- Email: [email protected]
16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia
FEMS Microbiology Letters 151 (1997) 145^153
16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia
Edward R.B. Moore a *, Annette S. Kruëger a , Lysiane Hauben a b , Susan E. Seal 1 c , Roland De Baere d , Rupert De Wachter d , Kenneth N. Timmis a , Jean Swings b ;
a
;
;
Bereich Mikrobiologie, GBF-Gesellschaft fu ë r Biotechnologische Forschung, Mascheroderweg 1, D-38124 Braunschweig, Germany
b c
Laboratorium voor Microbiologie, Universiteit Gent, B-9000 Gent, Belgium
The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
d
Department Biochemie, Universitaire Instelling Antwerpen, B-2610 Antwerpen, Belgium
Received 18 March 1997 ; accepted 28 March 1997
Abstract
The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage within the Q-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species. Keywords : Xanthomonas
;
Stenotrophomonas
; 16S rRNA gene; Phylogeny
1. Introduction
Species of the genus Xanthomonas [1] are plantassociated bacteria responsible for a variety of plant bacterioses. The ¢rst such disease noted, which today would be attributed to a xanthomonad, was `yellow disease' of hyacinth plants; the causative agent was named Bacterium hyacinthi [2]. Since that ¢rst report, * Corresponding author. Tel.: (49) 531-6181-557; Fax: (49) 531-6181-411; E-mail: [email protected] 1
species have been implicated in diseases of practically all major taxa of higher plants, occurring ubiquitously throughout the world and thereby attaining considerable agricultural importance [3]. Xanthomonas species are Gram-negative rods, with polar £agella and an obligately oxidative metabolism. They are oxidase negative and do not reduce nitrates. They produce characteristic water-insoluble yellow pigments: xanthomonadins; and extracellular heteropolysaccharides: xanthan gums. The xanthan gums, due to their high speci¢c viscosity and their stability under extreme environmental conditions, Xanthomonas
Present address : Natural Resources Institute, Chatham Maritime, Kent ME4 4TB, UK.
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 5 2 - 3
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
146
Xanthomonas maltophilia, and phenotypically simXanthomonas species in many respects, occurs
have attained an importance in diverse biotechnolog-
as
ical applications [4].
ilar to
Until
recently
has
widely in soils and water and, unlike the other species listed above, is not plant-pathogenic, presenting
scribed
a
within
[5], the
Xanthomonas
comprised as many as 10 taxospecies, as validly dethe
genus
Approved
Lists
of
Bacterial
recognizably
distinct
taxon.
Originally
isolated
as an attempt to order the pathogens according to
Bacterium bookerii, this organism was reclassi¢ed as Pseudomonas maltophilia [14] before being renamed Xanthomonas maltophilia on the basis of DNA^rRNA hybridization re-
their host speci¢cities when they could not be distin-
sults,
guished reliably from one another by other criteria
cellular fatty acid composition [7]. However, incor-
Names [6] and later references [7^10]. Subsequent revisions
produced
di¡erent
taxonomic
tions, including 125 pathovars of
from pleural £uid and named
organiza-
X. campestris
[11]
quinone
type,
P. maltophilia
[12]. On the basis of extensive phenotypic character-
poration of
izations, Van den Mooter and Swings [13] de¢ned
nas
eight distinct xanthomonad phena corresponding to
reclassi¢cation of
X. campestris (type species of the genus), X. campestris pv. graminis, X. albilineans, X. axonopodis, X. fragariae, X. maltophilia, X. oryzae, and X. populi. Stenotrophomonas maltophilia, formerly classi¢ed
nas maltophilia,
recognizable species :
enzyme
characterization
into the genus
X. maltophilia,
to
Stenotrophomo-
has been proposed [16].
This study presents the results of 16S rRNA gene sequence determinations and comparisons among the type and reference strains representing the xanthomonad phena of Van den Mooter and Swings [13],
List of bacterial strains examined
X. campestris pv. campestris X. campestris pv. campestris X. campestris pv. campestris X. campestris pv. barbareae X. campestris pv. musacearum X. albilineans X. albilineans X. axonopodis pv. axonopodis X. axonopodis pv. citri X. axonopodis pv. malvacearum X. axonopodis pv. manihotis X. fragariae X. fragariae X. oryzae pv. oryzae X. oryzae pv. oryzicola X. populi pv. populi X. populi pv. populi X. translucens pv. translucens X. translucens pv. graminis X. translucens pv. graminis S. maltophilia S. maltophilia
Strain LMG
EMBL accession number
T 568
X95917
LMG 567 8004 LMG 547 R536
T
LMG 494
X95918
LMG 482
T
LMG 538
X95919
LMG 682 NCPPB 633 3172
T
LMG 708 LMG 706
X95920
T
X95921
T
X95922
LMG 5047 LMG 665
Xanthomo-
was not universally accepted [15] and a further
Table 1
Organism
and
LMG 5743 LMG 974
T
LMG 876
LMG 726
X99298
NCPPB 3040
T
LMG 958
X95923
LMG 11114
X95925
LMG : Laboratorium voor Microbiologie Gent Culture Collection, Gent, Belgium. NCPPB : National Collection of Plant Pathogenic Bacteria, Harpenden, UK. Strain 8004 is a derivative of strain NCPPB 1145. Strain 3172 provided by Dr. J.M. Clarkson, Bath University, Bath, UK. Strain R536 provided by Dr. S. Eden-Green, Rothamsted Experimental Station, Harpenden, UK. EMBL Accession Numbers are provided for nearly complete gene sequences.
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
147
Fig. 1. Evolutionary distances derived from pairwise sequence dissimilarities were calculated using the DNADIST program included in the PHYLIP package. The least-squares distance algorithm of the FITCH program, included in the PHYLIP package, was used to generate the dendrogram depicting the inferred phylogenetic positions of the species of the genus
Xanthomonas
and
a : Figure demonstrates the monophyletic position of the xanthomonads within representative species of the
ria (C. testosteroni,
a species of the
L-subclass
of the
Proteobacteria,
was included as outgroup). Additional treeing methods (i.e., neigh-
bor-joining and maximum-likelihood) supported the phylogenetic relationships depicted between er representatives of the of the genus
Q-subclass
of the
Xanthomonas (S. maltophilia
Proteobacteria
Stenotrophomonas maltophilia. Q-subclass of the Proteobacte-
Xanthomonas, Stenotrophomonas
and oth-
(not shown). b : Figure demonstrates the intrageneric relationships of the species
was included as outgroup).
the determination of sequence signatures and infer-
ously [19]. Nearly complete 16S rRNA genes were
ences of intra- and intergeneric taxonomic relation-
ampli¢ed by PCR [20] using a forward primer hy-
ships for species of
monas.
Xanthomonas
and
Stenotropho-
bridizing at the complements of positions 8^27, and a reverse primer hybridizing at positions 1541^1525 (
E. coli
was 2. Materials and methods
16S rRNA gene sequence numbering). PCR
carried
out
using
Perkin-Elmer
Cetus
DNA
thermal cyclers (Perkin Elmer Corp., Norwalk, CO) and
2.1. Bacterial strains and culture conditions
conditions
as
reported
previously
[21].
PCR-
DNAs were puri¢ed with Centricon-100 microconcentrators (Amicon GmbH, Witten, Germany) and
The bacterial strains used in this study, listed in
Taq
sequenced directly using a protocol for `
Cycle
Table 1, were cultured on GYCA medium [17] or on
Sequencing' and £uorescent dye-labeled dideoxynu-
NYGB medium [18] with shaking.
cleotides
(Applied
Biosystems
Division,
Perkin-El-
mer Corp., Weiterstadt, Germany).
2.2. Genomic DNA isolation, PCR ampli¢cation and sequence determination of 16S rDNA Genomic DNA was isolated as described previ-
In the case of some strains, a segment of the 16S
E. coli 16S rRNA gene sequence numbering), was PCRrRNA gene, corresponding to positions 40^337 (
ampli¢ed and sequenced as described previously [22].
