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

doi:10.4236/ns.2010.212167

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Vol.2, No.12, 1369-1374 (2010)

doi:10.4236/ns.2010.212167

Natural Science

Assessment of a short phylogenetic marker based on

comparisons of 3' end 16S rDNA and 5' end 16S-23S ITS

nucleotide sequences on the genus Xanthomonas

Sabarimatou Yakoubou1,2, Jean-Charles Côté1*

1Agriculture and Agri-Food Canada, Research Centre, Québec, Canada; *Corresponding Author: [email protected]

2Département des Sciences Biologiques, Université du Québec à Montréal, Québec, Canada

Received 13 August 2010, revised 15 September 2010, accepted 20 September 2010.

ABSTRACT

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 com-

parison purposes. A bootstrapped neighbor-

joining 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 Steno-

trophomonas 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 dif-

ferent Xanthomonadacea genera: Stenotro-

phomonas, Xylella and Xanthomonas. At the

level of the Xanthomonas genus, distant spe-

cies 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.

Keywords: 16S rRNA; 16S-23S ITS; Phylogeny;

Xanthomonas

1. 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 gel-

ling agent in food, pharmaceutics, cosmetics and oil in-

dustries [1,2]. Most Xanthomonas species are plant

pathogens [3]. They cause diseases on several economi-

cally 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 spe-

cies [4-6].

Up to the mid-90’s, the classification of Xanthomonas

species and isolates was based on phenotypic data. The

main criteria for the creation of new species rested on

host specificity. The taxa “pathovar” was added to dis-

tinguish Xanthomonas species at the infrasubspecific

level. Some species, e.g. X. axonopodis, X. transluscens

or X. campestris, comprised more than ten, 40 and 125

pathovars, respectively [3]. Several methods were used

in an attempt to classify Xanthomonas species and

pathovars: restriction fragment-length polymorphism

(RFLP) [7,8], protein profiles [9] and fatty acid methyl

ester profiles [10-11]. Vauterin et al. reorganized the

classification of the genus Xanthomonas based on

DNA-DNA hybridization [12]. They revealed 20 DNA

homology groups which they considered genomic spe-

cies. Since then, other approaches based on different

nucleotide sequences have been used to study the phy-

logeny of Xanthomonas: the 16S rRNA gene [13], a

multilocus sequence typing (MLST), [14], the 16S-23S

intergenic spacer [15], the repetitive palindromic-based

S. Yakoubou et al. / Natural Scienc e 2 (2010) 1369-1374

1370

polymerase chain reaction fingerprinting (Rep-PCR)

[16], the gyrB gene [17] and a multilocus sequence

analysis (MLSA) [18]. Seven additional Xanthomonas

species have now been described [19-25].

In a recent study [26], a short 232 bp nucleotide se-

quence “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 discriminat-

ing power among species from same genus and closely

related genera from same family. This marker could re-

construct the phylogeny of the species, genera, families

and Orders within the Class γ-proteobacteria in accor-

dance 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.

2. MATERIALS AND METHODS

2.1. 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 Bio-

technology Information (NCBI) completed microbial ge-

nomes 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.

2.2. 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 nucleo-

tide 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 spe-

cies and related genera under study.

2.3. 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 neighbor-

joining tree was drawn using TreeView (version 1.6.6)

[30,31].

3. RESULTS AND DISCUSSION

A bootstrapped neighbor-joining tree based was in-

ferred from the alignment of the 224 bp sequence of all

25 Xanthomonas species and pathovars, four Xylella

fastidiosa strains and Stenotrophomonas maltophila un-

der 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 in-

cludes five Xanthomonas species: X. theicola, X. sac-

chari, 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 spe-

cies 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 undistinguish-

able. These species share 100% nucleotide sequence

identities. The grouping of these six species is in agree-

ment 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 to-

gether, 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 undistin-

guishable. These species share 100% nucleotide se-

quence identities. The three X. campestris strains appear

undistiguishable. They share 100% nucleotide sequence

identities. The three X. campestris strains appear un-

S. Yakoubou et al. / Natural Scienc e 2 (2010) 1369-1374

137

1371

distiguishable. They share 100% nucleotide sequence

identities.

Of the 23 Xanthomonas species under study, 15 spe-

cies 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 dinstin-

guished. Pathovars could not be distinguished, as exem-

plified by the three X. camp estris pathovars. The overall

Table 1. Bacterial species used in this study.

