cepacia (Palleroni and Holmes 1981) comb. nov.

Transcription

1 Microbiol. Immunol. Vol. 36 (12), , 1992 Proposal of Burkholderi a gen. nov. and Transfer of Seven Species of the Genus Pseudomonas Homology Group II to the New Genus, with the Type Species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Eiko YABUUCHI,*,1 Yoshimasa KOSAKO,2 Hiroshi OYAIZU,3 Ikuya YANO,1 Hisako HOTTA,1 Yasuhiro HASHIMOTO,4 Takayuki EZAKI,4 and Michio ARAKAWA5 Department of Bacteriology, Osaka City University Medical School, Osaka, Osaka 545, Japan, 2Japan Collection of Microorganismș The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama , Japan, 3Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, 4Department of Microbiology, and 5Second Department of Internal Medicine, Gifu University School of Medicine, Gifu, Gifu 500, Japan (Accepted for publication, October 15, 1992) Abstract Based on the 16S rrna sequences, DNA-DNA homology values, cellular lipid and fatty acid composition, and phenotypic characteristics, a new genus Burkholderia is proposed for the RNA homology group II of genus Pseudomonas. Seven species in this group were transfered to the new genus. Thus seven new combinations, Burkholderia cepacia (Palleroni and Holmes 1981), Burkholderia mallei (Zopf 1885), Burkholderia pseudomallei (Whitmore 1913), Burkholderia caryophylli (Burkholder 1942), Burkholderia gladioli (Severini 1913), Burkholderia pickettii (Ralston et al 1973) and Burkholderia solanacearum (Smith 1896) were proposed. The genus Pseudomonas has been heterogeneous since Migula first named it in 1894 (12), and designated and described the species associated with the genus in 1895 (13). Gradually, Gram-positives, fermenters, or peritrichous flagellates were removed from the genus (3). In 1971, Palleroni and Doudoroff (14) had already noted remote relationship of Pseudomonas solanacearum to strains of other Pseudomonaspecies, based on the results of phenotypic characterization and DNA- DNA homologies. Palleroni et al (15) divided the genus into five groups of species according to the results of rrna-dna hybridization. In this report, Pseudomonas aeruginosa (Schroeter 1872) Migula 1900, the type species of the genus, was placed in homology group I. P. acidovorans and P. testosteroni, in homology group III (15), were transferred by Tamaoka et al (27) to the revived genus Comamonas De Vos et al 1985 (2). P. maltophilia Hugh 1981 in homology group V is now Xanthomonas maltophilia (Hugh 1981) Swings et al 1983 (25). Pseudomonas paucimobilis Holmes et al 1977 (AL) was removed from the genus, and a new genus Sphingomonas and a new combination Sphingomonas paucimobilis were proposed together with three other 125 1

2 125 2 E. YABUUCHI ET AL new species and two genospecies (29). Phylogenetic analysis of 16S rrna sequence of Proteobacteria demonstrated supportive evidence to make certain homology groups of the genus Pseudomonas as separate genera (18, 28). Thus we herein propose a new genus Burkholderia constituted of seven species which have been placed in the Pseudomonas homology group II. MATERIALS AND METHODS Bacterial strains used. The type strains for the species of P. cepacia, P. mallei, P. pseudomallei, P. pickettii, P. solanacearum, and reference strains of P. gladioli and P. caryophylli were used. Histories and corresponding number of strains are listed in Table 1. Type strain of P. aeruginosa EY 274 was used as taxonomic control. Strain(s) of P. gladioli (8), P. caryophylli and P. solanacearum were grown at 30 C. Cultures of the 5 other strains including P. aeruginosa EY 274 were incubated at 35 C unless otherwise stated. Phenotypic characteristics. Morphological, physiological and biochemical characteristics were examined as described previously (29). Active motility was microscopically observed in wet mount preparation. Defect of motility was determined after three passages of non-motile strain on semisolid motility agar plate. The semisolid agar used here was composed with Bacto-casitone 5 g, Bacto-yeast extract 3 g and Bacto-agar 3 g in 1,000 ml distilled water. Each 20 ml of sterilized and cooled medium was poured into Petri dish and solidified into semisolid agar plate. Each plate was inoculated at its center with one loop-full broth culture of non-motile strain at room temperature. If any diffuse spreading growth did not appear within seven days, organism at the edge of growth on semisolid agar plate was transferred to the same new plate and the culture proceeded as described above. Flagellar morphology was determined by Leifson flagella stain. Deep-browning of eculin agar slant was recorded as positive in esculin hydrolysis. Nutrient gelatin medium (Difco) was inoculated with test strain by stabbing with straight wire and incubated at 20 C. Whenever small depression of the medium at inoculation site was observed, reaction was read as positive. In addition to decarboxylase base Moeller (Difco) supplemented with 1% L-lysine monohydrochloride, Carlquist ninhydrin test was used to detect lysine-decarboxylase activity of the test strains. If the color of inoculated and incubated Oxidation-Fermentation (OF) basal medium (Difco) became definitely alkaline, and those supplemented with a carbohydrate of any kind remained unchanged, the acid production from supplemented carbohydrate was recorded as positive. Other physiological and biochemical tests were performed as described previously (29). Assimilation of carbon and energy sources. The 147 substrates as carbon and energy sources were tested by using API 50CH (carbohydrates), LRA 50A0 (organic acids), LRA 50AA (amino acids), and LRA 150 medium, according to the instruction manual from the manufacturer (biomerieux S.A., Marcy-l'Etoile, France). The results were read after 24 hr, 48 hr and 6 days incubation. Cellular lipids analysis. Cellular lipids of wet cells of seven strains from 24 hr