FEMSLE 7580 2-9-97
148
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
Sequence data were aligned with reference rRNA (and rRNA gene) sequences [23,24] using evolutionarily conserved primary sequence and secondary structure as reference [25,26]. Evolutionary distances were calculated from sequence-pair similarities [27] and dendrograms were generated using a weighted, least-squares, distance method [28]. 3. Results and discussion
Nearly complete 16S rRNA gene sequences have been determined for the type and reference strains of the eight xanthomonad phena discerned by Van den Mooter and Swings [13]. PCR, using a primer pair annealing at the 5P- and 3P-termini of the 16S rRNA gene, enabled the ampli¢cation and sequence determination of 1502 nucleotide positions (W97% of the entire gene, as estimated by comparison with the 16S rRNA gene sequence of E. coli). The sequence data have been deposited with the EMBL Nucleotide Sequence database [29] under accession numbers listed in Table 1. Sequence data from each strain were compared, by cluster analysis, to reference sequence data, and supported what had been observed previously from DNA^rRNA hybridization data [30,31]: that Xanthomonas species and Stenotrophomonas maltophilia form a monophyletic cluster within the Q-subclass of
the Proteobacteria [32]. Fig. 1a,b present the estimated phylogenetic/taxonomic positions of species of the genus Xanthomonas and Stenotrophomonas maltophilia with respect to each other and within a small representation of the Q-subclass of the Proteobacteria. A primary cluster, including the species X. oryzae pv. oryzae, X. populi pv. populi and X. fragariae, was collected around the type species of the genus, X. campestris pv. campestris. The sequences of these species are too similar to be able to con¢dently predict the exact phylogenetic branching order, although the closest similarities were observed between the sequences of X. campestris pv. campestris and X. oryzae pv. oryzae (one nucleotide di¡erence) and between X. campestris pv. campestris and X. populi pv. populi (two nucleotide di¡erences). The sequences of X. oryzae pv. oryzae and X. populi pv. populi possessed three nucleotide di¡erences. The sequences of X. fragariae and X. axonopodis pv. axonopodis branched outside the primary cluster with ¢ve and ten, respectively, nucleotide di¡erences from the sequence of X. campestris. The sequence of X. albilineans was observed to branch even farther outside the X. campestris primary cluster, possessing 35 nucleotide di¡erences (2.3% sequence di¡erence) from that of X. campestris pv. campestris. The sequence di¡erence observed among all species of Xanthomonas ranged from, approximately, 0.1% (between X. campestris pv. campestris and X. oryzae
Table 2 Similarity values determined from pairwise comparisons among 16S rRNA gene sequences of Xanthomonas, Stenotrophomonas, and other Proteobacteria Q-subclass species Organism Strain % Sequence similarity 01 02 03 04 05 06 07 08 09 10 11 01. X. campestris pv. campestris LMG 568T 02. X. campestris pv. graminis LMG 726R 97.3 03. X. albilineans LMG 494T 97.5 99.5 04. X. axonopodis pv. axonopodis LMG 538T 99.3 97.2 97.9 99.7 96.9 97.3 99.1 05. X. fragariae LMG 708T 06. X. oryzae pv. oryzae LMG 5047T 99.9 97.1 97.5 99.2 99.7 07. X. populi pv. populi LMG 5743T 99.9 97.1 97.5 99.2 99.5 99.7 08. S. maltophilia LMG 958T 96.9 95.4 95.1 96.3 96.7 96.8 96.8 09. Xylella fastidiosa ATCC 35880 95.0 93.5 95.3 94.7 94.7 94.8 94.8 94.1 10. Pseudomonas aeruginosa DSM 50071T 85.2 84.9 84.9 84.8 84.9 85.3 85.1 85.1 84.3 11. Escherichia coli 83.3 82.5 83.1 83.3 83.2 83.4 83.2 81.9 82.4 84.6 T = type strain of species; R =reference strain of pathovar. 16S rRNA sequences of X. fastidiosa, P. aeruginosa and E. coli obtained from the EMBL Database of Nucleotide Sequences.
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
149
Fig. 2. Sequences for the hypervariable region V-1, including helix 6, of species and pathovars of the genus Xanthomonas and S. malto. Only those positions wherein di¡erent nucleotides were observed from those in the sequence of X. campestris pv. campestris are shown. Hyphens depict sequence gaps. philia
pv.
) to 3% (between X. albilineans and X. ) (Table 2). It is noteworthy that the species clustering most closely with X. campestris (i.e., X. oryzae and X. populi) were observed to possess approximately only 0.1% sequence di¡erence from that of X. campestris. Although no species de¢nition based upon 16S rRNA similarity exists, such a high sequence similarity has been more generally observed between strains of a species, rather than di¡erent species of a genus. Yet, during the course of the study reported here, these species were con¢rmed to constitute different DNA^DNA hybridization groups [5], thus de¢ning these organisms as di¡erent genospecies, distinct from X. campestris pv. campestris and from each other. Other examples have been reported previously wherein species of a genus are clearly recognizable by their DNA^DNA similarities below the accepted molecular criterion of 70% [33], yet which possess no, or very few, nucleotide di¡erences in their 16S rRNA (gene) primary sequences [34^37]. Such observations have important implications for the use of 16S rRNA sequence data in bacterial oryzae fragariae
taxonomy. These implications have been expressed previously by Fox et al. [35], and more recently by Stackebrandt and Goebel [38], who have pointed out certain limitations in the use of 16S rRNA sequence data for de¢ning bacterial identities. It should be recognized that, while 16S rRNA sequence comparisons are very powerful for determining intergeneric relationships across the taxonomic spectrum, at the uppermost levels of sequence similarities (i.e., 99% or higher) the resolving power of 16S rRNA to re£ect the phylogenetic relationships may not be adequate for guaranteeing that two organisms are not di¡erent species. Stenotrophomonas maltophilia, which has been proposed to constitute a genus taxonomically separated from Xanthomonas, was observed to possess approximately 97% sequence similarity to X. campestris pv. campestris. The sequence similarities observed between S. maltophilia and all Xanthomonas species analyzed ranged from 95.1% to 96.9%. While S. maltophilia clearly was seen to branch outside the cluster formed by the Xanthomonas (sensu stricto) species, the question arises as to whether the level
FEMSLE 7580 2-9-97
150
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
of 16S rRNA gene sequence dissimilarity observed between S. maltophilia and Xanthomonas species (45 to 68 nucleotide di¡erences) supports the placement of S. maltophilia in a separate genus. It may be pointed out that within the group of six Xanthomonas species analyzed in this study, X. albilineans was also observed to cluster clearly distant from X. campestris, relative to the species forming the primary cluster around X. campestris, and the question may be raised as to whether X. albilineans should also be separated from the other species of Xanthomonas in a new genus. Such observations demonstrate an additional limitation in the use of 16S rRNA sequence comparisons for analyzing bacterial taxonomic units. That is, 16S rRNA sequence comparisons clearly demonstrate that S. maltophilia is more distantly related to X. campestris (the type species of the genus Xanthomonas) than is X. albilineans, which is more distantly related to X. campestris than is X. axonopodis, etc. However, the precise borders de¢ning the `true' Xanthomonas (i.e., Xanthomonas, sensu stricto) species (necessarily including those species clustering with X. campestris pv. campestris) from species of closely related genera, presently may not be de¢ned by sequence comparisons alone. Given the inferred close phylogenetic relationships, as well as the additional molecular characterizations showing many similarities, between S. maltophilia and species of the genus Xanthomonas [39^41], an obvious question arises as to whether the ability to produce disease in plants, per se, provides the proper basis for precluding S. maltophilia from the genus Xanthomonas. In any case, the sequence data must be correlated with other characteristics, shedding light upon the relative importance of phenotypic features which, until the phylogenetic relationships have been deduced, might not be recognized as `de¢nitive' characteristics. Although this may be an `obvious' deduction, it should be recognized that the systematic value of phenotypic characteristics, such as the type of pigment or production of extracellular polysaccharide, cannot be assessed con¢dently without correlating such data with the relationships inferred through molecular analyses. Ultimately, it can be seen that the `drawing' of the borders delineating the di¡erent genera must be, necessarily, de¢ned by the overall biology of the organisms. In light of the limited number of nucleotide di¡er-
ences observed between the 16S rRNA gene sequences of the di¡erent species of Xanthomonas, the question arises as to whether the nucleotide di¡erences observed are characteristic of the respective species, and thus may able to be considered `signatures', or are merely random base changes. Therefore, at least one additional strain for each species, determined to belong to the same DNA^DNA hybridization group as the type strain of that species [5], has been analyzed by sequencing the regions of the 16S rRNA gene wherein the majority of sequence di¡erences were observed among the species of the genus Xanthomonas. Among the type strains analyzed in this study, 84 nucleotide positions, or 5.4% of the complete sequence, were seen to vary. The majority of the variable positions were observed to occur in speci¢c, more-or-less concentrated, regions: positions 75^94 includes 18 variable positions; positions 200^ 227 includes nine variable positions; positions 631^ 672 includes 11 variable positions; positions 721^743 includes seven variable positions; positions 999^1041 includes eight variable positions. At all positions given to variation, the nucleotide observed in the type strain was con¢rmed, as well, by the determination, in all cases, of the `signature' in the second strain of the respective species. Thus, at least for the limited number of species analyzed in this study, the nucleotide di¡erences observed among the species of the genus Xanthomonas appear to be species-speci¢c. This observation suggests a potential for the use of 16S rRNA gene sequence data in diagnostic analyses of Xanthomonas phytopathogens. While, in this study, we have concentrated upon the 16S rRNA gene sequence characterizations of the eight xanthomonad phena described by Van den Mooter and Swings [13], additional DNA^DNA groupings for both Xanthomonas and Stentotrophomonas have been recently described [5]. Particularly within the X .campestris complex, comprising phenotypically indistinguishable pathovars, distinct DNA^ DNA groupings have been observed, suggesting the existence of many more species than are now recognized. We have included in this study sequence determinations of the 16S rRNA area VI [42], including helix 6, which is known to be hypervariable and was seen to be the region of the most concentrated variability among Xanthomonas species, as a screening method for distinguishing the di¡erent species of
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
151
the genus Xanthomonas. Fig. 2 presents a compari-
Union for High-Resolution Microbial Identi¢cation
son of the sequence data for the region of nucleotide
(BIOT-CT-0294-SSMA).
