Microbial. Immunol.

, 39(11), 897-904, 1995

Transfer of Two Burkholderia and An

Alcaligenes Species to Ralstonia Gen. Nov.:
Proposal of Ralstonia pickettii (Ralston, Palleroni
and Doudoroff 1973) Comb. Nov., Ralstonia
solanacearum (Smith 1896) Comb. Nov. and
Ralstonia eutropha (Davis 1969) Comb. Nov.
Eiko Yabuuchi*,1, Yoshimasa Kosako2, Ikuya Yano1, Hisako Hotta1, and Yukiko Nishiuchi1
1DepartmentofBacteriology , Osaka CityUniversityMedicalSchool,Osaka, Osaka 545,Japan, and 2JapanCollection
of
Microorganisms, The Institute of Physical and Chemical Research (RIKEN),Wako,Saitama 351-01, Japan

Received August 4, 1995. Accepted August 22, 1995

Abstract: Based on the results of phenotypic characterization, cellular lipid and fatty acid analysis, phy-
logenetic analysis of 16S rDNA nucleotide sequences and rRNA-DNA hybridization, Burkholderia picket-
tii, Burkholderia solanacearum and Alcaligenes eutrophus are transferred to the new genus Ralstonia, and
Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896)
comb. nov., and R. eutropha (Davis 1969) comb. nov. are proposed. The type species of the new genus is R.
pickettii. Type strain of R. pickettii is ATCC 27511T, of R. solanacearum is ATCC 10696T, and of R.
eutropha is ATCC 17697T.

Key words: Ralstonia gen. nov., Raistonia pickettii, Ralstonia solanacearum, Ralstonia eutropha

Based on sequence similarity of 16S rRNA genes, posed a new species Burkholderia vietnamiensis and
Li et al (9) suggested two related genera within the two new combinations,Burkholderia andropogonis and
homology group II pseudomonads. The generic name Burkholderia cocovenenans. They also emended the
BurkholderiaYabuuchiet al, 1993 (4, 19) was proposed descriptionof the genus accompanied with the removal
for the seven speciesof RNA homologygroup II (10) of of B. pickettii and B. solanacearum from the genus
genus Pseudomonas Migula 1894. It was based on the Burkholderia.
results of cellular lipid and fatty acid analyses and 16S The homologies between the type strains of labeled
rRNA nucleotide sequence analysis of five type and DNA from B. pickettii to unlabeled DNA from B.
two reference strains of seven species. Kosako con- solanacearumand viceversa were 41% and 45%,respec-
firmed previous results of DNA-DNA homologies of tively (19). The Tm(e) between P solanacearum ATCC
each type strain of B. caryophylli and B. gladioli to the 10696Tto P cepacia Ballard 717Twas 76.1 (3), those of
remaining fiveBurkholderia species (Kasako, personal B. cepacia LMG 1222Tto [B.] solanacearum LMG
communication). Urakami et al (18) added two new 2299Tand [B.] pickettii ATCC 27511Twere 75.2 and
combinations,B. plantarii and B. glumae, and a new 75.3, respectively (6).
speciesB. vandii. Davis (1) validly transferred illegitimately named
As Palleroni(10) has demonstratedtwo DNA homol- species Hydrogenomonaseutropha to the genus Alcali-
ogy groupswithinRNA homologygroup II, Yabuuchiet genes. Alcaligenes eutrophus Davis was listed in the
al (19) also noted B. pickettii (12) and B. solanacearum Approved Lists of Bacterial Names. Gillis et al (6)
(14, 15) are similar to each other and different from the demonstrateda close relationshipamong the strainsof A.
remainingfiveBurkholderia species in DNA homology, Abbreviations: AL-X, unknown aminolipid; ca., circa; comb.
cellular lipid composition,and phylogeneticanalysis of nov., combinatio nova, new combination; gen. nov., genus nova,
16S rRNA nucleotide sequences. Gillis et al (6) pro- new genus; GL, glycolipid; OL, ornithine-lipid; PE, phos-
*Address correspondence to Dr . Eiko Yabuuchi, Department of phatidylethanolamine; PG, phosphatidylglycerol; rRNA, ribo-
Bacteriology, Osaka City University Medical School, 1-4-54, somal ribonucleic acid; T, type strain for the species; TLC, thin-
Abeno-ku, Asahi-machi, Osaka, Osaka 545, Japan. layer chromato-graphy or -gram.