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4 125 4 E. YABUUCHI ET AL HI agar plate culture were extracted with chloroform methanol (C-M, 1 : 2, v/v), and sonicated with Branson sonifier for 3 min. The homogenate thus obtained was separated by the addition of 1/4 vol. of distilled water and the lipids in lower phase were dried with a rotary evaporator. The dried lipids were dissolved in C-M (2: 1, v/v) at a concentration of 1 mg/ml. Lipids were separated by two-dimensional thin-layer chromatography (TLC) on silica gel G plate (UniplateTM, Analtec Inc., Newark, Del., U.S.A.) with solvent systems, C-M-Water (W) (65: 25 : 4, v/v) for the first dimension, and C-M-Acetic acid(a)-w (100 : 20 : 12 : 5, v/v) for the second. Spots of the plates were visualized with 0.1% ninhydrin solution in ethanol, Dittmer reagent, of 50% sulfuric acid, and compared with standard substances. In order to determine the positional distribution of fatty acids in the two major ninhydrin-positive phospholipids, PE-1 and PE-2, these spots were eluted from plate and digested with phospholipase A2 from porcine pancreas. The resultant lysophospholipids and fatty acids were analyzed as described below. Cellular fatty acids analysis. For the analysis of cellular fatty acid composition, wet cells (24 hr culture) of seven strains on HI agar (Difco) plates were hydrolyzed with 10 times volume of 15% NaOH in 50% methanol at 95 C for 20 hr. Fatty acids were extracted from acidified reaction mixtures with n-hexane, and methylated. with diazomethane. Fatty acid methyl esters were analyzed with a gas chromato- graph (5890 series II, Hewlett-Packard Co., Avondale Division, Penn., U.S.A.) equipped with a flame ionization detector and a 30 m x 0.25 mm fused silica capillary column with 5TM 2380 (Superco Inc., Bellafonte, Penn., U.S.A.) as a stationary phase. Injection and detector were maintained at 250 C. The column temperature was 120 C for 2 min at first, then raised to 200 C at 2 C/min without final holding. Fatty acid methyl esters were identified by retention time comparison and further confirmed by gas chromatography/mass spectrometry (GC/MS), by using JEOL model SX-102 and the same HP gas chromatograph described above. Fatty acid methyl esters were quantitated with HP3396 series II integrator connected to HP 5890 series II gas chromatograph. Base ratio and DNA-DNA hybridization. DNAs of test strains were extracted by Marmur's method (11) with minor modification, and ribonuclease A (Sigma) was used for enzymatic hydrolysis of RNA. G+ C contents of DNAs were determined by nuclease P1 and alkaline phosphatase reversed-phase high-performance liquid chromatography (HPLC) method described by Tamaoka and Komagata (26). Enzymatic hydrolyses of nucleotides into nucleosides were performed as described previously (29). DNAs from the type strain of P. aeruginosa and type and reference strains of seven Burkholderia species were hybridized with each other by fluorometric method in microdilution wells according to the description by Ezaki et al (5), since evidence showed the usefulness of this non-isotopic method as an alternative to membrane filter hybridization with radioisotopes to determine genetic relatedness among bacterial strains. Hybridization was performed under the presence of 50% formamide at 50 C.