positions 75^94 (E. coli 16S rRNA gene sequence
recipient of a fellowship through the Human Capital
positions). While a sequence in this region was ob-
Mobility
served which was characteristic of all X. campestris
by
pathovars analyzed, this sequence was also observed
the GBF and the Universiteit Gent. S. Seal thanks
in other species and their pathovars. Many di¡erent
the Overseas Development Administration for fund-
species, as de¢ned by DNA^DNA similarities, could
ing, commissioned by the Natural Resources Insti-
not be characterized by the primary sequences of this
tute (Project X0082).
the
Programme
Commission
Lysiane
Hauben
(CHRX-CT93-0194) of
European
was
the
awarded
Communities
to
hypervariable region alone, although three general groups could be de¢ned as comprised of those species clustering with X. campestris, those
clustering
References
with X. albilineans, and those clustering with S. maltophilia. This limited potential to use the hypervariable region of helix 6 to distinguish Xanthomonas species re£ects the overall close relationship of these organisms, and suggests only a limited potential for developing species- or pathovar-speci¢c gene probes or PCR primers for diagnostic analyses. However, this study analyzed a limited number of species and strains and only one hypervariable region was analyzed in any depth. Maes [43] described a screening method,
applied
to
nucleic
acid
extracts
of
wheat
seeds, using PCR to distinguish Xanthomonas species from
S.
maltophilia
and
other
species
of
bacteria
known to be phytopathogenic. Seal et al. [44] showed
[1] Dowson, W.J. (1939) On the systematic position and generic names of the Gram-negative bacterial plant pathogens. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2 100, 177^193. [2] Wakker, J.H. (1883) Vorla ë u¢ge mittheilungen u ë ber Hyacinthenkrankheiten. Bot. Zentralblatt 14, 315^317. [3] Star,
M.
(1981)
The
genus
Xanthomonas.
In :
The
Prokar-
yotes : a Handbook on Habitats, Isolation, and Identi¢cation of
Bacteria
(M.
Starr,
H.
Stolp,
H.G.
Tru ë per,
A.
Balows,
H.G. Schlegel, Eds.), p. 742, Springer, Berlin. [4] Jeanes, A. (1974) Applications of extracellular microbial polysaccharide-polyelectrolytes : review of literature, including patents. J. Polymer Sci. 45, 209^227. [5] Vauterin, L., Hoste, B., Kersters, K. and Swings, J. (1995) Reclassi¢cation of Xanthomonas. Int. J. Syst. Bacteriol. 45, 472^489.
that PCR-ampli¢cation targeting a region of the 16S
[6] Skerman, V.B.D., McGowan, V., and Sneath, P.H.A. (1980)
rRNA gene allowed detection of Ralstonia (`Pseudo-
Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30,
monas') solanacearum at levels of 100 cfu/ml. Thus, the use of 16S rRNA gene sequence targeted sites for PCR
ampli¢cation
or
gene
probing
in
diagnostic
analyses possesses a strong potential, if intrageneric (i.e.,
species
or
species
clusters)
characteristic
225^420. [7] Swings, J., De Vos, P., Van den Mooter, M. and De Ley, J. (1983) Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. Int. J. Syst. Bacteriol. 33, 409^413.
pri-
[8] Gabriel, D.W., Kingsley, M.T., Hunter, J.E. and Gottwald, T.
mary sequence regions are able to be de¢ned. Addi-
(1989) Reinstatement of Xanthomonas citri (ex Hasse) and X.
tional analyses correlating the 16S rRNA gene sequences
of
all
currently
recognized,
validly
described, species of the genus Xanthomonas are currently being carried out.
phaseoli (ex Smith) to species and reclassi¢cation of all X. campestris pv. citri strains. Int. J. Syst. Bacteriol. 39, 14^22. [9] Swings,
J.,
Van
den
Mooter,
M.,
Vauterin,
L.,
Hoste,
B.,
Gillis, M., Mew, T.W. and Kersters, K. (1990) Reclassi¢cation of the causal agents of bacterial blight (Xanthomonas campestris pv. oryzae) and bacterial leaf streak (Xanthomonas campestris pv. oryzicola of rice as pathovars of Xanthomonas or-
Acknowledgments
yzae
(ex
Ishiyama
1922)
sp.
nov.,
nom.
ref.
Int.
J.
Syst.
Bacteriol. 40, 309^311. è , M. and Ride è , S. (1992) Xanthomonas populi (ex Ride è [10] Ride
The authors gratefully acknowledge the technical
1958) sp. nov., nom. rev. Int. J. Syst. Bacteriol. 42, 652^653.
assistance of A. Arnscheidt and L. Jackson. We are
[11] Bradbury, J.F. (1984) Xanthomonas Dowson 1939, 187. In :
indebted to Gary Olsen and the Ribosomal Database Project for generously providing the computer soft-
Bergey's Manual of Systematic Bacteriology, Vol. I (Krieg, N.R. and Holt, J.C., Eds.), pp. 199^210. Williams and Wilkins, Baltimore, MD.
ware used for the sequence analysis. This study was
[12] Dye, D.W. and Lelliott, R.A. (1974) Xanthomonas. Dowson
supported, in part, by a grant from the European
1939, 187. In : Bergey's Manual of Determinative Bacteriol-
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
152
ogy, 8th edn. (Buchanan, R.E. and Gibbons, N.E., Eds.), pp.
[13] Van den Mooter, M. and Swings, J. (1990) Numerical analysis of 295 phenotypic features of 226
è, P., Stoehr, P.J., Cameron, G.N. and Flores, [29] Rodriguez-Tome T.P. (1996) The european bioinformatics institute (EBI) data-
243^249. Williams and Wilkins, Baltimore, MD.
Xanthomonas
strains and
bases. Nucl. Acids Res. 24, 6^12. [30] [ Palleroni, N.J., Kunisawa, R. Contopoulou, R. and Douder-
related strains and an improved taxonomy of the genus. Int.
o¡, M. (1973) Nucleic acid homologies in the genus
J. Syst. Bacteriol. 40, 348^369.
monas.
[14] Hugh, R.
philia,
and
an
Ryschenkow,
Alcaligenes-like
E.
Pseudomonas malto-
(1961)
species.