897
898 E. YABUUCHI ET AL

Table 1. Phenotypic features of 6 type strains of 3 Ralstonia sp., 2 Burkholderia sp. and A. faecalis
 NOTES
Table 2. Carbohydrate assimilation tests to differentiate Ralstonia sp. from Burkholderia sp. and Alcaligenes faecalis
eutrophus, B. pickettii and B. solanacearum by dendro-
grams obtained from 137 auxanographic tests and Tm(e).
In the dendrogramof auxanographictests, the cluster of
A. eutrophus showed 73% similarity with B. pickettii
cluster, furthermore the Tm(e) value of the hybrid with
rRNAfrom the type strainsof A. eutrophusto B. cepacia
and B. solanacearum were 75.0 and 75.5 C, respective-
ly. These facts indicate a close relationship among the
strainsof B. pickettii,B. solanacearum,andA. eutrophus,
phenotypicallyand phylogenetically.
In additionto furtherstudies on morphological,phys-
iological and biochemical characterization, cellular
lipids-fatty acids analysis, and phylogenetic analysis
were carried out to clarify the taxonomic position of
these three species.
Phenotypic characteristics of B. pickettii and B.
solanacearum have been described previously (19).
Cells of the type strain of A. eutrophusis Gram-negative,
well-flagellatedperitrichous straight rod with rounded
ends. They are single,in pairs, and often in chainsof five
or more than 10 cells. The strain is able to grow at 37 C
and 41 C. Though the organism is non-fermentative, it
grows well ca. 7 mm beneath the surface of OF sugar
media and growth also occurs up to the bottom of the
tube along the stab line similar to the growth of Kleb-
siella pneumoniae stabbed in a semisolid agar tube.
Such growth behavior was seen in OF sugar tubes neg-
ative for oxidative acid production. Davis described
that the organismdoes not ferment any carbohydrate. In
OF basal medium (Difco) added with 1% carbohydrate,
the organism oxidized fructose, glucose, glycerol, D-
xylose, lactose, and inulin. Because some other strains
899
simultaneously tested were unable to grow at higher
temperatures, these were tested at 28 C and oxidative
acid production was usually observed on the 3rd to 5th
day of incubation. Decarboxylasebase Moeller (Difco)
supplemented with 1% monochlorate of L-lysine or L-
omithine became yellow,together with control medium
without amino acid. The argininedehydrolasetest in this
medium was weakly but definitely positive on the 3rd
day of incubation. The organism hydrolyzed Tween
80, but not gelatin,esculin and urea. The featuresof the
type strainsof three Ralstonia species are listed in Table
1, in comparison with those of B. cepacia, B. andro-
pogonis and Alcaligenes faecalis, the type species of
genusAlcaligenes. As describedbelow, the type strainof
B. andropogonis is included in this table, because the
two-dimensionalthin-layerchromatogram(TLC)revealed
PE (phosphatidylethanolamine)-1 and -2 spots which
were thought to be characteristicfor the strains of Burk-
holderia species. Although it was not possible to find
any suitablefeature to differentiateRalstonia from Burk-
holderia at the generic level (Table 1), four kinds of
carbohydrates,galactose,mannitol,mannoseand sorbitol,
not assimilatedby three Ralstonia specieswere utilized
by eleven Burkholderia species including B. andro-
pogonis (Table2).
As described above, the presence of two kinds of
ornithine-lipids,OL-1 and OL-2, were characteristic of
cellular lipids of Burkholderia species (19) (Fig. 1E),
while cellular lipids of R. pickettii, R. solanacearum
and R. eutropha failed to reveal these spots (Fig. 1,
A,B,C) (Table3). In contrast to the other Burkholderia
species, the cellular lipids chromatogram of B. andro-
900 E. YABUUCHI ET AL

