FEMS Microbiology Letters 173 (1999) 359^364
Lipids and fatty acids of Burkholderia and Ralstonia species
Lesley Galbraith, Martina H. Jonsson, L. Charlotta Rudhe, Stephen G. Wilkinson *
Department of Chemistry, University of Hull, Hull HU6 7RX, UK
Received 15 December 1998; accepted 20 January 1999
Abstract
Contrary to previous reports, ornithine amide lipids are produced by some strains of Ralstonia eutropha and Ralstonia
pickettii under some growth conditions, thus eroding one of the characteristic differences from Burkholderia spp. The
proportion of phosphatidylethanolamine containing 2-hydroxy acids also varies with growth conditions, but not significantly
with growth temperature (30 or 37³C) for shake-flask cultures, unlike chemostat cultures of Burkholderia cepacia. Several fatty
acid profiles were represented among the isolated lipopolysaccharides (LPS). Burkholderia spp. typically had 14:0, 3-OH-14:0
and 3-OH-16:0 as major components, but in two strains of B. cepacia the LPS was relatively rich in 12:0 (rather than 14:0) or
contained 2-OH-14:0 (but little 3-OH-16:0). In two strains each of R. eutropha and R. pickettii, the LPS contained mainly 14:0
and 3-OH-14:0 with some 2-OH-14:0. In another strain of R. pickettii the LPS contained 2-OH-16:0 but not 2-OH-14:0. The
data may have chemotaxonomic potential. z 1999 Federation of European Microbiological Societies. Published by Elsevier
Science B.V. All rights reserved.
Keywords : Burkholderia ; Ralstonia; Fatty acid; Lipopolysaccharide; Lipid
1. Introduction
Species in the genera Burkholderia and Ralstonia
characteristically produce two forms of phosphatidylethanolamine (PE), di¡erentiated by the absence
(PE1) or presence (PE2) of 2-hydroxy fatty acids
[1^5]. In addition, Burkholderia spp. produce two
comparable forms of ornithine amide lipid (OL) in
which an amide-bound 3-hydroxy acid is itself esteri¢ed by a non-hydroxy acid (OL1) or a 2-hydroxy
acid (OL2) [2^6]. The two types of zwitterionic lipid
* Corresponding author.
Tel.: +44 (1482) 46 5484; Fax: +44 (1482) 46 6410;
E-mail: [email protected]
(PE and OL) appear to be functionally interchangeable in at least some bacteria [7^9].
Lipid pro¢les have been used extensively in bacterial chemotaxonomy and the analysis of component
fatty acids is a popular method for the rapid, computer-assisted identi¢cation of strains. Hydroxy
acids, normally derived from lipopolysaccharide
(LPS) in Gram-negative bacteria, are of particular
diagnostic value [10,11]. However, the existence of
other sources of hydroxy acids (such as PE2 and
OL) can mask signi¢cant LPS-based di¡erences in
cellular fatty acids, and growth of organisms under
controlled, de¢ned conditions may be necessary to
avoid complications from phenotypic variation [12].
This study was carried out to clarify these issues for
Ralstonia eutropha and R. pickettii (for which no
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 0 9 8 - 1
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direct data on LPS fatty acids were available) and to
permit comparisons with R. solanacearum and Burkholderia spp.
2. Materials and methods
2.1. Bacteria, growth conditions, and preparation of
cell walls
Batch fermenter cultures of the following strains
were grown in nutrient broth CM67 (Oxoid, 20 l)
with aeration at 20 l min31 and stirring at 300 rev
min31 : Burkholderia cepacia CIP 8236, B. cocovenenans LMG 11626, B. gladioli pv. agaricicola
NCPPB 3580, B. glumae LMG 1277, R. eutropha
NCIMB 40529, R. eutropha NCIMB 11842, R. pickettii A93/71 (all 24 h at 30³C); B. plantarii LMG
10908 (30 h at 30³C); B. vandii LMG 16020 (40 h
at 30³C); B. cepacia CDC 86, B. cepacia CIP 8238,
B. vietnamiensis LMG 6998, B. vietnamiensis LMG
6999, B. vietnamiensis LMG 10926 (all 24 h at 34³C);
R. pickettii CL468/83, CL391/83, CL348/84 and
T104/72 (all 24 h at 37³C) ; B. gladioli pv. gladioli
NCPPB 1891 (27 h at 37³C). In all cases the cells
were disintegrated (Dyno Mill KDL) and the wall
fractions were puri¢ed and freeze-dried. Both R. eutropha strains and R. pickettii strain A93/71 were
also grown as shake-£ask cultures (1 l) for 17 h at
30 or 37³C and the washed cells were freeze-dried.