J.
Gen.
Microbiol.
26,
[31] De
Pseudo-
Int. J. Syst. Bacteriol. 23, 333^339.
Vos,
P.
and
similarities of
De
Ley,
Pseudomonas
J.
(1983)
and
Intra-
and
Xanthomonas
intergeneric
ribosomal ribo-
nucleic acid cistrons. Int. J. Syst. Bacteriol. 33, 487^509.
123^132. [15] Van Zyl, E. and Steyn, P.L. (1992) Reinterpretation of the
Xanthomonas maltophilia
[32] Stackebrandt, E., Murray, R.G.E. and Tru ë per, H.G. (1988)
and taxonom-
Proteobacteria classis nov., a name for the phylogenetic taxon
ic criteria in this genus. Request for an opinion. Int. J. Syst.
that includes the `purple bacteria and their relatives'. Int. J.
taxonomic position of
Syst. Bacteriol. 38, 321^325.
Bacteriol. 42, 193^198.
Stenotrophomonas, a Xanthomonas maltophilia (Hugh 1980)
[16] Palleroni, N.J. and Bradbury, J.F. (1993) new bacterial genus for
[17] Swings, J.G. and Civerolo, E.L. (1993) Taxonomy of the Ge-
Xanthomonas.
Xanthomonas
In :
(Swings, J.G. and Civer-
[18] Daniels, M.J., Barber, C.E., Turner, P.C., Cleary W.G. and
campestris
Krichevsky,
M.I.,
E.,
Moore,
Starr,
M.R.
W.E.C., and
Murray,
Tru ë per,
H.G.
(1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37,
[34] Ash, C., Farrow, J.A.E., Wallbanks, S. and Collins, M.D.
Xanthomonas cam-
(1991)
Phylogenetic
showing altered pathogenicity. J. Gen.
vealed
by
Sawczyc, M. (1984) Isolation ofmutants of pv.
O.,
Stackebrandt,
463^464.
olo, E.L., Eds.), p. 130. Chapman and Hall, London.
pestris
Kandler, R.G.E.,
Swings et al. 1983. Int. J. Syst. Bacteriol. 43, 606^609.
nus
[33] Wayne, L.G., Brenner, D.J., Colwell, R.R., Grimont, P.A.D.,
heterogeneity
comparative
analysis
of
of
the
Bacillus
genus
re-
small-subunit-ribosomal
RNA sequences. Lett. Appl. Microbiol. 13, 202^206.
Microbiol. 130, 2447^245. [19] Marmur, J. (1961) A procedure for the isolation of deoxyri-
[35] Fox, G.E., Wisotzkey, J.D. and Jurtshuk, P., Jr. (1992) How
bonucleic acid from micro-organisms. J. Mol. Biol. 3, 208^
close is close : 16S rRNA sequence identity may not be su¤-
218.
cient to guarantee species identity. Int. J. Syst. Bacteriol. 42,
[20] Mullis, DNA
K.B. in
and
vitro
Faloona,
via
a
F.
(1987)
Speci¢c
polymerase-catalyzed
synthesis
chain
of
reaction.
Phylogenetic
Methods Enzymol. 155, 335^350. [21] Karlson,
U.,
Dwyer,
D.F.,
Hooper,
S.W.,
Moore,
E.R.B.,
Timmis, K.N. and Eltis, L.D. (1993) Two independently regulated cytochromes p-450 in a
Rhodococcus rhodochrous
strain
that degrades 2-ethoxyphenol and 4-methoxybenzoate. J. Bac-
Aeromonas
interrelationships
Plesiomonas
and
of
members
of
the
Genera
as determined by 16S ribosomal
DNA sequencing : lack of congruence with results of DNA^ DNA hybridizations. Int. J. Syst. Bacteriol. 42, 412^421. [37] Bennasar,
A.,
Rossello-Mora,
R.,
Lalucat,
J.
and
Moore,
E.R.B. (1996) 16S rRNA gene sequence analysis relative to
teriol. 175, 1467^1474. [22] Young, J.P.W., Downer, H.L. and Eardly, B.D. (1991) Pylogeny of the phototrophic
166^170. [36] Martinez-Murcia, A.J., Benloch, S. and Collins, M.D. (1992)
Rhizobium
strain BTAil by polymer-
ase chain reaction-based sequencing of a 16S rRNA gene seg-
Pseudomonas stutzeri and proposal domonas balearica sp. nov. Int. J. Syst. Bacteriol. genomovars of
Pseu-
of
46, 200^
206. [38] Stackebrandt, E. and Goebel, B.M. (1994) Taxonomic note : a
ment. J. Bacteriol. 173, 2271^2277. R.,
place for DNA^DNA reassociation and 16S rRNA sequence
McCaughey, M.J. and Woese, C.R. (1996) The ribosomal da-
analysis in the present species de¢nition in bacteriology. Int. J.
[23] Maidak,
B.L.,
Olsen,
G.J.,
Larsen,
N.,
Overbeek,
Syst. Bacteriol. 44, 846^849.
tabase project (RDP). Nucl. Acids Res. 24, 82^85. [24] Van De Peer, Y., Nicola| ë, S., De Rijk, P. and De Wachter, R.
[39] Vauterin, L., Swings, J., Kersters, K., Gillis, M., Mew, T.W.,
(1996) Database on the structure of small ribosomal subunit
Schroth,
RNA. Nucl. Acids Res. 24, 86^91.
D.E.,
[25] Gutell, R.R., Weiser, B., Woese, C.R. and Noller, H.F. (1985) Comparative
anatomy
of
16S-like
ribosomal
RNA.
Prog.
M.N.,
Civerolo,
Palleroni, E.L.,
[26] Woese, C.R., Gutell, R., Gupta, R. and Noller, H.G. (1983)
Xanthomonas.
improved taxonomy of
E.L., Swings,
Xanthomonas campestris
Evolution
of protein
molecules. In : Mammalian Protein Metabolism (H.N. Munro,
Stead,
H.,
Stall,
Int. J. Syst. Bacteriol.
J.
and Kersters, K. (1991)
sulfate^polyacrylamide
pv.
gel
citri
Di¡erentiation of
strains by sodium dodecyl
electrophoresis
of
proteins,
fatty
acid analysis, and DNA^DNA hybridization. Int. J. Syst. Bacteriol. 41, 535^542.
Ed.), pp. 21^132. Academic Press, New York. [28] Olsen, G.J. (1987) The earliest phylogenetic branchings : com-
[41] Yang, P., De Vos, P., Kersters, K. and Swings, J. (1993) Poly-
paring rRNA-based evolutionary trees inferred with various
amine
techniques. Cold Spring Harbor Symp. Quant. Biol. 52, 825^
Xanthomonas.
838.
D.C.,
ête, Mara|
[40] Vauterin, L., Yang, P., Hoste, B., Vancanneyt, M., Civerolo,
bosomal ribonucleic acids. Microbiol. Rev. 47, 621^669. Cantor, C.R. (1969)
A.C.,
R.E., Vidaver, A.K. and Bradbury, J.F. (1990) Towards and
Detailed analysis of the higher-order structure of 16S-like ri-
T.H. and
Hildebrand,
40, 312^316.
Nucl. Acid Res. Mol. Biol. 32, 155^216.
[27] Jukes,
N.J.,
Hayward,
patterns
as
chemotaxonomic
markers
for
the
genus
Int. J. Syst. Bacteriol. 43, 709^714.
[42] Neefs, J.-M., Van De Peer, Y., Hendriks, L. and De Wachter,
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153 R. (1990) Copilation of small ribosomal subunit RNA sequences. Nucl. Acids Res. 16, 2237^2317. [43] Maes, M. (1993) Fast classi¢cation of plant-associated bacteria in the
Xanthomonas
genus. FEMS Microbiol. Lett. 113,
Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the blood disease (1993) Di¡erentiation of
bacterium by partial 16S rRNA sequencing : construction of olionucleotide primers for sensitive detection by polymerase chain reaction. J. Gen. Microbiol. 139, 1587^1594.
161^166.
153
[44] Seal, S.E.., Jackson, L.A., Young, J.P.W. and Daniels, M.J.
