protein residues, such as amino acid substitu-tions or specific deletions or insertions, observed in all membersof one or more taxa but not in others, make it possible to establishcommon ancestry and identify major groups that share the samesignature (Brocchieri, 2001; Gupta, 1997).

There are numerous mechanisms by which specific insertionsand deletions (herein referred to as INDELs) may be formed, suchas, for example, DNA recombination, expansion of repetitive DNAsequences and insertion sequence (IS)-mediated events. As theyare, in general, the product of one specific event, INDELs were pro-posed as reliable signature sequences with certain advantages overtraditional phylogenetic analyses based on gene or protein se-quences (Griffiths and Gupta, 2007; Gupta, 1998, 2006; Guptaand Mok, 2007). Phylogenetic relationships assume constant muta-tion frequencies and this may be incorrect over long periods oftime, ultimately leading to the identification of incorrect speciesrelationships (Philippe et al., 2005). In contrast, conserved INDELsof defined sizes are not greatly affected by such differences in evo-lutionary rates (Gupta, 1998).

lacement of Xanthomonadales based on conserved protein signature se-

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Based on a 16S rRNA tree, the proteobacteria phylum has beendivided into five phylogenetically distinct groups (gamma, beta, al-pha, delta and epsilon subdivisions or classes) (Olsen et al., 1991;Stackebrandt and Woese, 1984). These subdivisions constitutewell-defined clades, their member species being clearly distin-guishable one from the other and from the other bacterial divisions(Eisen, 1995; Ludwig and Schleifer, 1994; Stoffels et al., 1999). Insupport of the use of signature sequences as evolutionary descrip-tors, all proteobacterial subdivisions contain specific INDELs insome of their protein sequences, confirming the evolutionary rela-tionships inside the proteobacteria group (Gupta, 2000, 2005,2006; Gupta and Mok, 2007).

The gamma subdivision is a well-defined group that probablydiverged from the rest of proteobacteria as early as 500 millionyears ago (Clark et al., 1999). It is one of the largest groups withinBacteria and includes at least 14 different subgroups, or orders(Acidithiobacillales, Aeromonadales, Alteromonadales, Cardiobac-teriales, Chromatiales, Enterobacteriales, Legionellales, Methylo-coccales, Oceanospirillales, Pasteurellales, Pseudomonadales,Thiotrichales, Vibrionales and Xanthomonadales), where we findfree-living and commensal species, intracellular symbionts andplant and animal pathogens, as well as species of medical and agri-cultural relevance. In one of the most basal group of gamma-prote-obacteria, the order Xanthomonadales, resides many significantpathogenic bacteria affecting humans, animals and plants. Thisclade, though it contains a single family (Xanthomonadaceae),comprises a diverse set of species differing both in their phenotypictraits and habitat. The group has considerable economic impact onagriculture, worldwide, and more than 350 different plant diseasescaused by xanthomonads have been reported (Leyns et al., 1984;Swings and Civerolo, 1993). Xanthomonas and Xylella are generawithin Xanthomonadales for which complete genome sequencesare available for several species and strains (da Silva et al., 2002;Lee et al., 2005; Qian et al., 2005; Simpson et al., 2000; Thiemeet al., 2005; Van Sluys et al., 2003). The genus Xanthomonas com-prises a diverse group of Gram-negative, obligate aerobic, non-fer-mentative rods, in which all reported strains are plant-associatedand most are reported as being pathogenic to a particular planthost (Van Sluys et al., 2002). Similarly, Xylella fastidiosa (Xf) isresponsible for causing economically important diseases in grape-vines, citrus fruits and many other plant species. Foremost amongthese are Pierces’s disease (PD) of grapevines, citrus variegatedchlorosis (CVC), and the leaf scorch diseases of almonds (ALS)and oleanders (OLS) (Davis et al., 1978; Purcell and Hopkins, 1996).