Fig. 1. Two-dimensional thin-layer chromatograms of cellular lipids of type strain of three Ralstonia and two Burkholderia species. Sol-
vent system for the 1st dimension: C-M-W=65:25:4(v/v), the 2nd C-M-A-W=100:20:12:5(v/v). {In solvent system, C: chloroform,
M: methanol, W: distilled water, A: acetic acid}. A: Ralstonia pickettii EY 3254T,B: Ralstonia solanacearum EY 2181T,C: Ralstonia
eutropha EY 3298T,D: Burkholderia andropogonis EY 3792T,and E: Burkholderia cepacia EY 645T. PE, phosphatidylethanolamine;
PG, phosphatidylglycerol; LPE, lysophosphatidylethanolamine, OL, ornithine-lipid; GL, glycolipid.

Table 3. Cellular lipid composition of type or reference
Table 4. Cellular fatty acid composition (%) of 3 Ralstonia sp., 2 Burkholderia species and A. faecalis
pogonis showed neither OL-1 nor OL-2 spots (Fig. 1D).
The TLC of A. faecalis showed spots of PG, PE-1, and
OL-1, while PE-2 and OL-2 were not demonstrated
(Fig. 1F).
Cellularfatty acid analysis by gas-liquid chromatog-
raphy was reported as useful for the grouping of
Pseudomonasspecies (16). The three Ralstonia species
failed to demonstratethe peaks of C19 cyclopropanoic
acid (CPA)and 20H-C19CPA.B. andropogonisis same
with threeRalstonia sp. and differentfrom B. cepacia in
the absence of 2-hydroxy acids of C16:1, C18:1 and
C19 cyclopropanoic(Table4).
As shown in Table 5, the almost complete 16S rDNA
sequence data of type strains of three species were
NOTES 901
strains of seven Burkholderia species
obtained from DDBJ (Institute of Genetics, Mishima,
Shizuoka, Japan). The CLUSTAL W program (13)
which is the improved version of the CLUSTAL V (7)
was used to align multiple sequences, to calculate
nucleotidesubstitutionrates (Kn. values)(8) and to con-
struct a neighbor-joining phylogenetic tree (13). To
determine the stability of our phylogenetic tree, the
sequence data were sampled 1,000 times for bootstrap
analysis (5). Alignment positions that included gaps
and/or unidentifiedbases were not taken into consider-
ation for the calculation. Brevundimonas diminuta,
Comamonas testosteroniand Pseudomonas aeruginosa
were included in our group of representativesof the a-,
β-,and Y-subclasses of Proteobacteria. As shown in Fig.
902 E. YABUUCHI ET AL

Table 5. Strains used in the 165 rDNA sequence analysis

Fig. 2. Phylogenetic tree derived from 16S rRNA sequence analysis. The relationship between Ralstonia gen. nov. and genus Burk-
holderia is demonstrated. A remote distance of R. eutropha comb. nov. from A. faecalis, type species of genus Alcaligenes is also shown.