FEMSLE 7580 2-9-97
16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia
Edward R.B. Moore a *, Annette S. Kruëger a , Lysiane Hauben a b , Susan E. Seal 1 c , Roland De Baere d , Rupert De Wachter d , Kenneth N. Timmis a , Jean Swings b ;
a
;
;
Bereich Mikrobiologie, GBF-Gesellschaft fu ë r Biotechnologische Forschung, Mascheroderweg 1, D-38124 Braunschweig, Germany
b c
Laboratorium voor Microbiologie, Universiteit Gent, B-9000 Gent, Belgium
The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
d
Department Biochemie, Universitaire Instelling Antwerpen, B-2610 Antwerpen, Belgium
Received 18 March 1997 ; accepted 28 March 1997
Abstract
The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage within the Q-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species. Keywords : Xanthomonas
;
Stenotrophomonas
; 16S rRNA gene; Phylogeny
1. Introduction
Species of the genus Xanthomonas [1] are plantassociated bacteria responsible for a variety of plant bacterioses. The ¢rst such disease noted, which today would be attributed to a xanthomonad, was `yellow disease' of hyacinth plants; the causative agent was named Bacterium hyacinthi [2]. Since that ¢rst report, * Corresponding author. Tel.: (49) 531-6181-557; Fax: (49) 531-6181-411; E-mail: [email protected] 1
species have been implicated in diseases of practically all major taxa of higher plants, occurring ubiquitously throughout the world and thereby attaining considerable agricultural importance [3]. Xanthomonas species are Gram-negative rods, with polar £agella and an obligately oxidative metabolism. They are oxidase negative and do not reduce nitrates. They produce characteristic water-insoluble yellow pigments: xanthomonadins; and extracellular heteropolysaccharides: xanthan gums. The xanthan gums, due to their high speci¢c viscosity and their stability under extreme environmental conditions, Xanthomonas
Present address : Natural Resources Institute, Chatham Maritime, Kent ME4 4TB, UK.
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 5 2 - 3
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
146
Xanthomonas maltophilia, and phenotypically simXanthomonas species in many respects, occurs
have attained an importance in diverse biotechnolog-
as
ical applications [4].
ilar to
Until
recently
has
widely in soils and water and, unlike the other species listed above, is not plant-pathogenic, presenting
scribed
a
within
[5], the
Xanthomonas
comprised as many as 10 taxospecies, as validly dethe
genus
Approved
Lists
of
Bacterial
recognizably
distinct
taxon.
Originally
isolated
as an attempt to order the pathogens according to
Bacterium bookerii, this organism was reclassi¢ed as Pseudomonas maltophilia [14] before being renamed Xanthomonas maltophilia on the basis of DNA^rRNA hybridization re-
their host speci¢cities when they could not be distin-
sults,
guished reliably from one another by other criteria
cellular fatty acid composition [7]. However, incor-
Names [6] and later references [7^10]. Subsequent revisions
produced
di¡erent
taxonomic
tions, including 125 pathovars of
from pleural £uid and named
organiza-
X. campestris
[11]
quinone
type,
P. maltophilia
[12]. On the basis of extensive phenotypic character-
poration of
izations, Van den Mooter and Swings [13] de¢ned
nas
eight distinct xanthomonad phena corresponding to
reclassi¢cation of
X. campestris (type species of the genus), X. campestris pv. graminis, X. albilineans, X. axonopodis, X. fragariae, X. maltophilia, X. oryzae, and X. populi. Stenotrophomonas maltophilia, formerly classi¢ed
nas maltophilia,
recognizable species :
enzyme
characterization
into the genus
X. maltophilia,
to
Stenotrophomo-
has been proposed [16].
This study presents the results of 16S rRNA gene sequence determinations and comparisons among the type and reference strains representing the xanthomonad phena of Van den Mooter and Swings [13],
List of bacterial strains examined
X. campestris pv. campestris X. campestris pv. campestris X. campestris pv. campestris X. campestris pv. barbareae X. campestris pv. musacearum X. albilineans X. albilineans X. axonopodis pv. axonopodis X. axonopodis pv. citri X. axonopodis pv. malvacearum X. axonopodis pv. manihotis X. fragariae X. fragariae X. oryzae pv. oryzae X. oryzae pv. oryzicola X. populi pv. populi X. populi pv. populi X. translucens pv. translucens X. translucens pv. graminis X. translucens pv. graminis S. maltophilia S. maltophilia
Strain LMG
EMBL accession number
T 568
X95917
LMG 567 8004 LMG 547 R536
T
LMG 494
X95918
LMG 482
T
LMG 538
X95919
LMG 682 NCPPB 633 3172
T
LMG 708 LMG 706
X95920
T
X95921
T
X95922
LMG 5047 LMG 665
Xanthomo-
was not universally accepted [15] and a further
Table 1
Organism
and
LMG 5743 LMG 974
T
LMG 876
LMG 726
X99298
NCPPB 3040
T
LMG 958
X95923
LMG 11114
X95925
LMG : Laboratorium voor Microbiologie Gent Culture Collection, Gent, Belgium. NCPPB : National Collection of Plant Pathogenic Bacteria, Harpenden, UK. Strain 8004 is a derivative of strain NCPPB 1145. Strain 3172 provided by Dr. J.M. Clarkson, Bath University, Bath, UK. Strain R536 provided by Dr. S. Eden-Green, Rothamsted Experimental Station, Harpenden, UK. EMBL Accession Numbers are provided for nearly complete gene sequences.
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
147
Fig. 1. Evolutionary distances derived from pairwise sequence dissimilarities were calculated using the DNADIST program included in the PHYLIP package. The least-squares distance algorithm of the FITCH program, included in the PHYLIP package, was used to generate the dendrogram depicting the inferred phylogenetic positions of the species of the genus
Xanthomonas
and
a : Figure demonstrates the monophyletic position of the xanthomonads within representative species of the
ria (C. testosteroni,
a species of the
L-subclass
of the
Proteobacteria,
was included as outgroup). Additional treeing methods (i.e., neigh-
bor-joining and maximum-likelihood) supported the phylogenetic relationships depicted between er representatives of the of the genus
Q-subclass
of the
Xanthomonas (S. maltophilia
Proteobacteria
Stenotrophomonas maltophilia. Q-subclass of the Proteobacte-
Xanthomonas, Stenotrophomonas
and oth-
(not shown). b : Figure demonstrates the intrageneric relationships of the species
was included as outgroup).
the determination of sequence signatures and infer-
ously [19]. Nearly complete 16S rRNA genes were
ences of intra- and intergeneric taxonomic relation-
ampli¢ed by PCR [20] using a forward primer hy-
ships for species of
monas.
Xanthomonas
and
Stenotropho-
bridizing at the complements of positions 8^27, and a reverse primer hybridizing at positions 1541^1525 (
E. coli
was 2. Materials and methods
16S rRNA gene sequence numbering). PCR
carried
out
using
Perkin-Elmer
Cetus
DNA
thermal cyclers (Perkin Elmer Corp., Norwalk, CO) and
2.1. Bacterial strains and culture conditions
conditions
as
reported
previously
[21].
PCR-
DNAs were puri¢ed with Centricon-100 microconcentrators (Amicon GmbH, Witten, Germany) and
The bacterial strains used in this study, listed in
Taq
sequenced directly using a protocol for `
Cycle
Table 1, were cultured on GYCA medium [17] or on
Sequencing' and £uorescent dye-labeled dideoxynu-
NYGB medium [18] with shaking.
cleotides
(Applied
Biosystems
Division,
Perkin-El-
mer Corp., Weiterstadt, Germany).
2.2. Genomic DNA isolation, PCR ampli¢cation and sequence determination of 16S rDNA Genomic DNA was isolated as described previ-
In the case of some strains, a segment of the 16S
E. coli 16S rRNA gene sequence numbering), was PCRrRNA gene, corresponding to positions 40^337 (
ampli¢ed and sequenced as described previously [22].
