International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2135–2139
NOTE
Printed in Great Britain
Pseudomonas antimicrobica Attafuah and
Bradbury 1990 is a junior synonym of
Burkholderia gladioli (Severini 1913) Yabuuchi
et al. 1993
Tom Coenye, Monique Gillis and Peter Vandamme
Author for correspondence : Tom Coenye. Tel : j32 9 264 51 14. Fax : j32 9 264 50 92.
e-mail : tom.coenye!rug.ac.be
Laboratory of Microbiology,
Universiteit Gent, K.-L.
Ledeganckstraat 35,
B-9000 Ghent, Belgium
Comparison of the 16S rDNA sequence of Pseudomonas antimicrobica LMG
18920T with published 16S rDNA sequences from other pseudomonads
indicated that Pseudomonas antimicrobica belongs to the genus Burkholderia,
with Burkholderia gladioli, Burkholderia glumae and Burkholderia plantarii as
its closest neighbours. DNA–DNA hybridizations confirmed that Pseudomonas
antimicrobica and Burkholderia gladioli represent the same species. Strain LMG
18920T and other Burkholderia gladioli strains were also indistinguishable by
SDS-PAGE of whole-cell proteins and had similar biochemical characteristics.
The whole-cell fatty acid composition, however, was different from that of
other Burkholderia gladioli strains. It is concluded that Pseudomonas
antimicrobica is a later synonym of Burkholderia gladioli. As Burkholderia
gladioli is known to cause infections in patients with cystic fibrosis and chronic
granulomatous disease, the eventual use of strain LMG 18920T as a biological
control agent should be approached with caution.
Keywords : Pseudomonas antimicrobica, Burkholderia gladioli, biocontrol,
Burkholderia cepacia, taxonomy
The name Pseudomonas antimicrobica was proposed in
1989 for a micro-organism isolated from the mealy bug
(Planococcoides njalensis) (Attafuah & Bradbury,
1989). The absence of arginine dihydrolase, failure to
grow at 40 mC and the fact that the organism did not
store poly-β-hydroxybutyrate in its cells suggested an
affinity with rRNA group III of the genus Pseudomonas, but its phylogenetic position was not determined (Kersters et al., 1996). Pseudomonas antimicrobica LMG 18920T showed antagonism against a
wide range of fungi and bacteria, including a number
of well-known phytopathogens (Attafuah & Bradbury,
1989) ; additional testing by Walker et al. (1996)
revealed that it was antagonistic to Botrytis cinerea, a
plant pathogen that causes grey mould on various host
plants, including numerous commercial crops. Tests
are under way to determine whether Pseudomonas
antimicrobica would be a suitable biological agent for
the control of Botrytis cinerea (Walker et al., 1996). We
compared the 16S rDNA sequence of Pseudomonas
antimicrobica LMG 18920T with the published 16S
rDNA sequences of reference strains of other pseudomonads. The high level of similarity to Burkholderia
gladioli, Burkholderia glumae, Burkholderia plantarii
and members of the Burkholderia cepacia complex
prompted the polyphasic taxonomic study described
herein.
Pseudomonas antimicrobica strain LMG 18920T (l
NCIB 9898T) and Burkholderia reference strains have
been described previously (Attafuah & Bradbury,
1989 ; Vandamme et al., 1997 ; Coenye et al., 1999a).
All strains were grown aerobically on Trypticase soy
agar (BBL) and incubated at 28 mC unless otherwise
indicated. The 16S rDNA sequence of Pseudomonas
antimicrobica LMG 18920T was retrieved from the
GenBank database (accession no. AB021384) and
compared with published 16S rDNA sequences of
other pseudomonads. A phylogenetic tree was constructed with the GeneCompar 2.1 software package
(Applied Maths), using the neighbour-joining method
(Saitou & Nei, 1987). Approximately 1460 bases were
used and all unknown bases were excluded from the
calculations.
DNA was prepared as described by Pitcher et al.
(1989) and DNA–DNA hybridizations were per-
01395 # 2000 IUMS
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T. Coenye, M. Gillis and P. Vandamme
.....................................................................................................
Fig. 1. Neighbour-joining phylogenetic tree
showing the position of Pseudomonas
antimicrobica LMG 18920T within the genus
Burkholderia, based on 16S rDNA sequence
comparisons. Pelistega europaea was used
as an outgroup. Bar, 10 % sequence dissimilarity.
.....................................................................................................