Although the gamma subdivision is a well-defined clade, theclassification of Xanthomonadales inside the group is quite prob-lematic. The 16S rRNA phylogenetic tree, by far the most fre-quently used sequence to assess phylogenetic placement of agiven bacterial group, places Xanthomonadales at the root of thegamma-proteobacteria (Lima et al., 2008). However, very oftenphylogenetic trees of highly conserved genes place this group inthe same branch as the beta subdivision (Martins-Pinheiro et al.,2004) or even as an outgroup of the beta/gamma-proteobacteria(Schneider et al., 2007). Moreover, these studies are complicatedby the high frequency of genes that are proposed to be acquiredby LGT, especially within the Xanthomonas genus. Due to frequentLGT, Xanthomonadales represents an extreme case of genomicmosaicism that prevents it from being assigned to any one of themajor proteobacterial clades (Comas et al., 2006; Lima et al.,2005, 2008). On considering the disagreements on the phyloge-netic placement of Xanthomonadales, the use of other reliable,non-phylogenetic approaches may allow us to better position thisgroup within the proteobacteria.

In this work, we were able to identify several specific INDELs,based on a sequence alignment of several highly conserved pro-teins, mostly those linked to DNA metabolism, that confirm the

Please cite this article in press as: Cutiño-Jiménez, A.M., et al. Evolutionary pquences. Mol. Phylogenet. Evol. (2009), doi:10.1016/j.ympev.2009.09.026

Xanthomonadales as a monophyletic, early-branching group with-in the gamma-proteobacteria subdivision.

2. Methodology

2.1. Phylogenetic analyses

Protein sequences were aligned by using Clustal X 2.0.9 pro-gram (Larkin et al., 2007), and regions of the alignments that wereambiguous, hypervariable or containing gaps were excluded fromsubsequent analysis (GeneDoc program, Nicholas et al., 1997).ProtTest was used to assess the best-fit amino acid substitutionmodel for maximum likelihood-based tree reconstruction (Abascalet al., 2005). Maximum likelihood (ML) trees were set up with RAx-ML 7.0.4 (Stamatakis, 2006), with the WAG (Whelan–Goldman)model and parameters for invariable sites (+I) and gamma-distrib-uted rate heterogeneity (+G, 4 categories). One-thousand bootstrapreplicates were executed and bootstrap values drawn up on thebest-scoring ML-tree. Trees were visualized using the TreeViewprogram (Page, 1996), and were arbitrarily rooted at midpoint(although trees should be fundamentally viewed as unrooted).The concatenated tree was based on the alignment of the concate-nated sequences of 11 evolutionarily conserved genes: arginyl-tRNA synthetase; LigA; DNA gyrase subunit A; Hsp70; isoleucyl-tRNA synthetase; DNA polymerase I; DNA polymerase III subunitalpha; DNA polymerase subunit epsilon; RecA; ribosomal L2 andS3 proteins. The proteins employed in phylogenetic analyses areshown in Table S01 (Supplementary material).

2.2. Identification of conserved signature sequences

DNA repair and replication-related protein sequences were ob-tained by similarity searches using the BlastP program (Altschulet al., 1997) as implemented in the NCBI server. The list of organ-isms used in this study, as well as the seed sequences used to per-form the similarity searches, is shown in Table S02. Sequenceswere aligned using Clustal X (Thompson et al., 1997), and align-ment parameters suggested by Hall (2005) were implemented(gap opening: 35.00 and gap extension: 0.75).

The identification of potential INDELs was performed as pro-posed before (Gupta, 1998). Briefly, this methodology is based onthe study of insertions and deletions present in a set of orthologousproteins. INDELs are detected as regions of defined length and se-quence, and flanked by highly conserved regions (which excludesmisleading INDEL identification due to improper alignment orsequencing errors) and found at precisely the same position inorthologs from different species (INDELs must be positional homo-logs). All sequence alignments are available upon request.