5 BURKHOLDERIA GEN. NOV. FOR THE PSEUDOMONAS HOMOLOGY Sequencing of 16S rrna. The sequences of 16S rrna of B. mallei, B. pseudomallei, B. gladioli, B. pickettii and B. solanacearum were determined by the polymerase chain reaction (PCR) (21) according to the following method. Total DNAs were extracted and purified by repeated phenol treatment ; this was followed by RNase treatment from ca. 50 mg wet weight cells. The 16S rrna coding region of DNA was amplified from the total DNA by the use of two primers attaching to positions from 10 to 25 (5'AGTTTGATCCTGGCTC OH 3') and 1541 to 1525 (5'AAGGA- GGTGATCCAGCC OH 3') (in the Escherichia coli numbering system) and Taq DNA polymerase (Cetus, Inc., U.S.A.). The amplified DNA was purified by the glass beads method by the use of Easytrap Kit (Takara Shuzo, Inc., Japan). Then, the purified DNA was sequenced by Sequenase Kit for 355 datp (United Biochemical, Inc., U.S.A.) with the primers 5'AGTTTGATCCTGGCTC OH 3' (same sequence to 10-25), 5'GTGTTACTCACCCGT OH 3' (complementary to ), 5'TACGGGAGGCAGCAG OH 3' (same to ), 5'CTGCTGCCT- CCCGTAG OH 3' (complementary to ), 5'GTGCCAGCAGCCGCGG OH 3' (same to ), 5'ACCGCGGCTGCTGGC OH 3' (complementary to ), 5'TCTACGCATTTCACC OH 3' (complementary to ), 5'ATTA- GATACCCTGGTA OH 3' (same to ), 5'GTCAATTCCTTTGAGTTT OH 3' (complementary to ), 5'GCAACGAGCGCAACCC OH 3' (same to ), 5'AGGGTTGCGCTCGTTG OH 3' (complementary to ), 5'TGTACACACCGCCCGT OG 3' (same to ), 5'ACGGGCGGTGTG- TAC OH 3' (complementary to ), 5'GGCTACCTTGTTACGA OH 3' (complementary to ), and 5'AAGGAGGTGATCCAGCC OH 3' (complementary to ). The sequences of positions from 27 to 997 and positions from 1149 to 1526 were determined by the above primers. Sequence of 16S rrna of B. solanacearum at 157 positions ( ) was determined by the method described previously (29). Phylogenetic analysis. Sequences of 16S rrna of Agrobacterium tumefaciens, E. coli, P. aeruginosa, Pseudomonas cepacia, Comamonas testosteroni, and Cytophaga heparina were cited from EMBL DNA data base for the comparison. The genetic distances between the sequences were estimated by Knuc value (10). Then, the phylogenetic tree was constructed by the neighbor joining method (22) using C. heparina as a root organism. Deleted and unknown positions of five species and positions of B. solanacearum were eliminated for the comparison of sequences. Positions , , , and of five species were eliminated from the comparison because the secondary structure of those parts differed between strains. Total length of sequence compared was The topology of the reconstructed phylogenetic tree was evaluated by bootstrap sampling method (6). RESULTS Phenotypic Characteristics Each of the strains of the seven species were Gram-negative rod-shaped organisms. In addition to B. mallei 2233, B. solanacearum 2181 was non-motile without

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8 125 8 E. YABUUCHI ET AL Table 3. Negative physiological and biochemical characteristics of the seven Burkholderia strains any flagellum after three passages on semisolid agar plates. B. cepacia 645, B. pseudomallei 2004, B. caryophylli 3257 and B. gladioli 3258 were motile with polar tuft of flagella. B. pickettii 3254 was motile with polar monotrichous flagellum. Bipolar staining of B. pseudomallei 2004 was not remarkable. Positive physiological and biochemical characteristics of P. aeruginosa 274T and five type and two reference strains of Burkholderia species are listed in Table 2; and those tests for which all the test strains including P. aeruginosa 274 gave negative results are shown in Table 3. Compared to the inability of P. aeruginosa 274, each strain