Strain 34, an arabinose-utilising environmental strain
of B. pseudomallei provisionally designated B. thailandensis [13], was grown on tryptone soy agar (48 h
at 37³C), cells were harvested in 0.4% formol, heated
(1 h at 100³C), and acetone-dried.
2.2. Extraction of lipids and LPS
Lipids were extracted from dried cells or cell walls
by stirring them with chloroform/methanol (2:1, v/v)
for 2 h at room temperature. When necessary, polyL-hydroxybutyrate (PHB) was removed by the addition of diethyl ether (4 vol) to a solution of the lipids
in chloroform, followed by centrifugation. LPS was
obtained from defatted cell walls by using hot aqueous phenol as described [14]. LPS was mainly recovered from the aqueous phase except in the case of R.
pickettii strain A93/71.
2.3. Examination of lipid composition
Polar lipids were analysed by TLC on silica gel 60
F254 (Merck) with chloroform/methanol/water
(65:25:4, v/v/v) and chloroform/methanol/acetic
acid (65:25:10, v/v/v) as solvents, and using ninhydrin, the Dittmer-Lester reagent, iodine vapour, and
K-naphthol/sulfuric acid for the detection of components [15]. Phospholipids were also identi¢ed by paper electrophoresis of their water-soluble deacylation
products at pH 5.3 and by 31 P-nuclear magnetic resonance (NMR) spectroscopy [2,3]. PHB was identi¢ed by its 1 H-NMR spectrum. All NMR spectra
were recorded with a JEOL JNM LA400 instrument.
Acid hydrolysates were checked for the presence of
ornithine by paper chromatography and electrophoresis [2,3].
2.4. Analysis of fatty acids
Fatty acids in lipid extracts were converted into
methyl esters by mild alkaline methanolysis [2,3].
Fatty acids in LPS (and amide-bound acids in
some wall lipids) were released by acid hydrolysis,
and esteri¢ed by using methanolic HCl [16]. The
methyl esters were identi¢ed and quanti¢ed by
GLC using a capillary column of BP1 or BP5 (25
mU0.25 mm) in a Perkin Elmer Autosystem XL
chromatograph, supported by GLC-MS using a Finnigan MAT GCQ instrument.
3. Results and discussion
3.1. Polar lipid pro¢les
Lipid extracts, from which PHB was removed by
precipitation with ether, were prepared from whole
cells of R. pickettii strain A93/71 grown as shake£ask cultures at 30 or 37³C. TLC showed the absence of OL, as previously reported for the type
strain NCTC 11149 (= ATCC 27511 = EY 3254) [3^
5] and strain GIFU 1943 [6]. In addition to both
forms of PE, strain A93/71 produced phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) in
similar proportions at both growth temperatures,
and the same phospholipids (but no OL) were
present in cell-wall extracts (Table 1). But whereas
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361
Table 1
Distribution of lipid phosphorusa between di¡erent classes in Ralstonia strains
Phospholipid
DPG
PG
PEb
a
b
R. pickettii A93/71
R. eutropha NCIMB 40529
Cells (30³C)
Cells (37³C)
Walls (30³C)
Cells (30³C)
Cells (37³C)
Walls (30³C)
8
25
67
11
24
65
8
16
76
13
21
66
15
20
65
15
15
70
Results are expressed as percentages of the total peak area for signals in the
Combined data for PE1 and (where present) PE2.
PE2 was a minor phospholipid in the whole-cell extracts, the ratio of PE2:PE1 in the wall lipids from
the fermenter culture (30³C) was W2:1. Similar
phospholipid pro¢les were obtained for the walls
from fermenter cultures (37³C) of strains CL391/83
and CL348/84, but in both cases trace amounts of
OL1 and OL2 were also present, while the ornithine
amide lipids were signi¢cant components (comparable with PE2, but less than PE1) in wall extracts
from strains T104/72 and CL468/83.