Fig. 2. Dendrogram derived from the
unweighted pair-group average linkage of
correlation coefficients between the protein
patterns of the strains studied. The correlation coefficient is expressed as percentage
similarity for convenience.
formed with photobiotin-labelled probes in microplate
wells, as described by Ezaki et al. (1989), using an
HTS7000 Bio Assay Reader (Perkin-Elmer) for the
fluorescence measurements. The hybridization temperature was 50 mC in 50 % formamide. For SDSPAGE of whole-cell proteins, strains were grown on
2136
nutrient agar (CM3 ; Oxoid) supplemented with 0n04 %
(w\v) KH PO and 0n24 % (w\v) Na HPO ;12H O
# %incubated aerobically at
# 28 %mC. The
#
(pH 6n8) and
preparation of whole-cell proteins and SDS-PAGE
were performed as described previously (Pot et al.,
1994). Densitometric analysis, normalization and
International Journal of Systematic and Evolutionary Microbiology 50
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For fatty acid methyl ester analysis, a loopful of wellgrown cells was harvested after an incubation period
of 24 h at 28 mC ; fatty acid methyl esters were then
prepared, separated and identified using the Microbial
Identification System (Microbial ID) as described
previously (Vandamme et al., 1992). Phytopathogenicity was tested as described by Gonzalez & Vidaver
(1979). Burkholderia gladioli strain LMG 2216T was
used as a positive control. Sterile distilled water was
used as a negative control.
B. plantarii LMG 10907
B. plantarii LMG 9035T
B. glumae LMG 1277
B. glumae LMG 2196T
B. gladioli LMG 18159
B. gladioli LMG 18158
B. gladioli LMG 6881
B. gladioli LMG 2216T
P. antimicrobica LMG 18920T
Reclassification of Pseudomonas antimicrobica
.................................................................................................................................................
Fig. 3. Electrophoretic protein patterns of a selection of the
strains investigated. The molecular mass markers used (lane 10)
were (from the bottom to the top) lysozyme (molecular mass,
14.5 kDa), trypsin inhibitor (20 kDa), trypsinogen (24 kDa),
carbonic anhydrase (29 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), egg albumin (45 kDa), bovine albumin
(60 kDa) and β-galactosidase (116 kDa).
interpolation of the protein profiles, as well as numerical analysis using the Pearson product moment
correlation coefficient, were performed using the
GelCompar 4.2 software package (Applied Maths).
The 16S rDNA sequence of Pseudomonas antimicrobica strain LMG 18920T was very similar to that
of Burkholderia gladioli LMG 2216T (X67038)
(97n8 %), Burkholderia glumae LMG 2196T (U96931)
(97n9 %) and Burkholderia plantarii LMG 9035T
(U96933) (98n2 %) (Fig. 1). Levels of similarity to
members of the Burkholderia cepacia complex (between 97n2 and 97n4 %) and to other Burkholderia
species (between 96n5 % and 93n2 %) were lower. Levels
of similarity to representatives of other taxa belonging
to the β-Proteobacteria were below 87n2 %. These data
unambiguously indicate that Pseudomonas antimicrobica belongs to the genus Burkholderia, with
Burkholderia gladioli, Burkholderia glumae and Burkholderia plantarii as its closest neighbours. The
DNA–DNA hybridization results revealed that the
DNA–DNA binding value between Pseudomonas antimicrobica LMG 18920T and Burkholderia gladioli
LMG 2216T was high (96 %). The levels of
DNA–DNA binding between Pseudomonas antimicrobica LMG 18920T and the type strains of Burkholderia glumae (14 %) and Burkholderia plantarii
(19 %) were low. These data unambiguously showed
that Pseudomonas antimicrobica LMG 18920T and
Burkholderia gladioli represent the same taxon. Numerical analysis of the protein patterns revealed that
the reference strains of Burkholderia glumae and
Burkholderia plantarii each formed a separate cluster
(Fig. 2). The protein patterns of the Burkholderia
gladioli reference strains and strain LMG 18920T were
very similar (Fig. 3), but, upon numerical analysis, two
separate clusters of Burkholderia gladioli strains (one
Table 1. Fatty acid composition (%) of the strains studied
.................................................................................................................................................................................................................................................................................................................
Those fatty acids for which the mean amount for all taxa was less than 1 % are not given. Mean percentages and standard
deviations are given. tr, Trace amount (less than 1 %).
Strain
14 : 0
16 : 0
17 : 0
cyclo
16 : 1
2-OH
16 : 0
2-OH
16 : 0
3-OH
18 : 0
19 : 0
cyclo ω8c
18 : 1
2-OH
Summed feature :*
3
Pseudomonas antimicrobica
LMG 18920T
Burkholderia gladioli (4)†
Burkholderia glumae (3)†
Burkholderia plantarii (3)†
4n7
20n4
4n9p0n2
4n7p0n1
4n5p0n1
29n0p1n1
27n0p2n4
33n6p2n7
8n2
17n2p1n9
11n8p0n7
25n2p0n1
1n9
1n5
5n5
1n9
1n5p0n2
1n1p0n2
tr
3n2p0n3
3n0p1n2
1n4p0n1
7n1p1n0
5n6p0n4
6n3p0n5
tr
tr
1n2p0n1
6n6
10n0p2n7
8n5p1n1
10n3p2n1
2n3
7n0
3n5p0n7
3n5p0n3
4n4p2n0
6n8p1n1
6n1p0n6
5n2p0n8
4
10n8
5n1p2n3
4n5p0n2
1n2p0n4
7
29n2
11n0p3n7
22n2p1n7
5n3p1n3
* Summed feature 3 comprises 14 : 0 3-OH, 16 : 1 iso I, an unidentified fatty acid with an equivalent chain-length value of 10n928, or
12 : 0 ALDE, or any combination of these fatty acids. Summed feature 4 comprises 16 : 1 ω7c or 15 iso 2-OH, or both. Summed feature
7 comprises 18 : 1 ω7c, 18 : 1 ω9t, or 18 : 1 ω12t, or any combination of these fatty acids.