9 BURKHOLDERIA GEN. NOV. FOR THE PSEUDOMONAS HOMOLOGY and of P. aeruginosa of the seven Burkholderia species was able to produce oxidative acidity from 2 to 8 carbohydrates of the following: cellobiose, lactose, maltose, sucrose, dulcitol, inositol, sorbitol, and salicin, which were never oxidized by strains of P. aeruginosa (Table 2). B. cepacia 645 had the largest number of positive tests 38 was obtained by B. cepaci a 645; and the 2181 had the smallest number 18. Acylamidase test was positive in only two strains, P. aeruginosa 274 and B. cepacia 645. The largest number of negative test 36, was obtained by B. solanacearum 2181; and the smallest number 17 was obtained by P. cepacia 645.

10 126 0 E. YABUUCHI ET AL Assimilation Profiles Results of positive assimilation tests on 40 carbohydrate substrates are summarized in Table 4, on 44 organic acids in Table 5 and 41 amino acids in Table 6. Type strain of more than three species of the genus Burkholderia assimilated the substrates of seven carbohydrates (adonitol, D-xylose, galactose, inositol, mannose, sorbitol, and sucrose), three organic acids (aconitate, citraconate and L-tartrate), and three amino acids (glucosamine, L-cysteine, and L-threonine), which were not assimilated by P. aeruginosa 274. Substrates of nine carbohydrates, five organic acids, and eight amino acids for which P. aeruginosa 274 and seven strains of Burkholderia species gave uniformly negative results are summarized in Table 7. Cellular Lipids Profile Two-dimensional TLC of lipids revealed, in addition to the spot of phosphatidylglycerol (PG), two major ninhydrin-positive phospholipids (PE-1 and PE-2) (Fig. 1, a, b and c). PE-1 was identical to egg phosphatidylethanolamine, while PE-2 migrated coincidedly with the polar phosphatidylethanolamine (1, 7). PE-1 possessed non-hydroxy fatty acids, while PE-2 possessed non-hydroxy and 2-hydroxy fatty acids in a ratio of 1: 1. These facts were supported by fast atom bombardment mass spectrometry (FAB/MS) of lysophosphatidylethanolamine, obtained after phospholipase A2 treatment of PE-1 and PE-2. Both lysophospholipids possessed non-hydroxy fatty acyl residue with the same carbon and double bond numbers. Phospholipase A2 digestion of PE-1 yielded non-hydroxy fatty acid, while that of PE-2 produced 20H fatty acid essentially. Therefore, it was demonstrated that 20H fatty acid existed in the 2-position of glycerol moiety. Two other ninhydrin-positive lipids (0L-1 and OL-2) (9) existed in five species i.e., except P. pickettii and P. solanacearum. FAB/MS analysis of the intact ornithine lipids showed that OL-1 possessed one 3-hydroxy (amide) fatty acid and nonhydroxylester fatty acid, while OL-2 possessed one 3-hydroxy (amide) fatty acid and 2-hydroxy (ester) fatty acid, respectively. An unidentified ninhydrin-positive spot (AL-X) was present in B. mallei 2233 and B. pseudomallei Two-dimensional chromatogram of P. aeruginosa 274 lipids (Fig. 1d) failed to demonstrate spots of PE-2, OL-1 and OL-2. Relative amount of polar lipids of the seven Burkholderia species is listed in Table 8. Cellular Fatty Acid Profiles The cellular fatty acid composition of seven species of the genus Burkholderia are shown in Table 9. Each strain contained C16: 0, C16: 1, C18: 111,12fatty acids as the major components. Six species other than B. pickettii contained C17-CPA and C 19CPA significantly, but not B. pickettii. However, the most characteristic profile was that these species possessed various 20H or 30H fatty acids in appreciable quantities. Among them, 3-hydroxy myristic acid (30H-C14: 0) was the most common in Burkholderia species and differs from P. aeruginosa. 3-Hydroxy palmitic acid (30H-C16 0) existed as the amide fatty acid of OL-1 or OL-2 in five species,