Genera Species Pathovars/Strain GenBank accession no.

Stenotrophomonas maltophilia R551-3 NC_01107

Xanthomonas albilineans LMG 494T X95918

alfalfae F1 AF442741

arboricola pv. juglandis LMG 747 T Y10757

axonopodis LMG 538 T X95919

bromi LMG 947 T AF209754

campestris pv. campestris ATCC 33913 T NC_003902

campestris pv. campestris B100 NC_010688

campestris pv. campestris 8004 NC_007086

cassavae LMG 673 T AF209756

codiaei LMG 8678 T Y10765

cucurbitae LMG 690 T Y10760

euvesicatoria 85-10 NC_007508

fragariae LMG 708 T X95920

gardneri CNPH496 AY288083

hortorum LMG 733 T Y10759

hyacinthi LMG 739 T Y10754

melonis LMG 8670 T Y10756

oryzae pv. oryzae MAFF 311018 NC_007705

perforans CNPH411 AY288081

pisi LMG 847 T Y10758

Sacchari LMG 471 T Y10766

Theicola LMG 8684 T Y10763

transluscens pv. graminis AY247064

vasicola LMG 736 T Y10755

vesicatoria LMG 911 T Y10761

Xylella fastidiosa 9a5c NC_002488

M12 NC_010513

M23 NC_010577

Temecula NC_004556

S. Yakoubou et al. / Natural Scienc e 2 (2010) 1369-1374

1372

0.01

Xanthomonas melonis

Stenotrophomonas maltophilia

Xylella fastidiosa 9a 5c

Xylella fastidiosa M1 2

Xylella fastidiosaM23

Xylella fastidiosaTemecula1

100

Xanthomonas theicola

Xanthomonas sacchari

Xanthomonas albilineans

Xanthomonas translucens

Xanthomonas hyacinthi

Xanthomonas axonopodis

Xanthomonas codiaei

Xanthomonas perforans

Xanthomonas euvesicatoria str 85-10

Xanthomonas alfalfae

Xanthomonas fragariae

Xanthomonas campestris campestris B100

Xanthomonas campestris campestris8004

Xanthomonas campestris campestris ATCC 33913

Xanthomonas hortorum

Xanthomonas vasicola

Xanthomonas oryzae

Xanthomonas bromi

Xanthomonas cassavae

Xanthomonas gardneri

Xanthomonas vesicatoria

Xanthomonas cucurbitae

Xanthomonas arboricola

Xanthomonas pisi

Grou ps

III

0.010.01

Xanthomonas melonis

Stenotrophomonas maltophilia

Xylella fastidiosa 9a 5c

Xylella fastidiosa M1 2

Xylella fastidiosaM23

Xylella fastidiosaTemecula1

100

Xanthomonas theicola

Xanthomonas sacchari

Xanthomonas albilineans

Xanthomonas translucens

Xanthomonas hyacinthi

Xanthomonas axonopodis

Xanthomonas codiaei

Xanthomonas perforans

Xanthomonas euvesicatoria str 85-10

Xanthomonas alfalfae

Xanthomonas fragariae

Xanthomonas campestris campestris B100

Xanthomonas campestris campestris8004

Xanthomonas campestris campestris ATCC 33913

Xanthomonas hortorum

Xanthomonas vasicola

Xanthomonas oryzae

Xanthomonas bromi

Xanthomonas cassavae

Xanthomonas gardneri

Xanthomonas vesicatoria

Xanthomonas cucurbitae

Xanthomonas arboricola

Xanthomonas pisi

Grou ps

III

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

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 spe-

cies. 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 recon-

structing the phylogenies of the genus Bacillus and

closely-related genera [32], the Order Bacillales [33] and

the Class γ-proteobacteria [26] in accordance with ac-

cepted phylogenies inferred from much more compre-

hensive datasets. This marker presented several advan-

tages 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

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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: St eno-

trophomonas, Xylella and Xanthomonas. At the level of

the Xanthomonas genus, distant species could be distin-

guished. 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.

4. CONCLUSION

A short DNA marker based on 3’ end 16S rDNA and

5’ end ITS, had been shown previously to be able to re-

construct 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 Xanthomo-

nadacea. Next, at the genus Xanthomonas level, we have

shown that although the short DNA marker could dis-

tinguish 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.

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