Comparable studies of R. eutropha strain NCIMB
40529 showed the absence of OL from whole-cell
lipids at 30 and 37³C and the presence of a trace
of PE2 only in the 30³C culture. However, the PE2
content in wall lipids was rather higher (ratio of
PE1:PE2W10:1) and small proportions of OL1
and OL2 were also detected. Otherwise, the phospholipid pro¢les were similar to those for R. pickettii
strain A93/71 (Table 1). Shake-£ask cultures of the
type strain of R. eutropha (NCIMB 11842 = ATCC
17697 = EY 3798) gave lipid pro¢les very similar to
those from strain NCIMB 40529 (little or no PE2 or
OL, whereas a ratio of PE1:PE2W4:1 has been reported [5]). Also as for strain NCIMB 40529, PE2,
OL1 and OL2 were minor but clear components of
wall lipids from a fermenter culture of strain
NCIMB 11842. DPG was not listed as a lipid component in Japanese studies of Burkholderia and Ralstonia spp. [4,5], but has been identi¢ed in R. eutropha [17], R. pickettii [3], R. solanacearum [1] and
various Burkholderia spp. [2,3]. The results of this
study show that the production of OL is not an
absolute distinction between Burkholderia and Ralstonia spp. But unlike chemostat cultures of B. cepacia [12], shake-£ask cultures of R. eutropha and R.
pickettii do not seem to adapt to an increase in
growth temperature by elevation of the ratio
PE2:PE1.
31
P-NMR spectra.
3.2. Fatty acid pro¢les of extractable lipids
Fatty acid pro¢les were determined for the cellular
lipids of R. pickettii strain A93/71 and R. eutropha
strain NCIMB 40529 grown as shake-£ask cultures
at 30³C (Table 2). As expected, 2-hydroxy acids (derived from PE2) were only minor or trace components. The major non-hydroxy acids (16:0, 16:1 and
18:1) were those reported in previous studies [3^
5,17^21]. The proportions of 2-hydroxy acids in the
wall lipids from the two strains were signi¢cantly
higher (e.g. 2-OH-16:1, 13%; 2-OH-18:1, 7% for
R. pickettii) as expected from the greater contents
of PE2. Also, 3-OH-16:0 (the amide-bound fatty
acid in OL) was detected in acid hydrolysates of
wall extracts from R. pickettii T104/72 and R. eutropha NCIMB 40529 shown to contain ornithine.
Table 2
Fatty acid compositiona of extractable cellular lipids in Ralstonia
strains grown at 30³C
Fatty acidb
14 :0
16 :0
16 :1
17 :cy
18 :0
18 :1
19 :cy
2-OH-16:0
2-OH-16:1
2-OH-18:1
a
R. pickettii A93/71
c
Tr
28
39
1
1
25
3
Tr
1
1
R. eutropha NCIMB 40529
1
34
42
Tr
Tr
23
Tr
Ndc
Nd
Nd
Data are for ester-bound acids and are expressed as percentages
of the total peak area on GLC of the methyl esters.
b
Shorthand: number before the colon is the number of carbons;
number after the colon is the number of double bonds; cy, cyclopropane; OH, hydroxy.
c
Tr, trace ; Nd, not detected.
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3.3. Fatty acid pro¢les of LPS
strains CIP 8238 and CDC 86 (Table 3). In the former case, the LPS was relatively rich in 12:0 rather
than 14:0, and in the latter case the LPS contained a
signi¢cant proportion of 2-OH-14:0 but little 3-OH16:0. Both patterns have been described for other
strains of B. cepacia [23], and 2-OH-14:0 has also
been reported as a component of LPS from B. pseudomallei [28]. The virtual absence of 3-OH-16:0
(known to be amide-bound in other strains of B.
cepacia and B. pseudomallei [28]) is unlikely to be
determined by the growth conditions used (which
were the same for B. cepacia strains CIP 8238 and
CDC 86).
Another pro¢le (mainly 14:0 and 3-OH-14:0, with
some 2-OH-14:0) was provided by two strains each
of R. eutropha and R. pickettii (Table 3). In a further
strain of R. pickettii (A93/71), 2-OH-16:0 was
present in place of 2-OH-14:0. The absence of
3-OH-16:0 from the cellular fatty acids has been
reported for both R. pickettii [4,5,20,21] and the
type strain of R. eutropha [18], although a contradictory claim has been made for the latter [5]. The
presence of 2-OH-14:0 (which should not be derived
from PE2 or OL2 [3,4]) among the cellular fatty
LPS was extracted from defatted cell walls of various Ralstonia and Burkholderia strains, and data on
fatty acid composition of aqueous-phase LPS were
obtained (Table 3). Where checked (R. pickettii
strains A93/71 and T104/72) LPS recovered from
the phenolic phase gave results very similar to those
shown for the products from the aqueous phase.
