Assessment of a short phylogenetic marker based on comparisons of 3 ' end 16 S rDNA and 5 ' end 16 S-23 S ITS nucleotide sequences on the genus Xanthomonas

A short phylogenetic marker previously used in the reconstruction of the Class γ-proteobacteria was assessed here at a lower taxa level, species in the genus Xanthomonas. This maker is 224 nucleotides in length. It is a combination of a 157 nucleotide sequence at the 3' end of the 16S rRNA gene and a 67 nucleotide sequence at the 5' end of the 16S-23S ITS sequence. A total of 23 Xanthomonas species were analyzed. Species from the phylogenetically related genera Xylella and Stenotrophomonas were included for comparison purposes. A bootstrapped neighborjoining phylogenetic tree was inferred from comparative analyses of the 224 bp nucleotide sequence of all 30 bacterial strains under study. Four major Groups were revealed based on the topology of the neighbor-joining tree, Group I to IV. Group I and II contained the genera Stenotrophomonas and Xylella, respectively. Group III included five Xanthomonas species: X. theicola, X. sacchari, X. albineans, X. transluscens and X. hyacinthi. This group of Xanthomonas species is often referred to as the hyacinthi group. Group IV contained the other 18 Xanthomonas species. The overall topology of the neighbor-joining tree was in agreement with currently accepted phylogenetic. The short phylogenetic marker used here could resolve species from three different Xanthomonadacea genera: Stenotrophomonas, Xylella and Xanthomonas. At the level of the Xanthomonas genus, distant species could be distinguished, and whereas some closely-related species could be distinguished, others were undistinguishable. Pathovars could not be distinguished. We have met the resolving limit of this marker: pathovars and very closely related species from same genus.


INTRODUCTION
The genus Xanthomonas comprises 27 species.These species are primarily characterized by the production of xanthomonadins, a water-insoluble yellow pigment, and the production of an exo-polysaccharide, the xanthan gum, which is used as a thickening, stabilizing and gelling agent in food, pharmaceutics, cosmetics and oil industries [1,2].Most Xanthomonas species are plant pathogens [3].They cause diseases on several economically important plants including crucifers, Solanaceae, citrus, cotton, cereals, ornamentals, fruit and nut trees [3,4].It is estimated that at least 124 monocotyledons and 268 dicotyledons are infected by Xanthomonas species [4][5][6].
In a recent study [26], a short 232 bp nucleotide sequence "marker" was used to reconstruct the phylogeny of the Class γ-proteobacteria.This 232 bp marker was a combination of the last 157 bp at the 3' end of the 16S rRNA gene and the first 75 bp at the 5' end of the 16S-23S rRNA internal transcribed spacer (ITS).We showed that the 157 bp sequence was highly conserved among closely related species.Owing to its higher rate of nucleotide substitutions, the 75 bp added discriminating power among species from same genus and closely related genera from same family.This marker could reconstruct the phylogeny of the species, genera, families and Orders within the Class γ-proteobacteria in accordance with the accepted classification.
In the current study, we further assess the resolving power of this marker at a much lower taxa level: species within the genus Xanthomonas.

Bacterial Species and Strains
A total of 25 Xanthomonas strains from 23 species were analyzed.Four Xylella fastidiosa strains and one Stenotrophomonas maltophila stain were added for comparison purposes.They were selected on the basis that their complete genome sequences were freely available in GenBank, at the National Center for Biotechnology Information (NCBI) completed microbial genomes database (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi,August 2009).All bacterial strains and their GenBank accession numbers are listed in Table 1.

Sequences Analysis
First, the 16S rRNA and the 16S-23S ITS sequences of the 30 bacterial strains under study were retrieved from GenBank.Second, the 16S rRNA gene nucleotide sequences were aligned using ClustalW [27] (data not shown).The length of the nucleotide sequence most conserved was determined at 157 bp.Third, the 16S-23S ITS were aligned using ClustalW (data not shown).The length of the nucleotide sequence most conserved was determined at 67 bp.These two most conserved nucleotide sequences, the 157 bp at the 3' end of 16S, and the 67 bp at the 5' end of 16S-23S ITS, were combined into a single 224 bp sequence for each bacterial species and strain under study.This 224 bp sequence will be used here as a phylogenetic marker for the Xanthomonas species and related genera under study.

Phylogenetic Analyses
A neighbor-joining tree was constructed [28] based on the alignment of the 224 bp sequence of the 30 bacterial strains under study.The tree was bootstrapped using 1,000 random samples of sites from the alignment, all using CLUSTAL W software [27] at the DNA Data Bank of Japan (DDBJ) (http://clustalw.ddbj.nig.ac.jp/tope.html),with the Kimura's parameter method [29].The neighborjoining tree was drawn using TreeView (version 1.6.6)[30,31].