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15 BURKHOLDERLI Table a 7. Substrates GEN. not assimilated NOV. FOR by strains THE PSEUDOMON. of 7 Burkholderia LS' HOMOLOGY species and P. aeruginosa C b d Fig. 1. Two-dimensional thin-layer chromatograms of four Burkholderia species. a, B. cepacia EY 645T; b, B. mallei EY 2233T; c, B. gladioli EY 3258; d, B. solanacearumey 2181T. Solvent 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). PE, phosphatidylethanolamine; PG, phosphatidylglycerol; LYE, lysophosphatidylethanolainine; 01., ornithine lipid; Al.-X, unknown amino lipid; LY, most probably poly-beta-hydroxybutyrate, GI.-X, unknown glycolipid; PIG, yellow pigment.

16 126 6 E. YABUUCHI ET AL Table 8. Cellular lipid composition of type or reference strains of seven Burkholderia species but there was no appreciable quantity in B. pickettii and B. solanacearum. On the other hand, 2-hydroxy fatty acids existed as ester fatty acid of PE-2 in Burkholderi a species. Among them, 20H-C16: 0, 20H-C16: 1, 20H-C18: 1 and 20H-C19CPA were the characteristic components. The most characteristic components in P. aeruginosa, 20H-C12 : 0, 30H-C10: 0 and 30H-C12 : 0, were not detected in any Burkholderia species. Nucleic Acid Information As shown in Table 10, G±C contents of Burkholderia species distributed from 64 to 68.3%, and DNA-DNA homology values divided seven species into two groups. Sequence alignments of 1174 nucleotides of 16S rrna coding DNA of five species are shown in Figs. 2 and 3, in comparison with that of E. coli. Phylogenetic tree derived from the sequences of five species is illustrated in Fig. 4. DISCUSSION Each of the seven Burkholderia species was characterized by their ability to oxidize and assimilate several disaccharides and polyalcohols which were never metabolized by strains of P. aeruginosa, type species of the genus Pseudomonas. Some differences in assimilation results from those which appeared in the literature may be due to the difference of methods used. Lipid composition of homology group II differed entirely from that of P. aeruginosa. Major spots of the two-dimensional chromatogram of P. aeruginos a EY 274 revealed phosphatidylethanolamine, PE-1, and phsophatidylglycerol. In addition to these, a polar phosphatidylethanolamine, PE-2, possessing non-hydroxy fatty acid at the 1-position and 20H fatty acid at the 2-position of glycerol existed in seven species of Burkholderia gen. nov. (Pseudomonas homology group II). Concerning the polar phospholipid of P. solanacearum, our result differs from an earlier report on this organism (4). However, previous paper (30) reported that the phospha-

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18 126 8 E. YABUUCHI ET AL Table 10. GC% and DNA homology among the type strains of P. aeruginosa and 7 Berkholderia sp. tidylethanolamine and phosphatidylglycerol of Gram-positive bacteria contained 20H fatty acid in the 2-position. In the present work, the same fact was observed in the Gram-negatives. Detailed description on molecular species of polar phospholipids of the genus will be published elsewhere. Two other ninhydrin-positive non-phospholipids (0L-1 and OL-2), chromatographically similar with the ornithine lipids produced by P. cepacia (9), existed in five species but not in B. pickettii and B. solanacearum, and also in P. aeruginosa. Non-hydroxy and hydroxy fatty acid compositions of Burkholderia species were characteristically different from those of P. aeruginosa. Thus, the polar lipid and fatty acid composition, as shown in Table 9, indicate that the new genus Burkholderia resides far from genus Pseudomonas. Readfearn et al (20) transferred Bacillus mallei to the genus Pseudomonas based on the nutritional and biochemical properties. The results of DNA-DNA hybridization well correspond to that of rrna-dna hybridization by Palleroni et al (15). B. mallei and B. pseudomallei were genetically recognized as a single species. However, from the epidemiological and zoonotic considerations, they remained as separate species. Bacillus solanacearum was named and described by Smith (23) as early as 1896, and was transferred to the genus Pseudomonas by himself in Close relationship with Pseudomonas pickettii has been reported by Ralston et al (19), just before the publication of five groups of Pseudomonas species based on the rrna- DNA homologies. Our results of cellular lipid analysis and DNA-DNA homology suggested the division of the seven species of Pseudomonas homology group II. However, as shown in phylogenetic tree (Fig. 4), two DNA groups were considered to be included in one genus, Burkholderia. Description of Burkholderia gen. nov. (Burkholder. ia. M.L. dim. ending-ia; M.L. fem. n. Burkholderia named after W.H. Burkholder, the American bacteriologist who first discovered the etiologic agent of rotten onion). The cells of the species of this genus are Gram-negative, non-fermentative, straight rods, that have a single polar flagellum or a tuft of polar flagella when motile. A single species, B. mallei, is atrichous and non-motile. Catalase is pro-