† Data are from Vandamme et al. (1997).
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2137
T. Coenye, M. Gillis and P. Vandamme
including strain LMG 18920T) were formed (Fig. 2).
The obvious reason for this subdivision is a highdensity protein band in the region between 35 and
43 kDa, which is present in some strains but absent in
others, as discussed previously (Coenye et al., 1999a).
The type or reference strains of other Burkholderia
species occupied separate positions. The following
fatty acids were present in strain LMG 18920T : 14 : 0,
16 : 0, 17 : 0 cyclo, 16 : 1 2-OH, 16 : 0 2-OH, 16 : 0 3-OH,
18 : 0, 19 : 0 cyclo ω8c, 18 : 1 2-OH, summed feature 3
(comprising 14 : 0 3-OH, 16 : 1 iso I, an unidentified
fatty acid with an equivalent chain-length value of
10.928, or 12 : 0 ALDE, or any combination of these
fatty acids), summed feature 4 (comprising 16 : 1 ω7c
or 15 iso 2-OH or both) and summed feature 7
(comprising 18 : 1 ω7c, 18 : 1 ω9t, or 18 : 1 ω12t, or any
combination of these fatty acids). Summed feature 3,
summed feature 4 and summed feature 7 probably
correspond to 14 : 0 3-OH, 16 : 1 ω7c and 18 : 1 ω7c,
respectively, as these fatty acids have been reported in
Burkholderia strains before (Stead, 1992 ; Vandamme
et al., 1997). The fatty acid profile of LMG 18920T is
most similar to those of Burkholderia gladioli, Burkholderia glumae and Burkholderia plantarii. When
compared with other Burkholderia gladioli strains,
strain LMG 18920T is characterized by lower amounts
of 16 : 0 and 17 : 0 cyclo and a higher amount of
summed feature 7 (Table 1). Strain LMG 18920T
caused a soft-rot in onions that was comparable to the
rot caused by Burkholderia gladioli LMG 2216T.
Comparison of the previously published biochemical
characteristics of strain LMG 18920T (Attafuah &
Bradbury, 1989) and Burkholderia gladioli (Palleroni,
1984 ; Gillis et al., 1995 ; Coenye et al., 1999a) indicated
that the phenotypic characteristics of strain LMG
18920T and Burkholderia gladioli were very similar, the
only differences being the inability of strain LMG
18920T to utilize adipate, malate and m-tartrate and to
accumulate poly-β-hydroxybutyrate, and the inability
of Burkholderia gladioli to hydrolyse aesculin. However, care should be taken when comparing these data,
as different methodologies have been used in these
different studies. Our polyphasic taxonomic study
unambiguously demonstrated that Pseudomonas antimicrobica and Burkholderia gladioli represent the same
taxon ; we conclude that Pseudomonas antimicrobica is
a junior synonym of Burkholderia gladioli.
Strain LMG 18920T has attracted attention as a
potentially useful organism in the biocontrol of
Botrytis cinerea, the causative agent of grey mould
(Walker et al., 1996). However, several Burkholderia
gladioli strains have been involved in infections in
compromised human hosts and patients with cystic
fibrosis and chronic granulomatuous disease are
especially prone to infection with this micro-organism
(Christenson et al., 1989 ; Ross et al., 1995 ; Hoare &
Cant, 1996 ; Khan et al., 1996). In addition, Burkholderia gladioli is a well-known plant pathogen that is
traditionally isolated from Gladiolus spp. and Iris sp.
(Palleroni, 1984). It is currently impossible to dis2138
tinguish between those Burkholderia gladioli isolates
that are potentially useful for biocontrol purposes and
those isolates capable of causing infections in compromised human hosts (Christenson et al., 1989 ;
Coenye et al., 1999a, b) ; consequently, the eventual
use of this organism as a biological control agent
should be approached with caution.
Acknowledgements
T. C. acknowledges the support received from the Vlaams
Instituut voor Bevordering van Wetenschappelijk-technologisch Onderzoek in de Industrie (Belgium) in the form of a
bursary for advanced study. P. V. and M. G. are indebted to
the Fund for Scientific Research–Flanders (Belgium) for a
postdoctoral fellowships and for research and personnel
grants, respectively. We acknowledge the financial support
received from the Cystic Fibrosis Trust (UK) (grant RS15)
and we wish to thank Dirk Dewettinck, Severine Laevens
and Urbain Torck for excellent technical assistance.
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