FEMSLE 7580 2-9-97
148
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
Sequence data were aligned with reference rRNA (and rRNA gene) sequences [23,24] using evolutionarily conserved primary sequence and secondary structure as reference [25,26]. Evolutionary distances were calculated from sequence-pair similarities [27] and dendrograms were generated using a weighted, least-squares, distance method [28]. 3. Results and discussion
Nearly complete 16S rRNA gene sequences have been determined for the type and reference strains of the eight xanthomonad phena discerned by Van den Mooter and Swings [13]. PCR, using a primer pair annealing at the 5P- and 3P-termini of the 16S rRNA gene, enabled the ampli¢cation and sequence determination of 1502 nucleotide positions (W97% of the entire gene, as estimated by comparison with the 16S rRNA gene sequence of E. coli). The sequence data have been deposited with the EMBL Nucleotide Sequence database [29] under accession numbers listed in Table 1. Sequence data from each strain were compared, by cluster analysis, to reference sequence data, and supported what had been observed previously from DNA^rRNA hybridization data [30,31]: that Xanthomonas species and Stenotrophomonas maltophilia form a monophyletic cluster within the Q-subclass of
the Proteobacteria [32]. Fig. 1a,b present the estimated phylogenetic/taxonomic positions of species of the genus Xanthomonas and Stenotrophomonas maltophilia with respect to each other and within a small representation of the Q-subclass of the Proteobacteria. A primary cluster, including the species X. oryzae pv. oryzae, X. populi pv. populi and X. fragariae, was collected around the type species of the genus, X. campestris pv. campestris. The sequences of these species are too similar to be able to con¢dently predict the exact phylogenetic branching order, although the closest similarities were observed between the sequences of X. campestris pv. campestris and X. oryzae pv. oryzae (one nucleotide di¡erence) and between X. campestris pv. campestris and X. populi pv. populi (two nucleotide di¡erences). The sequences of X. oryzae pv. oryzae and X. populi pv. populi possessed three nucleotide di¡erences. The sequences of X. fragariae and X. axonopodis pv. axonopodis branched outside the primary cluster with ¢ve and ten, respectively, nucleotide di¡erences from the sequence of X. campestris. The sequence of X. albilineans was observed to branch even farther outside the X. campestris primary cluster, possessing 35 nucleotide di¡erences (2.3% sequence di¡erence) from that of X. campestris pv. campestris. The sequence di¡erence observed among all species of Xanthomonas ranged from, approximately, 0.1% (between X. campestris pv. campestris and X. oryzae
Table 2 Similarity values determined from pairwise comparisons among 16S rRNA gene sequences of Xanthomonas, Stenotrophomonas, and other Proteobacteria Q-subclass species Organism Strain % Sequence similarity 01 02 03 04 05 06 07 08 09 10 11 01. X. campestris pv. campestris LMG 568T 02. X. campestris pv. graminis LMG 726R 97.3 03. X. albilineans LMG 494T 97.5 99.5 04. X. axonopodis pv. axonopodis LMG 538T 99.3 97.2 97.9 99.7 96.9 97.3 99.1 05. X. fragariae LMG 708T 06. X. oryzae pv. oryzae LMG 5047T 99.9 97.1 97.5 99.2 99.7 07. X. populi pv. populi LMG 5743T 99.9 97.1 97.5 99.2 99.5 99.7 08. S. maltophilia LMG 958T 96.9 95.4 95.1 96.3 96.7 96.8 96.8 09. Xylella fastidiosa ATCC 35880 95.0 93.5 95.3 94.7 94.7 94.8 94.8 94.1 10. Pseudomonas aeruginosa DSM 50071T 85.2 84.9 84.9 84.8 84.9 85.3 85.1 85.1 84.3 11. Escherichia coli 83.3 82.5 83.1 83.3 83.2 83.4 83.2 81.9 82.4 84.6 T = type strain of species; R =reference strain of pathovar. 16S rRNA sequences of X. fastidiosa, P. aeruginosa and E. coli obtained from the EMBL Database of Nucleotide Sequences.
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
149
Fig. 2. Sequences for the hypervariable region V-1, including helix 6, of species and pathovars of the genus Xanthomonas and S. malto. Only those positions wherein di¡erent nucleotides were observed from those in the sequence of X. campestris pv. campestris are shown. Hyphens depict sequence gaps. philia
pv.
) to 3% (between X. albilineans and X. ) (Table 2). It is noteworthy that the species clustering most closely with X. campestris (i.e., X. oryzae and X. populi) were observed to possess approximately only 0.1% sequence di¡erence from that of X. campestris. Although no species de¢nition based upon 16S rRNA similarity exists, such a high sequence similarity has been more generally observed between strains of a species, rather than di¡erent species of a genus. Yet, during the course of the study reported here, these species were con¢rmed to constitute different DNA^DNA hybridization groups [5], thus de¢ning these organisms as di¡erent genospecies, distinct from X. campestris pv. campestris and from each other. Other examples have been reported previously wherein species of a genus are clearly recognizable by their DNA^DNA similarities below the accepted molecular criterion of 70% [33], yet which possess no, or very few, nucleotide di¡erences in their 16S rRNA (gene) primary sequences [34^37]. Such observations have important implications for the use of 16S rRNA sequence data in bacterial oryzae fragariae
taxonomy. These implications have been expressed previously by Fox et al. [35], and more recently by Stackebrandt and Goebel [38], who have pointed out certain limitations in the use of 16S rRNA sequence data for de¢ning bacterial identities. It should be recognized that, while 16S rRNA sequence comparisons are very powerful for determining intergeneric relationships across the taxonomic spectrum, at the uppermost levels of sequence similarities (i.e., 99% or higher) the resolving power of 16S rRNA to re£ect the phylogenetic relationships may not be adequate for guaranteeing that two organisms are not di¡erent species. Stenotrophomonas maltophilia, which has been proposed to constitute a genus taxonomically separated from Xanthomonas, was observed to possess approximately 97% sequence similarity to X. campestris pv. campestris. The sequence similarities observed between S. maltophilia and all Xanthomonas species analyzed ranged from 95.1% to 96.9%. While S. maltophilia clearly was seen to branch outside the cluster formed by the Xanthomonas (sensu stricto) species, the question arises as to whether the level
FEMSLE 7580 2-9-97
150
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
of 16S rRNA gene sequence dissimilarity observed between S. maltophilia and Xanthomonas species (45 to 68 nucleotide di¡erences) supports the placement of S. maltophilia in a separate genus. It may be pointed out that within the group of six Xanthomonas species analyzed in this study, X. albilineans was also observed to cluster clearly distant from X. campestris, relative to the species forming the primary cluster around X. campestris, and the question may be raised as to whether X. albilineans should also be separated from the other species of Xanthomonas in a new genus. Such observations demonstrate an additional limitation in the use of 16S rRNA sequence comparisons for analyzing bacterial taxonomic units. That is, 16S rRNA sequence comparisons clearly demonstrate that S. maltophilia is more distantly related to X. campestris (the type species of the genus Xanthomonas) than is X. albilineans, which is more distantly related to X. campestris than is X. axonopodis, etc. However, the precise borders de¢ning the `true' Xanthomonas (i.e., Xanthomonas, sensu stricto) species (necessarily including those species clustering with X. campestris pv. campestris) from species of closely related genera, presently may not be de¢ned by sequence comparisons alone. Given the inferred close phylogenetic relationships, as well as the additional molecular characterizations showing many similarities, between S. maltophilia and species of the genus Xanthomonas [39^41], an obvious question arises as to whether the ability to produce disease in plants, per se, provides the proper basis for precluding S. maltophilia from the genus Xanthomonas. In any case, the sequence data must be correlated with other characteristics, shedding light upon the relative importance of phenotypic features which, until the phylogenetic relationships have been deduced, might not be recognized as `de¢nitive' characteristics. Although this may be an `obvious' deduction, it should be recognized that the systematic value of phenotypic characteristics, such as the type of pigment or production of extracellular polysaccharide, cannot be assessed con¢dently without correlating such data with the relationships inferred through molecular analyses. Ultimately, it can be seen that the `drawing' of the borders delineating the di¡erent genera must be, necessarily, de¢ned by the overall biology of the organisms. In light of the limited number of nucleotide di¡er-
ences observed between the 16S rRNA gene sequences of the di¡erent species of Xanthomonas, the question arises as to whether the nucleotide di¡erences observed are characteristic of the respective species, and thus may able to be considered `signatures', or are merely random base changes. Therefore, at least one additional strain for each species, determined to belong to the same DNA^DNA hybridization group as the type strain of that species [5], has been analyzed by sequencing the regions of the 16S rRNA gene wherein the majority of sequence di¡erences were observed among the species of the genus Xanthomonas. Among the type strains analyzed in this study, 84 nucleotide positions, or 5.4% of the complete sequence, were seen to vary. The majority of the variable positions were observed to occur in speci¢c, more-or-less concentrated, regions: positions 75^94 includes 18 variable positions; positions 200^ 227 includes nine variable positions; positions 631^ 672 includes 11 variable positions; positions 721^743 includes seven variable positions; positions 999^1041 includes eight variable positions. At all positions given to variation, the nucleotide observed in the type strain was con¢rmed, as well, by the determination, in all cases, of the `signature' in the second strain of the respective species. Thus, at least for the limited number of species analyzed in this study, the nucleotide di¡erences observed among the species of the genus Xanthomonas appear to be species-speci¢c. This observation suggests a potential for the use of 16S rRNA gene sequence data in diagnostic analyses of Xanthomonas phytopathogens. While, in this study, we have concentrated upon the 16S rRNA gene sequence characterizations of the eight xanthomonad phena described by Van den Mooter and Swings [13], additional DNA^DNA groupings for both Xanthomonas and Stentotrophomonas have been recently described [5]. Particularly within the X .campestris complex, comprising phenotypically indistinguishable pathovars, distinct DNA^ DNA groupings have been observed, suggesting the existence of many more species than are now recognized. We have included in this study sequence determinations of the 16S rRNA area VI [42], including helix 6, which is known to be hypervariable and was seen to be the region of the most concentrated variability among Xanthomonas species, as a screening method for distinguishing the di¡erent species of
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
151
the genus Xanthomonas. Fig. 2 presents a compari-
Union for High-Resolution Microbial Identi¢cation
son of the sequence data for the region of nucleotide
(BIOT-CT-0294-SSMA).