Several fatty acid pro¢les are apparent. Pro¢les for
the LPS from B. cepacia strain CIP 8236 and all
other Burkholderia spp. examined were constructed
mainly from 14:0 (19^27%), 3-OH-14:0 (26^35%)
and 3-OH-16:0 (31^43%). This is in accord with
data for LPS from other strains of B. cepacia
[22,23], suggesting the absence of qualitative phenotypic variation despite di¡erences in the period and
temperature of growth. The presence of the two 3hydroxy acids is also suggested by surveys of cellular
fatty acids in di¡erent Burkholderia spp. (e.g.
[4,5,10,20,21,24^26]), subject to caution about their
additional derivation from OL [2,3,6] or glycolipids
[27].
Deviant pro¢les were observed for B. cepacia
Table 3
Fatty acid compositiona of LPS in Burkholderia and Ralstonia spp.
Organism
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
B.
R.
R.
R.
R.
R.
cepacia CIP 8236
cocovenenans
gladioli pv. gladioli
gladioli pv. agaricicola
glumae
plantarii
thailandensis
vandii
vietnamiensis LMG 6998
vietnamiensis LMG 6999
vietnamiensis LMG 10296
cepacia CIP 8238
cepacia CDC 86
pickettii CL468/83
pickettii T104/72
eutropha NCIMB 40529c
eutropha NCIMB 11842c
pickettii A93/71
Fatty acid
12:0
14:0
14:1(2)b
16:0
16:1(2)b
2-OH-14 :0
3-OH-14 :0
2-OH-16 :0
3-OH-16:0
Tr
0
Tr
Tr
0
Tr
Tr
Tr
Tr
Tr
2
16
3
Tr
Tr
2
2
Tr
20
27
24
25
20
22
21
33
23
19
24
2
12
28
32
18
15
19
1
Tr
Tr
Tr
Tr
Tr
1
Tr
1
2
1
Tr
3
4
Tr
3
2
3
2
12
4
3
13
2
3
Tr
2
6
1
3
11
Tr
Tr
Tr
Tr
2
2
Tr
2
2
3
2
3
3
3
4
3
3
1
0
0
0
0
0
1
0
0
0
0
0
Tr
Tr
Tr
2
Tr
0
12
9
4
14
16
0
33
30
33
35
26
30
32
32
33
29
30
37
56
59
64
59
59
56
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
40
31
37
35
38
43
40
32
38
37
39
38
1
0
0
0
0
0
a
Data are expressed as percentages of the total peak area on GLC of the methyl esters.
2-Enoic acids (dehydration products of 3-hydroxy acids).
c
Also contains 16 :1 (NCIMB 40529, 4%; NCIMB 11842, 6%).
b
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L. Galbraith et al. / FEMS Microbiology Letters 173 (1999) 359^364
acids has also been described for some strains of R.
pickettii [20,21] but not the type strain [4,5], and
again there are con£icting reports for R. eutropha
[5,18]. In neither Ralstonia species did the LPS contain 2-OH-18:1 which is present in R. solanacearum
[29,30], together with 3-OH-14:0, the only LPS-speci¢c hydroxy acid [4,5,10,24]. Di¡erent pro¢les have
been described for LPS from R. solanacearum biotype II [31].
As indicated by recent nomenclatural changes (e.g.
[4,5,13,25,26]), the classi¢cation of the bacteria included in this study is in a state of £ux, and the
data on the lipid and fatty acid pro¢les (particularly
the 3-hydroxy acids in LPS) may contribute to future
developments.
[6]
[7]
[8]
[9]
[10]
Acknowledgments
We thank Dr T.L. Pitt and Dr H.M. Aucken
(Central Public Health Laboratory, Colindale, London) for most cultures and for the cells of B. thailandensis strain 34, Dr A.J. Anderson (Department
of Biological Sciences, University of Hull) for the
culture of R. eutropha strain TRON, and Mrs B.
Worthington for NMR spectra.
[11]
[12]
[13]
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