J. Gen. App!. Mierobiol., 24, 199-213 (1978)

CELLULAR FATTY ACID COMPOSITION IN SPECIES

SHIGEAKI IKEMOTO,* HIROSHI KURAISHI,** KAZUO KOMAGATA,* RYOZO AZUMA,*** TSUNEJI SUTO,*** AND HARUYOSHI MUROOKA****

*The Institute of Applied Microbiology , University of Tokyo, Bunkyo-ku, Tokyo **Faculty of Agriculture , Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo ***National Institute of Animal Health , Kodaira-shi, Tokyo * * * * College of Agriculture and Veterinary Medicine , Nihon University, Setagaya-ku, Tokyo

(Received January 27, 1978)

Cellular fatty acid composition of 50 strains of the genus Pseudomonas was determined by gas-liquid chromatography. Straight-chain saturated acid of C16:0 and straight-chain unsaturated acids of C16.1 and C18:1 with a double bond were commonly found in all the strains tested. The pres- ence of hydroxy acids, cyclopropane acids, and branched-chain acids showed the characteristics for the groups and species in the genus Pseudo- monas. Similarity values calculated on the basis of fatty acids exhibited a clear correlation to the taxonomic groups of this genus. Bacterial fatty acid composition was considered to be useful for the study of inter- relation and for rapid identification of the .

Taxonomic studies on bacteria have been mainly carried out on the basis of morphological, biochemical, and physiological characteristics. Recently, chemi- cal constituents of bacterial cells have become of interest from the point of taxon- omy, and DNA base composition, cell wall composition, and type of co-enzyme Q have been used for taxonomic criteria in some taxa. The progress of instru- mental analyses has contributed to the development of chemotaxonomy, and the results involved have been useful not only for the bacterial classification, but also for rapid identification of the bacteria. Since bacterial fatty acids were found to be located in the cell membrane and play a part in the transport system, cellular fatty acid composition is of interest in bacterial , and its taxonomic significance has been recognized in some

Address reprint requests : Dr. K. Komagata, The Institute of Applied Microbiology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan.

199 200 IKEMOTO, KURAISHI, KOMAGATA, AZUMA, SUTO, and MUROOKA VOL. 24 1978 Fatty Acid Composition in Pseudomonas Species 201 202 IKEMOTO, KURAISHI, KOMAGATA, AZUMA, SUTO, and MUROOKA VOL. 24 genera (1-7). In the previous paper (8), two of the present authors (S. I. and K. K.) reported the cellular fatty acid composition of methanol-utilizing bacteria, and pointed out the difference in the composition between gram-negative polarly flagellated methanol-utilizing bacteria and Pseudomonas fluorescens. It seemed of interest to investigate the cellular fatty acid composition of a wide variety of the members of the genus Pseudomonas and the possibility of employing the composition as an aid to rapid identification of the strains of this genus.

MATERIALSAND METHODS Organisms. Bacterial strains obtained from culture collections and those iso- lated from activated sludge by two of the present authors (H. K. and H. M.) were used for this study, and they comprised 50 strains and 24 species. Their sources and corresponding designation numbers of other culture collections are listed in Table 1. The species names were used according to their designation when they were received from the culture collections. Cell preparation. Cells used for fatty acid analysis were harvested from the stationary period of the culture in nutrient broth (meat extract 10 g, peptone 10 g, and NaCI 5 g, distilled water 1,000 ml, pH 7.2) after 40-hr incubation at 30 °. Cells were separated by centrifugation, followed by being washed twice with distilled water, and the washed cells were lyophilized. Fatty acid analysis. Procedure for fatty acid analysis was reported previously (8). Lyophilized cells were methylated with 5 % HCl-methanol at 100° for 3 hr. After methanolysis, the reaction mixture was extracted with petroleum ether. The solvent fraction was concentrated under nitrogen current, and subjected to analysis of fatty acids by using a Shimadzu Model 4B gas chromatograph. Fatty acids were primarily identified by comparison of relative retention time of their methyl esters with the standard fatty acids. Further, the equivalent chain length (ECL) was determined from the logarithm of the retention time of methyl esters of saturated straight-chain fatty acids plotted against their carbon number, and some of fatty acids were presumed on the basis of ECL. For analysis of hydroxy acids in some strains, gas chromatography was adopted after separation of 2- hydroxy acids and 3-hydroxy acids from non-polar fatty acids by using thin-layer chromatography and extraction of the acids from the chromatogram. The chromatograms on a Silica Gel G (Merk, Darmstadt) plate were developed with a solvent system of hexane and ether (85:15, v fv). The percentage of each acid was estimated from the ratio of the peak area to the total area. Similarity values based on fatty acid composition. Similarity values of the strains were calculated on the basis of fatty acid composition by the procedure similar to that of ROZETTand PETERSON(9) which was adopted in the classification of organic compounds by factor analysis of mass spectra, and the dendrogram was 1978 Fatty Acid Compositionin PseudomonasSpecies 203

obtained by the unweighted group method (10).