19 BURKHOLDERIA GEN. NOV. FOR THE PSEUDOMONAS HOMOLOGY Fig. 2-1.

20 127 0 E. YABUUCHI ET AL Fig. 2-2.

21 BURKHOLDER1A GEN. NOV. FOR THE PSEUDOMONAS HOMOLOGY Fig Fig. 2. Sequence alignments of 16S rrna coding DNA (positions from ) for Escherichia coli, Burkholderia mallei (8z. B. pseudomallei), B. cepacia, B. gladioli, B. solanacearum and P. pickettii. Sequence of B. pseudomallei 16S rrna was the same as that of B. mallei. duced, oxidase activity is variable by species. In addition to monosaccharides, disaccharides and polyalcohols are oxidized and assimilated as sole source of carbon and energy. Cellular lipids are characterized by the presence of phosphatidylglycerol possessing hydroxy fatty acid at the 2-position of glycerol. Fatty acid compositions of cellular lipids are characterized by the presence of 20H acids of C16: 0, 16: 1, 18: 1, 19CPA and 30H acids of C14: 0 and 16: 0; and, characteristically absent are 20H-C12: 0, 30H-C10: 0 and 30H-C12: O. Species are pathogenic to either human and animal or plant. Guanine-plus-cytosine content of DNA was mol%. Type species is Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. The authors for Burkholderia gen. nov. should be cited as Yabuuchi Kosako, Oyaizu, Yano, and Ezaki. Description of the type or reference strains of seven species assigned to Burkholderia gen. nov. are given below. Further description on each species appeared in Bergey's manual (1984). The authors for the following new combinations are the same as those for the genus. 1) Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. In addition to the description by Palleroni and Holmes (16), the type strain is characterized by lysine decarboxylase activity and oxidative acid production from several disaccharides and polyalcohols. Oxidase activity was positive but rather slow and weak. Cellular lipid and fatty acid composition is as the description on the genus. Type strains: Ballard 717=ATCC 25416=NCTC G+C content was 66.6 mol%. 2) Burkholderia mallei (Zopf 1885) comb. nov. A sole non-motile species in

22 127 2 E. YABUUCHI ET AL Fig. 3. Sequence alignments of 16S rrna coding DNA (positions from ) for E. coll., B. mallei (& B. pseudomallei), B. cepacia, B. gladioli, B. solanacearum, and B. pickettii. Sequence of B. pseudomallei 16S rrna was the same as that of B. mallei.

23 BURKHOLDERIA GEN. NOV. FOR THE PSEUDOMONAS HOMOLOGY Fig. 4. Phylogenetic tree derived from the 16S rrna sequences. Cytophaga heparina was used as a root organism. Positions (in E. coli numbering system) , , , and were eliminated for the comparison because the secondary structure of those parts varied among strains compared. The numbers on the branches refer to the confidence (%) estimated by bootstrap analysis (100 replications). the genus without flagellum. Probably because of its parasitic nature, the organism is less active than B. pseudomallei, physiologically and biochemically. In addition to the ornithine lipids, cellular lipid contained unknown amino lipid. Cellular lipid and fatty acid composition, DNA-DNA homology value and 16S rrna sequence indicate the genetic similarity of B. mallei and B. pseudomallei. Because of epidemiological and zoonotic significance, the two species were left separate. Type strain: ATCC G+ C content of DNA was 68.3 mol%. 3) Burkholderia pseudomallei (Whitmore 1913) comb. nov. Type strain produces rather smooth colonies compared to other isolates. Gas was not produced from nitrate. Physiologically and biochemically more active than B. mallei. Contrary to the description by Stanier et al (24), beta-hydroxybutyrate was not detected when cellular lipids were analyzed by two-dimensional TLC, while unknown amino lipid similar to that of B. mallei was detected. Type strain: ATCC 23343=WRAIR 286. GH-C content of DNA was 67.9 mol%. 4) Burkholderia caryophylli (Burkholder 1942) comb. nov. Because the type strain was extremely difficult to obtain, the description here is based on a reference strain MAFF Motile with polar tuft of flagella. Sequence of 16S rrna at 157 positions ( ) is the same as those of B. mallei and B. pseudomallei, one difference with B. gladioli, and two differences with B. cepacia. GH-C content of test strain DNA was 64.6 mol%. Type strain: ATCC ) Burkholderia gladioli (Severini 1913) comb. nov. Because the type strain of the species was difficult to obtain, the description here is based on a reference strain MAFF In addition to the description in Bergey's manual (1984), the test strain oxidized dissacharides and polyalcohols, assimilated many kinds of