3. Results

Phylogenetic analyses of proteobacterial groups were con-ducted with the concatenated sequences of 11 conserved proteins,including the sequences employed in the search for conserved IN-DELs (Fig. 1). The use of concatenated sequences for tree genera-tion gave more consistent trees and better bootstrap supportthan single genes. In this concatenated tree, the Xanthomonadalesform a monophyletic group within the same clade as the beta-pro-teobacteria and at the base of the gamma-proteobacteria, close toother orders that are also difficult to correctly classify, such asthe Legionelalles, Chromatiales, Methylococcales, Thiotrichalesand Cardiobacteriales (Fig. 1). Phylogenetic trees for individual se-quences (including 16S rRNA, RecA, TopA and all the 11 genes em-ployed in the concatenated tree; see MM for the list) were alsogenerated. As a general trend, proteobacterial groups are clearly



Fig. 1. Maximum likelihood tree of the proteobacteria based on the concatenated sequences of 11 conserved proteins. Branch lengths are proportional to the number ofamino acid substitutions per site. Numbers at the nodes represent the percentage of bootstrap support (only values > 50% are shown). See Table S01 for the accession codes,and Table S02 for abbreviations of species names.

A.M. Cutiño-Jiménez et al. / Molecular Phylogenetics and Evolution xxx (2009) xxx–xxx 3

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distinguished (betas and gammas always forming a monophyleticgroup with high bootstrap support; data not shown). Nevertheless,depending on the sequence analyzed, the gamma-proteobacteriamay or may not form a monophyletic group (as in the case of TopA,where the group is split into at least two, Fig. 1S, in Supplementarymaterial) and the branching order for betas, gammas and Xantho-monadales, as seen in the topology obtained for the concatenatedtree, is supported in most of the cases, but not always (7 out of


11 single gene trees have the same topology as the concatenatedtree; data not shown).

These results confirm that the evolutionary history of these bac-teria is quite complex and underline the need to use novel tools toinvestigate the taxonomic position of Xanthomonadales. Therefore,we sought additional protein signature sequences, the INDELs. Theassumption behind this approach is that when a conserved INDEL(conserved in terms of length, sequence and residual position, and




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flanked by a region sharing high levels of sequence similarity overall organisms analyzed) is found in an alignment, the simplest andmost parsimonious explanation for its presence is that it was intro-duced only once during the course of evolution into a hypotheticalancestral organism and then kept in all its progeny. Thus, INDELsshared by two organisms indicate a common ancestry. To assessthe phylogenetic relationship of the Xanthomonadales group with-in the proteobacteria, we selected evolutionarily conserved pro-teins with a role in DNA metabolism processes. The proteinsselected participate in informational processes and their functionis dependent upon interaction with other proteins, thereby mini-mizing the chance of LGT (Jain et al., 1999). Table 1 summarizesthe major INDELs that characterize distinct proteobacterial groups,especially the Xanthomonadales. Each protein containing an INDELused in this study is further described below.

3.1. Gamma-proteobacteria specific signature sequences

3.1.1. DNA polymerase III, alpha subunitThe DNA polymerase III holoenzyme is a complex that contains

10 different types of subunits. The alpha subunit, encoded by thepolC gene, is a 130-kDa polypeptide endowed with 50 to 30 DNApolymerase activity but none for proofreading (Patel and Loeb,2001; Steitz, 1999). A signature sequence gene that consists of a5-amino acid insert is shared by homologs from gamma-proteo-bacteria, but is absent from Xanthomonadales, Methylococcales,Legionellales, Cardiobacteriales, part of the Thiotrichales (Francisel-la novicida), and from other proteobacterial subdivisions (Fig. 2). Aswith many other INDELs, this separates the Xanthomonadales (andother groups) from the core group of gamma-proteobacteria.