cos ~ -

Where x, is the peak area of a fatty acid, and ys is the peak area of the fatty acid which corresponds to that of x~.

RESULTS AND DISCUSSION

Cellular fatty acids of Pseudomonas strains mainly consisted of straight- chain acids, branched-chain acids, hydroxy acids, and cyclopropane acids. Even- numbered straight-chain saturated acids of Ci2:o and Cls:o, and even-numbered straight-chain unsaturated acids of Cis;l and Cis:i were found in all the strains

Fig. 1. Fatty acid patterns of Pseudomonas species. A, KS 0025 (fluorescent group); B, Pseudomonas acidovorans KS 0057 (achromogenic group); C, Pseudomonas lacunogenes KS 0036 (chromogenic group). 204 IKEMOTO, KURAISHI, KOMAGATA, AZUMA, SUTO, and MUROOKA VOL. 24 1978 Fatty Acid Composition in Pseudomonas Species 205 206 IKEMOTO, KURAISHI, KOMAGATA, AZUMA, SUTO, and MUROOKA VOL. 24 1978 Fatty Acid Composition in Pseudomonas Species 207 tested, but the distribution of branched-chain acids, hydroxy acids, and cyclo- propane acids was different among the groups within the genus, species, or strains. The genus Pseudomonas had been divided into three groups; fluorescent group, achromogenic group, and chromogenic group, on the basis of pigmentation, and biochemical and physiological characteristics by IizuKA and KoMAGATA(11). In Table 2 the fatty acid composition of the species is arranged according to their grouping, and those of Pseudomonas cepacia, Pseudomonas maltophilia, Pseudo- monas putrefaciens, and Pseudomonas rubescens, which are not considered to be included in the grouping, are added to these groups separately. Typical patterns of the fatty acid composition of Pseudomonas species are shown in Fig, 1.

Fluorescent group Fatty acids of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudo- monas ovalis, and other species mainly consisted of straight-chain acids of C16:o, C16:1,and Ci8:1,and these three acids accounted for approximately 50% of a total of fatty acids. Further, a small amount of 3-OH C10:o, 2-OH 012:0, 3-OH C12:o, cyclopropane C17:o,and cyclopropane C19:0were found in almost all the strains, and the presence of 2-OH C12:0,cyclopropane C17:0,and cyclopropane C19:0seemed to be characteristic for this group. The fluorescent group is considered to be homogenous on the basis of fatty acid composition. However, Pseudomonas fulva (12) and Pseudomonas straminea (12) produced both water-soluble and water- insoluble yellow pigments, and they seemed to be somewhat different from the typical species in this group in the point of cellular fatty acid composition in which cyclopropane Cl9:owas not found.

Achromogenic group Fatty acids of Pseudomonas acidovorans, Pseudomonas diminuta, Pseudomonas testosteroni, and other species in this group mainly consisted of straight-chain satu- rated and unsaturated acids of C16:0,016:1, and C18:1. Further, 3-OH C10:0and cyclo- propane C17:0were found in almost all the species, and cyclopropane C19:0was rec- ognized in a small number of the species. Distribution of hydroxy acids and cyclo- propane acids seemed to be characteristic for the species. Almost the same cellular fatty acid composition was found in P. acidovorans, Pseudomonas cruciviae, Pseudo- monas dacuhnae, Pseudomonas desmolytica, P. testosteroni, and Comamonas terri- gena, and acids of C16:o,C16:1, and C18:1accounted for 70 % of total acids. Although these species did not produce acid from glucose, and some nomenclature problems have been reported (13, 14), the cellular fatty acid composition would be useful for a better understanding of the species. Previously, Moss et al. (5) reported the identical fatty acid profiles for P. acidovorans and P. testosteroni. A small amount of branched-chain acids of iso-C13.0and iso-C15.0was found in Pseudomonas alcali- genes. P. diminuta has been considered to be peculiar in the genus Pseudomonas in the point of requirement of biotin and pantothenic acid, and it demonstrated 208 IKEMOTO, KURAISHI, KOMAGATA, AZUMA, SUTO, and MUROOKA VoI„ 24