positions 75^94 (E. coli 16S rRNA gene sequence
recipient of a fellowship through the Human Capital
positions). While a sequence in this region was ob-
Mobility
served which was characteristic of all X. campestris
by
pathovars analyzed, this sequence was also observed
the GBF and the Universiteit Gent. S. Seal thanks
in other species and their pathovars. Many di¡erent
the Overseas Development Administration for fund-
species, as de¢ned by DNA^DNA similarities, could
ing, commissioned by the Natural Resources Insti-
not be characterized by the primary sequences of this
tute (Project X0082).
the
Programme
Commission
Lysiane
Hauben
(CHRX-CT93-0194) of
European
was
the
awarded
Communities
to
hypervariable region alone, although three general groups could be de¢ned as comprised of those species clustering with X. campestris, those
clustering
References
with X. albilineans, and those clustering with S. maltophilia. This limited potential to use the hypervariable region of helix 6 to distinguish Xanthomonas species re£ects the overall close relationship of these organisms, and suggests only a limited potential for developing species- or pathovar-speci¢c gene probes or PCR primers for diagnostic analyses. However, this study analyzed a limited number of species and strains and only one hypervariable region was analyzed in any depth. Maes [43] described a screening method,
applied
to
nucleic
acid
extracts
of
wheat
seeds, using PCR to distinguish Xanthomonas species from
S.
maltophilia
and
other
species
of
bacteria
known to be phytopathogenic. Seal et al. [44] showed
[1] Dowson, W.J. (1939) On the systematic position and generic names of the Gram-negative bacterial plant pathogens. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2 100, 177^193. [2] Wakker, J.H. (1883) Vorla ë u¢ge mittheilungen u ë ber Hyacinthenkrankheiten. Bot. Zentralblatt 14, 315^317. [3] Star,
M.
(1981)
The
genus
Xanthomonas.
In :
The
Prokar-
yotes : a Handbook on Habitats, Isolation, and Identi¢cation of
Bacteria
(M.
Starr,
H.
Stolp,
H.G.
Tru ë per,
A.
Balows,
H.G. Schlegel, Eds.), p. 742, Springer, Berlin. [4] Jeanes, A. (1974) Applications of extracellular microbial polysaccharide-polyelectrolytes : review of literature, including patents. J. Polymer Sci. 45, 209^227. [5] Vauterin, L., Hoste, B., Kersters, K. and Swings, J. (1995) Reclassi¢cation of Xanthomonas. Int. J. Syst. Bacteriol. 45, 472^489.
that PCR-ampli¢cation targeting a region of the 16S
[6] Skerman, V.B.D., McGowan, V., and Sneath, P.H.A. (1980)
rRNA gene allowed detection of Ralstonia (`Pseudo-
Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30,
monas') solanacearum at levels of 100 cfu/ml. Thus, the use of 16S rRNA gene sequence targeted sites for PCR
ampli¢cation
or
gene
probing
in
diagnostic
analyses possesses a strong potential, if intrageneric (i.e.,
species
or
species
clusters)
characteristic
225^420. [7] Swings, J., De Vos, P., Van den Mooter, M. and De Ley, J. (1983) Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. Int. J. Syst. Bacteriol. 33, 409^413.
pri-
[8] Gabriel, D.W., Kingsley, M.T., Hunter, J.E. and Gottwald, T.
mary sequence regions are able to be de¢ned. Addi-
(1989) Reinstatement of Xanthomonas citri (ex Hasse) and X.
tional analyses correlating the 16S rRNA gene sequences
of
all
currently
recognized,
validly
described, species of the genus Xanthomonas are currently being carried out.
phaseoli (ex Smith) to species and reclassi¢cation of all X. campestris pv. citri strains. Int. J. Syst. Bacteriol. 39, 14^22. [9] Swings,
J.,
Van
den
Mooter,
M.,
Vauterin,
L.,
Hoste,
B.,
Gillis, M., Mew, T.W. and Kersters, K. (1990) Reclassi¢cation of the causal agents of bacterial blight (Xanthomonas campestris pv. oryzae) and bacterial leaf streak (Xanthomonas campestris pv. oryzicola of rice as pathovars of Xanthomonas or-
Acknowledgments
yzae
(ex
Ishiyama
1922)
sp.
nov.,
nom.
ref.
Int.
J.
Syst.
Bacteriol. 40, 309^311. è , M. and Ride è , S. (1992) Xanthomonas populi (ex Ride è [10] Ride
The authors gratefully acknowledge the technical
1958) sp. nov., nom. rev. Int. J. Syst. Bacteriol. 42, 652^653.
assistance of A. Arnscheidt and L. Jackson. We are
[11] Bradbury, J.F. (1984) Xanthomonas Dowson 1939, 187. In :
indebted to Gary Olsen and the Ribosomal Database Project for generously providing the computer soft-
Bergey's Manual of Systematic Bacteriology, Vol. I (Krieg, N.R. and Holt, J.C., Eds.), pp. 199^210. Williams and Wilkins, Baltimore, MD.
ware used for the sequence analysis. This study was
[12] Dye, D.W. and Lelliott, R.A. (1974) Xanthomonas. Dowson
supported, in part, by a grant from the European
1939, 187. In : Bergey's Manual of Determinative Bacteriol-
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153
152
ogy, 8th edn. (Buchanan, R.E. and Gibbons, N.E., Eds.), pp.
[13] Van den Mooter, M. and Swings, J. (1990) Numerical analysis of 295 phenotypic features of 226
è, P., Stoehr, P.J., Cameron, G.N. and Flores, [29] Rodriguez-Tome T.P. (1996) The european bioinformatics institute (EBI) data-
243^249. Williams and Wilkins, Baltimore, MD.
Xanthomonas
strains and
bases. Nucl. Acids Res. 24, 6^12. [30] [ Palleroni, N.J., Kunisawa, R. Contopoulou, R. and Douder-
related strains and an improved taxonomy of the genus. Int.
o¡, M. (1973) Nucleic acid homologies in the genus
J. Syst. Bacteriol. 40, 348^369.
monas.
[14] Hugh, R.
philia,
and
an
Ryschenkow,
Alcaligenes-like
E.
Pseudomonas malto-
(1961)
species.
J.
Gen.
Microbiol.
26,
[31] De
Pseudo-
Int. J. Syst. Bacteriol. 23, 333^339.