Table 1Major distinctive INDELs of proteobacterial groups and, in special, the Xanthomonadales o

Protein Description Present

DNA polymerase IIIalpha subunit

5-amino acid lengthinsertion (Fig. 2)

Enterobacteriales, VibrionalesPseudomonadales, AlteromonOceanospirillales, ChromatialThiomicrospira crunogena (fro

DNA Topoisomerase I 29–41-amino acid lengthinsertion (Fig. 2S)

Enterobacteriales, VibrionalesAlteromonadales, Aeromonadand Pseudomonadales

4–5-amino acid insertion(Fig. 3)

Xanthomonadales, LegionellaChromatiales, Cardiobacterial(Francisella novicida specie), aspecies belonging to the betaand a small number of delta-(Desulfuromonadales group)

DNA polymerase I 7-amino acid lengthinsertion (Fig. 3S)

Enterobacteriales, VibrionalesAlteromonadales, Aeromonadand Pseudomonadales orders

1-amino acid lengthinsertion (Fig. 3S)

Pseudomonadaceae family (rePseudomonas and Azotobacter

DNA polymerase IIIepsilon subunit

4–5-amino acid lengthinsertion (Fig. 4S)

Enterobacterialles, PasteurellaAeromonadales, CardiobacterShewanellaceae family (Alter

DNA ligase NAD-dependent

57–65-amino acid lengthinsertion (Fig. 4)

Xanthomonadales

MutS 5-amino acid lengthinsertion (Fig. 5S)

Xanthomonadales

RecA 2-amino acid lengthinsertion (Fig. 6S)

Xanthomonadales

DNA polymerase IIIalpha subunit

4-amino acid lengthinsertion (Fig. 7S)

Xanthomonadales


3.1.2. DNA topoisomerase IDNA topoisomerase I plays essential roles in DNA transcription

and replication, by altering the supercoiling of the double-strandedDNA molecule (Feinberg et al., 1999). It possesses several interest-ing INDELs, which indicates an atypical evolution and confirms anearly separation of two distinct branches among proteobacteria(Fig. 1S). One of them is a conserved 29–41-amino acid insertion(Fig. 2S, Supplementary material) shared by various speciesbelonging to the gamma-proteobacteria (Enterobacteriales, Vibrio-nales, Pasteurellales, Alteromonadales, Aeromonadales, Oceanospi-rillales and Pseudomonadales), but absent in homologs fromXanthomonadales, Methylococcales, Legionellales, Chromatiales,Cardiobacteriales, Thiotrichales, as well as the other proteobacteri-al groups (beta, alpha, delta and epsilon).

Another 4–5-amino acid INDEL in this protein is absent in mostof the gamma-proteobacteria, but present in Xanthomonadales,Legionellales, Methylococcales, Chromatiales, Cardiobacteriales,Thiotrichales (Francisella novicida), as well as proteobacteria ofthe beta and alpha subdivisions, and a small number of delta-pro-teobacteria (Fig. 3). Curiously, this INDEL is absent from most ofthe delta subdivision and is completely absent from the epsilonsubdivision. This heterogeneity suggests that delta-proteobacteriamay not be a monophyletic group, as part of it is more closely re-lated to alpha-proteobacteria, as already previously suggested byGupta (2005). This insertion is also absent outside of the proteo-bacteria clade (represented here by some firmicutes and chlamy-diae, Fig. 3). Interestingly, these inferences from signaturesequence are supported by phylogenetic analyses based on Topoi-somerase I (Fig. 1S): the beta-proteobacteria, most of the deepbranching gamma-proteobacteria groups, and Geobacter branch to-gether and independently from the other proteobacteria. Thus, the

rder.

Absent

, Pasteurellales,adales, Aeromonadales,es orders andm Thiotrichales group)

Xanthomonadales, Methylococcales, Legionellales,Cardiobacteriales, Francisella novicida; and in the othergroups of bacteria

, Pasteurellales,ales, Oceanospirillales

Xanthomonadales, Legionellales, Methylococcales,Chromatiales, Cardiobacteriales, Thiotrichales andhomologs from other bacteria

les, Methylococcales,es and Thiotrichaless well as proteobacteriaand alpha subdivisions,

proteobacteria

Other gamma-proteobacteria orders andThiomicrospira crunogena (from Thiotrichalesgroup).This insertion is also absent in most of thedelta, and completely absent from epsilon subdivision