RESULTS AND DISCUSSION
A bootstrapped neighbor-joining tree based was inferred from the alignment of the 224 bp sequence of all 25 Xanthomonas species and pathovars, four Xylella fastidiosa strains and Stenotrophomonas maltophila under study (Figure 1).Four Groups, Group I to IV, were revealed at the 95% nucleotide sequence identities.Group I contains Stenotrophomonas maltophila.Group II encompasses all four Xylella fastidiosa strains.They share 99% nucleotide sequence identities.Group III includes five Xanthomonas species: X. theicola, X. sacchari, X. albineans, X. transluscens and X. hyacinthi.They share at least 96% nucleotide sequence identities.This group of Xanthomonas species is often referred to as the hyacinthi group [15].Our results are in agreement with the first identification of the hyacinthi group based on the homology of their 16S rRNA [13], 16S-23S ITS [15] and gyrB nucleotide sequences [17] and MLSA [18].Group IV contains 18 Xanthomonas species.These species share at least 95% nucleotide sequence identities.Six species can be distinguished: X. axonopodis, X. codiaei, X. fragariae, X. campestris, X. cassavae and X. melonis.Xanthomonas perforans, X. euvesicatoria and X. alfalfae are grouped together and appear undistinguishable.These species share 100% nucleotide sequence identities.The grouping of these six species is in agreement with the work of Parkinson et al. [18] based on comparison of gyrase B gene sequences.Furthermore, the grouping of X. perforans, X. euvesicatoria and X. alfalfae is in agreement with the work of Young et al. [18] based on MLSA.Xanthomonas hortorum and X. vasicola, and X. oryzae and X. bromi are grouped together, respectively, and appear undistinguishable.Both pair of species share 99% and 100% nucleotide sequence identities, respectively.Five other Xanthomonas species, X. gardneri, X. vesicatoria, X. cucurbitae, X. arboricola and X. pisi are grouped together and appear undistinguishable.These species share 100% nucleotide sequence identities.The three X.campestris strains appear undistiguishable.They share 100% nucleotide sequence identities.The three X.campestris strains appear un-distiguishable.They share 100% nucleotide sequence identities.
Of the 23 Xanthomonas species under study, 15 species or group of species could be distinguished by the 224 bp sequence used as marker.Very closely related species, such as those in Group IV could not be dinstinguished.Pathovars could not be distinguished, as exemplified by the three X.campestris pathovars.The overall  topology of the neighbor-joining tree was, however, in agreement with phylogenetic trees based on the 16S rRNA [13] and the 16S-23S ITS [15].
In previous studies, we showed that a DNA sequence from 3' end 16S rRNA gene and 5' end 16S-23S ITS could be used as a marker in the reconstruction of phylogenies in the Gram-positive genus Bacillus and closely-related genera [32], the Gram-positive Order Bacillales [33], and the Gram-negative Class γ-proteobacteria [26].This maker ranged in size from 220 bp to 232 bp.It contained 150-157 bp from the 3' end of the 16S rRNA gene and 67-75 bp from the 5' end of the 16S-23S ITS.The 150-157 bp from the 3' end of the 16S rRNA gene was often sufficient to distinguish bacterial Orders, families, and species from different genera.This sequence was, however, highly conserved among closely related species.Owing to its higher rate of nucleotide substitutions, the 67-75 bp from the 5' end of the 16S-23S ITS added resolving power among closely related species from same genus.This marker had proven useful in reconstructing the phylogenies of the genus Bacillus and closely-related genera [32], the Order Bacillales [33] and the Class γ-proteobacteria [26] in accordance with accepted phylogenies inferred from much more comprehensive datasets.This marker presented several advantages over the use of the entire 16S rRNA gene or the 16S-23S ITS or the generation of extensive phenotypic and genotypic data in phylogenetic analyses.We showed that the method was simple, rapid, suited to large screening programmes and easily accessible to most laboratories.It also proved very valuable in revealing bacterial species which appeared misassigned and for which additional characterization appeared warranted.The resolving power of this marker has been further analyzed here in a much deeper branch of the Class γ-proteobacteria: the genus Xanthomonas.As expected, we have shown here that this marker could resolve species from three different Xanthomonadacea genera: Stenotrophomonas, Xylella and Xanthomonas.At the level of the Xanthomonas genus, distant species could be distinguished.However, although some closely-related species could be distinguished, others were grouped together and some were undistinguishable.Clearly, pathovars could not be distinguished.We have met the resolving limit of this marker: pathovars or very closely-related species.

CONCLUSION
A short DNA marker based on 3' end 16S rDNA and 5' end ITS, had been shown previously to be able to reconstruct the phylogeny of the Class γ-proteobacteria at the Orders, families, genera and distantly-related species levels This marker was analyzed here at a lower taxa level.First, we have shown that this marker could cluster species from same genera within the family Xanthomonadacea.Next, at the genus Xanthomonas level, we have shown that although the short DNA marker could distinguish several species, very closely-related species and pathovars could not be distinguished.We have reached the limit of the resolving power of the 224 bp sequence as a phylogenetic marker: very closely-related species and pathovars.

Figure 1 .
Figure 1.Bootstrapped neighbor-joining tree of the genus Xanthomonas species inferred from the alignment of the 224 bp marker.

Table 1 .
Bacterial species used in this study.