Vos,
P.
and
similarities of
De
Ley,
Pseudomonas
J.
(1983)
and
Intra-
and
Xanthomonas
intergeneric
ribosomal ribo-
nucleic acid cistrons. Int. J. Syst. Bacteriol. 33, 487^509.
123^132. [15] Van Zyl, E. and Steyn, P.L. (1992) Reinterpretation of the
Xanthomonas maltophilia
[32] Stackebrandt, E., Murray, R.G.E. and Tru ë per, H.G. (1988)
and taxonom-
Proteobacteria classis nov., a name for the phylogenetic taxon
ic criteria in this genus. Request for an opinion. Int. J. Syst.
that includes the `purple bacteria and their relatives'. Int. J.
taxonomic position of
Syst. Bacteriol. 38, 321^325.
Bacteriol. 42, 193^198.
Stenotrophomonas, a Xanthomonas maltophilia (Hugh 1980)
[16] Palleroni, N.J. and Bradbury, J.F. (1993) new bacterial genus for
[17] Swings, J.G. and Civerolo, E.L. (1993) Taxonomy of the Ge-
Xanthomonas.
Xanthomonas
In :
(Swings, J.G. and Civer-
[18] Daniels, M.J., Barber, C.E., Turner, P.C., Cleary W.G. and
campestris
Krichevsky,
M.I.,
E.,
Moore,
Starr,
M.R.
W.E.C., and
Murray,
Tru ë per,
H.G.
(1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37,
[34] Ash, C., Farrow, J.A.E., Wallbanks, S. and Collins, M.D.
Xanthomonas cam-
(1991)
Phylogenetic
showing altered pathogenicity. J. Gen.
vealed
by
Sawczyc, M. (1984) Isolation ofmutants of pv.
O.,
Stackebrandt,
463^464.
olo, E.L., Eds.), p. 130. Chapman and Hall, London.
pestris
Kandler, R.G.E.,
Swings et al. 1983. Int. J. Syst. Bacteriol. 43, 606^609.
nus
[33] Wayne, L.G., Brenner, D.J., Colwell, R.R., Grimont, P.A.D.,
heterogeneity
comparative
analysis
of
of
the
Bacillus
genus
re-
small-subunit-ribosomal
RNA sequences. Lett. Appl. Microbiol. 13, 202^206.
Microbiol. 130, 2447^245. [19] Marmur, J. (1961) A procedure for the isolation of deoxyri-
[35] Fox, G.E., Wisotzkey, J.D. and Jurtshuk, P., Jr. (1992) How
bonucleic acid from micro-organisms. J. Mol. Biol. 3, 208^
close is close : 16S rRNA sequence identity may not be su¤-
218.
cient to guarantee species identity. Int. J. Syst. Bacteriol. 42,
[20] Mullis, DNA
K.B. in
and
vitro
Faloona,
via
a
F.
(1987)
Speci¢c
polymerase-catalyzed
synthesis
chain
of
reaction.
Phylogenetic
Methods Enzymol. 155, 335^350. [21] Karlson,
U.,
Dwyer,
D.F.,
Hooper,
S.W.,
Moore,
E.R.B.,
Timmis, K.N. and Eltis, L.D. (1993) Two independently regulated cytochromes p-450 in a
Rhodococcus rhodochrous
strain
that degrades 2-ethoxyphenol and 4-methoxybenzoate. J. Bac-
Aeromonas
interrelationships
Plesiomonas
and
of
members
of
the
Genera
as determined by 16S ribosomal
DNA sequencing : lack of congruence with results of DNA^ DNA hybridizations. Int. J. Syst. Bacteriol. 42, 412^421. [37] Bennasar,
A.,
Rossello-Mora,
R.,
Lalucat,
J.
and
Moore,
E.R.B. (1996) 16S rRNA gene sequence analysis relative to
teriol. 175, 1467^1474. [22] Young, J.P.W., Downer, H.L. and Eardly, B.D. (1991) Pylogeny of the phototrophic
166^170. [36] Martinez-Murcia, A.J., Benloch, S. and Collins, M.D. (1992)
Rhizobium
strain BTAil by polymer-
ase chain reaction-based sequencing of a 16S rRNA gene seg-
Pseudomonas stutzeri and proposal domonas balearica sp. nov. Int. J. Syst. Bacteriol. genomovars of
Pseu-
of
46, 200^
206. [38] Stackebrandt, E. and Goebel, B.M. (1994) Taxonomic note : a
ment. J. Bacteriol. 173, 2271^2277. R.,
place for DNA^DNA reassociation and 16S rRNA sequence
McCaughey, M.J. and Woese, C.R. (1996) The ribosomal da-
analysis in the present species de¢nition in bacteriology. Int. J.
[23] Maidak,
B.L.,
Olsen,
G.J.,
Larsen,
N.,
Overbeek,
Syst. Bacteriol. 44, 846^849.
tabase project (RDP). Nucl. Acids Res. 24, 82^85. [24] Van De Peer, Y., Nicola| ë, S., De Rijk, P. and De Wachter, R.
[39] Vauterin, L., Swings, J., Kersters, K., Gillis, M., Mew, T.W.,
(1996) Database on the structure of small ribosomal subunit
Schroth,
RNA. Nucl. Acids Res. 24, 86^91.
D.E.,
[25] Gutell, R.R., Weiser, B., Woese, C.R. and Noller, H.F. (1985) Comparative
anatomy
of
16S-like
ribosomal
RNA.
Prog.
M.N.,
Civerolo,
Palleroni, E.L.,
[26] Woese, C.R., Gutell, R., Gupta, R. and Noller, H.G. (1983)
Xanthomonas.
improved taxonomy of
E.L., Swings,
Xanthomonas campestris
Evolution
of protein
molecules. In : Mammalian Protein Metabolism (H.N. Munro,
Stead,
H.,
Stall,
Int. J. Syst. Bacteriol.
J.
and Kersters, K. (1991)
sulfate^polyacrylamide
pv.
gel
citri
Di¡erentiation of
strains by sodium dodecyl
electrophoresis
of
proteins,
fatty
acid analysis, and DNA^DNA hybridization. Int. J. Syst. Bacteriol. 41, 535^542.
Ed.), pp. 21^132. Academic Press, New York. [28] Olsen, G.J. (1987) The earliest phylogenetic branchings : com-
[41] Yang, P., De Vos, P., Kersters, K. and Swings, J. (1993) Poly-
paring rRNA-based evolutionary trees inferred with various
amine
techniques. Cold Spring Harbor Symp. Quant. Biol. 52, 825^
Xanthomonas.
838.
D.C.,
ête, Mara|
[40] Vauterin, L., Yang, P., Hoste, B., Vancanneyt, M., Civerolo,
bosomal ribonucleic acids. Microbiol. Rev. 47, 621^669. Cantor, C.R. (1969)
A.C.,
R.E., Vidaver, A.K. and Bradbury, J.F. (1990) Towards and
Detailed analysis of the higher-order structure of 16S-like ri-
T.H. and
Hildebrand,
40, 312^316.
Nucl. Acid Res. Mol. Biol. 32, 155^216.
[27] Jukes,
N.J.,
Hayward,
patterns
as
chemotaxonomic
markers
for
the
genus
Int. J. Syst. Bacteriol. 43, 709^714.
[42] Neefs, J.-M., Van De Peer, Y., Hendriks, L. and De Wachter,
FEMSLE 7580 2-9-97
E.R.B. Moore et al. / FEMS Microbiology Letters 151 (1997) 145^153 R. (1990) Copilation of small ribosomal subunit RNA sequences. Nucl. Acids Res. 16, 2237^2317. [43] Maes, M. (1993) Fast classi¢cation of plant-associated bacteria in the
Xanthomonas
genus. FEMS Microbiol. Lett. 113,
Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the blood disease (1993) Di¡erentiation of
bacterium by partial 16S rRNA sequencing : construction of olionucleotide primers for sensitive detection by polymerase chain reaction. J. Gen. Microbiol. 139, 1587^1594.
161^166.
153
[44] Seal, S.E.., Jackson, L.A., Young, J.P.W. and Daniels, M.J.
FEMSLE 7580 2-9-97