, Pasteurellales,ales, Oceanospirillales

Other gamma-proteobacteria orders(Xanthomonadales, Chromatiales, Methylococcales,Legionellales, Cardiobacteriales and Thiotrichales).This insertion is also absent in some members fromPseudomonadales (Acinetobacter sp.) and other groupsof bacteria

presenting by)

In all other bacteria

les, Vibrionales,iales and members fromomonadales)

Other gamma-proteobacteria orders(Xanthomonadales, Methylococcales, Legionellales,Chromatiales, Thiotrichales, Oceanospirillales,Pseudomonadales and in the other Alteromonadales:Pseudoalteromonadaceae and Alteromonadaceaefamilies); and in all other bacteriaIn all other bacteria






Fig. 2. Partial sequence alignment of the DNA polymerase III epsilon subunit showing a 5-amino acid insert specific for the core gamma-proteobacteria groups, but absent inXanthomonadales, several deep branching bacteria of this class, and all other bacteria. Dots (.) in this and other alignments denote identity with the amino acid on the top lineand gaps are indicated by empty spaces. Accession numbers are given after each species name. See Table S02 for abbreviations of species names. The numbers on the leftindicate the amino acid position for each protein sequence. Sequence information for only representative species is shown, but the sequences from other available speciesfrom these groups behave similarly.


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Please cite this article in press as: Cutiño-Jiménez, A.M., et al. Evolutionary placement of Xanthomonadales based on conserved protein signature se-quences. Mol. Phylogenet. Evol. (2009), doi:10.1016/j.ympev.2009.09.026



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evolutionary history of this gene is not typical and indicates anearly divergence of the two groups (one of them with the inser-tion). The possibility that this gene underwent an ancient LGTevent cannot be excluded.

Fig. 3. Partial sequence alignment of Topoisomerase I, showing a 4–5-amino acid insert imost of the other deep branching bacteria of this class, as well as in the beta and alphaproteobacteria and also other bacteria (the Firmicutes/Chlamydiae group are presented


The atypical grouping for Thiotrichales (represented here byFrancisella novicida and Thiomicrospira crunogena, which are dis-criminated here by both signatures) is in agreement with earlierwork by the group of Gupta, who found the two genera sepa-

s absent from the core gamma-proteobacteria, but present in Xanthomonadales and-proteobacteria, but only a few of the delta-proteobacteria. For most of the delta-as outgroups), the insert is absent. Details as described in Fig. 2.




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rated in protein signature and phylogenetic analyses (Gao et al.,2009).

3.1.3. DNA polymerase IIn addition to 50 to 30 DNA polymerase activity, DNA polymerase

I exhibits 30 to 50 exonuclease activity, which mediates proofread-ing, and 50 to 30 nick translation during DNA repair (Patel et al.,2001). A 7–8-amino acid insertion is present in homologs fromthe core gamma-proteobacteria, but is not found in homologs fromXanthomonadales, Chromatiales, Methylococcales, Legionellales,Cardiobacteriales and Thiotrichales, neither in homologs from thebeta, alpha, delta and epsilon subdivisions (Fig. 3S, Supplementarymaterial).

Moreover, a small 1-amino acid insertion in the Pseudomona-dales order is also observed. This INDEL is present as well in theOceanospirillales Chromohalobacter salexigens, thus indicating aclose relationship with the Pseudomonadales, as already demon-strated in previous studies (Quillaguaman et al., 2004). The ab-sence of both INDELs in two Acinetobacter species (belonging tothe Pseudomonadales group) points to an ancient divergence ofthese bacteria within the Pseudomonadales.