24 127 4 E. YABUUCHI ET AL organic acids and amino acids. 20H-C19CPA and 30H-C16: 0 were not detected by cellular fatty acid analysis. GH-C content of test strain DNA was 67.9 mol%. Type strain: NCPPB ) Burkholderia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov. In addition to the description in Bergey's manual (1984), type strain of the species was positive in urease activity. Ornithine lipids (OL-1 and OL-2), commonly seen in five species of the genus, were not detected from cellular lipids analysis by two-dimensional TLC. Most probable spots of poly-beta-hydroxybutyrate were positive. Cellular fatty acid does not contain detectable amount of C19CPA, 20H-C19CPA and 30H-C16: 0. G+C content was 64.0 mol%. Type strain: ATCC ) Burkholderia solanacearum (Smith 1896) comb. nov. In spite of the description in the literature (17), type strain of the species was non-motile without any flagellum. Cellular lipid does not contain ornithine lipids (OL-1, OL-2). Cellular fatty acid contains 20H-C19CPA and 30H-C16: 0 at a concentration of each 1% of total fatty acids. G+C content was 66.6 mol%. Type strain: ATCC Although the two species B. pickettii and B. solanacearum demonstrated differences in certain phenotypic characteristics, cellular lipid composition and DNA-DNA homology value, the differences in rrna sequences of these two species against five other species were 5.5% and phylogenetic tree (Fig. 4) supported the inclusion of the seven species in one genus, Burkholderia. B. cepacia was designated as the type species of the genus Burkholderia. REFERENCES 1) Cox, A.D., and Wilkinson, S.G Polar lipids and fatty acids of Pseudomonas cepacia. Biochim. Biophys. Acta 1001: ) De Vos, P., Kersters, K., Falsen, E., Pot, B., Gillis, M., Segers, P., and De Ley, J Comamonas Davis and Park 1962 gen. nov., nom. rev, emend., and Comamonas terrigena Hugh 1962 sp. nov., nom. rev. Int. J. Syst. Bacteriol. 35: ) Doudoroff, M., and Palleroni, N.J Genus I. Pseudomonas Migula 1894, p In Buchanan, R.E., and Gibbons, N.E. (eds), Bergey's manual of determinative bacteriol, 8th ed, Williams & Wilkins, Baltimore. 4) Drigues, P.D., Lafforgue, D., and Asselineau, J Etude des phospholipides de Pseudomonas solanacearum. Presence d'acides gras a-hydroxyles monoethyleniques. Biochim. Biophys. Acta 666: ) Ezaki, T., Hashimoto, Y., and Yabuuchi, E Fluorometric DNA-DNA hybridization in microdilution wells as an alternative of membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39: ) Felsenstein, J Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: ) Galbraith, L., and Wilkinson, S.G Polar lipids and fatty acids of Pseudomonas caryophylli, Pseudomonas gladioli and Pseudomonas pickettii. J. Gen. Microbiol. 137: ) Hildebrand, D.C., Palleroni, N.J., and Doudoroff, M Synonymy of Pseudomonas gladioli Severini 1913 and Pseudomonas marginata (McCulloch 1921) Stapp Int. J. Syst. Bacteriol. 23: ) Kawai, Y. Yano, I., Kaneda, K., and Yabuuchi, E Ornithine-containing lipids of some

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