2, we obtained two clearly separate branches
β-subclass. The first is the Burkholderia branch
includes B. cepacia, the type species for the genus Burk-
holderia, the second is the Ralstonia branch composed of
R. pickettii, R. solanacearum, and R. eutropha. As
shown in Table 6, the Kn. value of R. pickettii to R.
solanacearum and R. eutropha are 0.023 and 0.037,
respectively, whereas Knucbetween R. eutropha and A.
faecalis is 0.103. Those of B. cepacia to R. pickettii, R.
solanacearum, and R. eutropha are 0.078, 0.077, and
0.080, respectively. These values clearly demonstrate
that R. pickettii, R. solanacearum, and R. eutropha are
three taxa belonging to the same genus. Thus, we here-
within the in propose Ralstonia gen. nov., Ralstonia pickettii
which (Ralston, Palleroni and Doudoroff 1973) comb. nov.,
Ralstonia solanacearum (Smith 1896) comb. nov., and
Ralstonia eutropha (Davis 1969) comb. nov.
Description of Ralstonia gen. nov. (Ralston. ia. M.L.
dim. ending-ia; M.L. fem. n. Ralstonia named after E.
Ralston, American bacteriologist who first described
Pseudomonas pickettii and suggested a taxonomicrela-
tionship to P solanacearum based on DNA homology
between them).
Cells of speciesof this genus are Gram-negativerod-
shaped, motile with single polar or peritrichous flagella
or non-motile without flagella. Oxidase activity is van-
 NOTES
Table 6. Kmucvalues among thirteen species of six genera
able by species. Monosaccharides, disaccharides and
polyalcohols are oxidized and assimilated as a sole
source of carbon and energy, as reported previously
(15). The absenceof assimilationof galactose,mannitol,
mannose, and sorbitol by the type strain of three Ral-
stonia speciesis a differentialphenotypicmarker to dis-
tinguish strains of Ralstonia sp. from those of Burk-
holderia species (Table 2). Cellular lipids of the, type
strainsof three Ralstonia species contain phosphatidyl-
ethanolaminepossessing2-hydroxy fatty acid at the sn-
2 position of the glycerol moiety, similar to those of
Burkholderiaspecies(19). As shown in Fig. 1, the type
strainsof three Ralstonia species failed to demonstrate
two kinds of ornithine-lipidson two-dimensionalthin-
layerchromatographyand to reveal C19 cyclopropanoic
acid in cellular fatty acids (Table 4). The presence of
these cellular lipids and fatty acid components were
clearly demonstrated in Burkholderia cepacia and the
other fiveBurkholderia species (19).
Flagellarmorphologyhas long been one of the impor-
tant features for the classification and identification of
Gram-negative bacterial strains. However, it is now
known that certain species lose their flagellawhen they
becomefacultativelyparasitic. For example,peritrichous
Escherichia coli might change to atrichous Shigella
speciesor polar multitrichousBurkholderiapseudomal-
lei to atrichousB. mallei. It is well-knownthat E. coli is
geneticallythe same speciesas Shigella sp., as well as B.
pseudomallei and B. mallei. Cells of certain species of
marine bacteria possess both sheathed polar monotri-
chous flagella and unsheathed lateral flagella when
grown on the surface of semisolid media incubated at
around 20 C, while they fail to produce lateral flagella
when grown in liquid media at 37 C. Thus, the signifi-
cance of flagellar morphology in the classification or
identificationat generic or familiar levels seems to be
reduced. Species with differentflagellar morphologyin
one genus, Ralstonia gen. nov. could not be eliminated
903
from the genus.
Strains of R. pickettii are isolated from various clini-
cal specimens and hospital environmentswith doubtful
pathogenicity. The heterogeneity of R. solanacearum
strains (11) remained to be solved taxonomically. R.
solanacearum is known to be pathogenic for Solanum
lycopersicum (tomato, potato, tobacco). Also isolated
from Casuarina, Strelitzia, ginger, banana, Heloconia,
peanut and Pelargonium (M. Gillis, personal communi-
cation). The guanineand cytosinecontentof DNA of the
type strainsof three species are 64.0, 66.6 (19) and 66.5
mol% determinedby HPLC method (17). Type species
is Ralstonia pickettii (Ralston, Palleroni and Doudoroff
1973) comb. nov.
Description of the type strains of Ralstonia pickettii
and Ralstoniasolanacearumare the same as those report-
ed previously (18). The phenotypic features, cellular
lipids-fatty acids composition, and the results of phylo-
genetic analysis of the type strain of Ralstonia eutropha
are describedin this report. The type strain of R. pickettii
is ATCC27511,of R. solanacearum is ATCC 10696,and
of R. eutropha is ATCC 17697.
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