3.1.4. DNA polymerase III, epsilon subunitThe DNA polymerase III epsilon subunit protein, encoded by the

dnaQ gene, is involved in DNA replication through its proofreading30 to 50 exonuclease activity (Patel and Loeb, 2001; Steitz, 1999). Asignature sequence consisting of a 4–5-amino acid insert is presentin dnaQ homologs of the gamma-proteobacteria, but absent inhomologs from Xanthomonadales, Methylococcales, Legionellales,Chromatiales, Thiotrichales, Oceanospirillales, Pseudomonadalesand Alteromonadales, as well in the other proteobacterial groups(Fig. 4S, Supplementary material). This indicates a late origin forthis insertion, and adds support to the assertion that groups notcarrying this insertion are related but diverge from the other gam-ma-proteobacteria. A smaller 1-amino acid insertion is also ob-served and shared by homologs from the Vibrionales andAlteromonadales (Shewanellaceae family). The fact that the Car-diobacteriales also carry this insertion points to a potential LGTevent among these bacteria.

3.2. Xanthomonadales specific signature sequences

3.2.1. NAD-dependent DNA ligase LigATwo distinct families of DNA ligase are found, one that is typical

of Bacteria and uses NAD+ as a cofactor, and the other that usesATP and is common to Archaea and Eukaryotes; both families ofDNA ligase, however, are present in Xanthomonas (Wilkinsonet al., 2001; Martins-Pinheiro et al., 2004). The NAD-dependentDNA ligase bears a distinctive 65-amino acid insertion, only foundin the Xanthomonadales group (Fig. 4). Recursive BLAST searchesusing this striking long INDEL as a seed sequence were performed,but only sequences from the Xanthomonadales group showedsimilarity.

3.2.2. Mismatch repair protein MutSThe MutS protein is involved in the repair of mismatches in

DNA. This protein forms a dimer (MutS2) that recognizes the mis-matched base on the daughter-strand and binds the mutated DNAwith feeble ATPase activity (Kunkel and Erie, 2005). This proteinreveals a conserved 5-amino acid insertion, exclusive to membersof the Xanthomonadales group (Fig. 5S, Supplementary material).

3.2.3. RecARecA plays an essential role in at least three distinct cellular

processes: homologous recombination, gene control of the SOSregulon and DNA damage induced mutagenesis (Cox, 2003; Eisen,


1995). Moreover, this protein has been widely used for phyloge-netic reconstruction in order to assess relationships within Bacte-ria (Eisen, 1995; Martins-Pinheiro et al., 2004). A 2-amino acidinsertion in a highly conserved region has been shown to distin-guish the Xanthomonadales order (Fig. 6S, Supplementarymaterial).

Other interesting signatures were also observed, such as a 4-amino acid insertion exclusive to Xanthomonadales in the alphasubunit of DNA polymerase III (Fig. 7S, Supplementary material),a 4-amino acid insertion in valyl-tRNA synthetase exclusive toXanthomonadales and a 37-amino acid insertion, also observedin valyl-tRNA synthetase, present in all gamma and beta-proteo-bacteria, but absent in the remaining groups, previously described(Gupta, 2000).

4. Discussion

An accurate classification of xanthomonads is not only impor-tant for the basic knowledge of the phylogeny of this group, butalso has practical implications for global regulation of severe dis-eases caused by these plant pathogens. Misidentification and/orunclear classification of xanthomonads may result in unnecessaryquarantine regulations and economic loss. In previous studiesusing different gene and protein sequences, the classification ofXanthomonadales was contradictory to the placement of thesebacteria as gamma-proteobacteria. For example, when analyzingthe phylogeny of the RecA protein (Martins-Pinheiro et al., 2004)or 16S rRNA (Lima et al., 2005, 2008), independent results placedthe Xanthomonadales in a branch close to the beta subdivision(as also shown herein, Fig. 1). Different genes and different meth-ods may sometimes result in contradicting trees. These problemsare typical in molecular phylogeny studies, even in those cases inwhich LGT can be ruled out. Other authors have previously high-lighted the incongruity between gene and presumed organismphylogeny (Poptsova and Gogarten, 2007).

Comas et al. (2006) analyzed whether incongruities apparent inprotein and/or gene trees from Xanthomonadales could have re-sulted from phylogenetic noise and/or LGT. Phylogenetic noise af-fects basal groups whose positions may have changed due tolimitations in phylogenetic reconstruction methods. They con-cluded that extensive transfer among Xanthomonadales and alpha,beta and even gamma-proteobacteria is the main source of incon-gruity in the proteobacterial tree. The results of Lima et al. (2008)corroborate this hypothesis through the identification of genomeislands as products of early and late LGT events, thus confirmingthe mosaic structure of Xanthomonas genomes. Phylogenetic treesof proteins not normally subject to LGT may help to solve thisproblem. However, as observed for 16S rRNA, RecA and other phy-logenetic reconstructions (Figs. 1 and 1S), the Xanthomonadalesgroup appears as paraphyletic with respect to the beta-proteobac-teria, branching at the base of the gamma-proteobacteria clade.

The identification of specific signature sequences is a reliableand consistent approach for determining relative branching andtaxonomic relationships, and has been used with success to deter-mine both broad and specific evolutionary relationships (Gupta,1997, 1998, 2000; Gupta and Griffiths, 2002). The main idea ofthese studies is based on the assumption that these signatures,mainly INDELs, are unique events within certain genes, and pro-vide powerful tools for inferring evolutionary relationships. How-ever, some of these INDELs do present some variation, whichappears as several small gaps within the insertion presented inFig. 2S. Although most of these gaps are clearly specific to certainclades, their origin indicates that the region of the protein affectedby the INDEL has some structural flexibility due to low selectivepressures.



Fig. 4. Partial sequence alignment of the DNA ligase NAD-dependent protein, showing a 65-amino acid insert specific for Xanthomonadales. Details as described in Fig. 2.


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In the present work, the search for INDELs was applied mainlyto uncover taxonomic relationships of the Xanthomonadales orderwithin the proteobacteria, by using a set of conserved proteins re-lated to core metabolic functions such as DNA repair and replica-tion. The main results of these analyses are summarized in aschematic genetic evolutionary flow of beta and gamma-proteo-bacteria, as shown in Fig. 5.

Interestingly, most of the described INDELs distinguish thegamma-proteobacteria group from other proteobacteria groups,as these INDELs are absent in beta, alpha or delta/epsilon subdivi-sions; however, the same profile of absence is shared by earlydivergent groups inside the gamma-proteobacteria, notably Xan-thomonadales, Chromatiales, Methylococcales, Legionellales, Car-diobacteriales and Thiotrichales. This strongly suggests a closerelationship among these groups, in agreement with the phyloge-netic trees shown in Figs. 1 and 1S. In fact, these INDELs were mostlikely to have been introduced after these groups branched fromthe rest of the gamma-proteobacteria. The profile of presence ingamma-proteobacterial species and absence in other proteobacte-rial groups indicates that these signatures constitute a synapomor-phy for this subdivision.


A detailed phylogenomic analysis of the gamma-proteobacteriawas recently published (Gao et al., 2009). Basically, a phylogenetictree based on concatenated protein sequences from conservedgenes placed Xanthomonadales as a deep branch (as well as Thio-trichales, Cardiobacteriales, Legionellales, Chromatialles and Meth-ylococcales) in the phylogenies of gamma-proteobacteria.Although the beta-proteobacteria were not included in those anal-yses, a specific 2-amino acid INDEL, in AIACR-transformylase(PurH), could differentiate all the other proteobacteria (includingbeta-proteobacteria) from Xanthomonadales and gamma-proteo-bacteria. This INDEL was included in the schematic genetic evolu-tionary flow shown in Fig. 5. Moreover, four conserved proteins, ofunknown functions, were identified as exclusive to the gamma-proteobacteria, including Xanthomonadales. These results are goodindications that Xanthomonadales share important features withthe gamma-proteobacteria, but similar searches for genes sharedexclusively between Xanthomonadales and beta-proteobacteriawould be important to understand the relationships among thesegroups. In fact, the INDEL found for topoisomerase I (Fig. 3) andits interesting phylogeny (Fig. 1S) place the Xanthomonadales (aswell as Legionellales, Chromatialles, Methylococcales, Cardiobacte-



Fig. 5. Simplified evolutionary scheme of gamma-proteobacteria, based on INDEL identification. Groups were placed in schematic representation according to sharedsignature sequences. INDELs (white circles) are depicted in the branches where they probably arose. Question marks indicate phylogenetic positions that are not clear,comprising the deep branching gamma-proteobacteria orders, including Xanthomonadales.


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riales and some of the Thiotrichales, Francisella genus) in the samebranch of beta-proteobacteria, but, in this case, independent ofgamma-proteobacteria. This can be explained by a duplication ofthis gene, followed by loss of one of the paralogues. The evolutionof the two paralogues place the Xanthomonadales as an outgroupfrom the core gamma-proteobacteria. An alternative explanationis that this insert was introduced in a common ancestor of the var-ious alpha (and a few delta), beta and gamma-proteobacteria, fol-lowed by a second genetic event leading to loss of this insert from acommon ancestor of the Enterobacteriales, Pasteurellales, Vibrio-nales, Alteromonadales Aeromonadales, Oceanospirillales, Pseudo-monadales and some from Thiotrichales group (Thiomicrospiragenus).

On the other hand, the INDELs exclusively shared by homologsfrom Xanthomonadales, and absent in any homolog from otherproteobacteria, are suggestive of the independent evolution of thisgroup and provide molecular signatures to distinguish and defineit. These signatures are likely recent molecular events in the Xan-thomonadales, which occurred after its divergence from the mainbranches of gamma- and beta-proteobacteria. In view of the highlyconserved and well-defined nature of these signatures, they clearlydefine the Xanthomonadales as monophyletic. Combining the dataof INDEL and phylogeny, these bacteria are more likely to haveevolved independently soon after, or concomitant to, the diver-gence of the beta and gamma-proteobacteria.

Molecular approaches are being used increasingly not only inevolutionary and taxonomic studies, but also in epidemiologicalscrutinies. Because of their specificity and conservation, signa-ture sequences for Xanthomonadales may provide a novel meansfor identifying known or new species within Xanthomonadalesby means of PCR-based and immunological methods. Althoughconserved inside Xanthomonas, Xylella and Stenotrophomonas,the insertion found in the DNA ligase NAD-dependent is hetero-geneous among the genera (Fig. 4). This characteristic is veryimportant and may be of practical significance, as PCR-basedprotocols could possibly contribute to pathogen detection andidentification at the genus level. Thus, knowledge of such spe-


cific INDELs may provide the means for developing specific, ra-pid and easy diagnostic protocols to identify the presence orabsence of Xanthomonadales in DNA samples from potentiallyinfected plants or animals.

5. Conclusions

The alignment of conserved protein sequences has led to theidentification of conserved INDELs, useful for assessing phyloge-netic placement of the Xanthomonadales group. The data pre-sented here delineate the evolution of the gamma lineage andprovide further evidence for the early gamma placement, indicat-ing that Xanthomonadales constitute one of the deeper branchinglineage within gamma-proteobacteria Moreover, the results con-firm the monophyly of Xanthomonadales and constitute interest-ing genetic markers to identify these bacteria.

Acknowledgments

The authors thank Dr. Radhey S. Gupta (McMaster University,Ontario, Canada) for helpful discussion. The authors are also indebt to the anonymous reviewers for their comments, criticismsand excellent suggestions. Financial support was obtained fromFAPESP (São Paulo, Brazil) and CNPq (Brasília, Brazil). During thiswork, A.M.C.J. received a fellowship from CAPES (Brasília, DF, Bra-zil), and M.M.P. and W.C.L. from FAPESP.

Appendix A. Supplementary data

ML phylogenetic tree of topoisomerase I (Fig. 1S) and partialprotein alignments (Figs. 2S–7S). List of proteins numbers usedin phylogenetic analyses (Table S01) and of organisms used to per-form similarity searches (Table S02). Supplementary data associ-ated with this article can be found, in the online version, atdoi:10.1016/j.ympev.2009.09.026.





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