Fig. 2. Fatty acid pattern of Pseudomonas iners KS 0047. the presence of approximately 20 % of cyclopropane acid of C19:oin total acids, and a small amount of iso-C15:0,and the absence of 3-OH C1o:owhich was com- monly found in the genus Pseudomonas. Pseudomonas iners (15) requires a small amount of NaCI for growth, and demonstrated a rather simple cellular fatty acid composition. Approximately 85 % of the acids consisted of straight-chain acids of C16:o,C16:1, and C18:1,and a small amount of acids of C10.0and 3-OH C1o:owere found but cyclopropane acid was not recognized, as shown in Fig. 2. Cellular fatty acid composition in this group was variable but characteristic for the species.

Chromogenic group Fatty acids of Pseudomonas lacunogenes mainly consisted of straight-chain acids of C16:o,C16:1, and C18:1,and these three acids accounted for approximately 90 % of total acids. The presence of a small amount of acids of 3-OH C10,0and 3-OH C12:owas recognized in this species but cyclopropane acid was not found. It was reported that the species of the chromogenic group produced water-insoluble yellow pigment, and that they were distributed widely in paddy rice and other cereals (11,12,16).

Fatty acid composition in Pseudomonas cepacia Pseudomonas cepacia was reported to be characterized by assimilation of a wide range of carbon compounds (17, 18). Straight-chain acids of C16,0and C18:1appeared as the main acids in this species, and hydroxy acids of 2-OH C16:0, 3-OH C14:o,and 3-OH C16:owere recognized in place of hydroxy acids of 2-OH C12:o,3-OH C10:o,and 3-OH C12:ocommonly found in the species of the genus Pseudomonas, as shown in Fig. 3. P. cepacia seems to be peculiar in cellular fatty acid composition of the genus Pseudomonas. SAMUELSet al. (19) analyzed the fatty acids of P. cepacia and Pseudomonas kingii, and concluded the identity of both species from the cellular fatty acid composition. Further, KURANEet al. (20) reported that they identified a strain as P, cepacia on the basis of cellular fatty acid composition, though it possessed several characteristics different from the typical strain of the species. 1978 Fatty Acid Composition in Pseudomonas Species 209

Fig. 3. Fatty acid pattern of Pseudomonas cepacia KS 0052,

Fatty acid composition in Pseudomonas maltophilia Pseudomonas maltophilia was reported to be peculiar in the point of require- ment of methionine, oxidase-negative, and DNase-positive in the genus Pseudo- monas. Cellular fatty acid composition of this species was remarkably different from those of other species, and iso-branched acid of C15:0accounted for approximately 50 % of total acids, as shown in Fig. 4. Strains previously named Pseudomonas melanogenum (sic) wererei dentified as P. maltophilia from bacteriological charac- teristics (21, 22), and the strains of both species demonstrated the same cellular fatty acid composition. Moss and DEES(23) reported the same conclusion on the basis of cellular fatty acid composition.

Fig. 4. Fatty acid pattern of Pseudomonas maltophilia KS 0001.

Fatty acid composition in Pseudomonas putrefaciens and Pseudomonas rubescens Fatty acids of P. putrefaciens and P. rubescens mainly consisted of iso-branched acid of C15:oand straight-chain acid of C16:1,as shown in Fig. 5. From this fact and other characteristics, P. putrefaciens and P, rubescens seem to be identical. DNA base composition of P. putrefaciens was reported to be about 20% lower than those of the species in the genus Pseudomonas, and this species has not been 210 IKEMOTO, KURAISHI, KOMAGATA, AZUMA, SUTO, and MUROOKA VOL. 24

Fig. 5. Fatty acid pattern of Pseudomonas putrefaciens KS 0045. considered to belong to the genus Pseudomonas (24). As DEES and Moss (25) pointed out previously, cellular fatty acid composition of this species was re- markably different from those of other species in the genus Pseudomonas. It is of interest that the cellular fatty acid composition of these species is similar to that of P. maltophilia in spite of the remarkable difference in DNA base com- position.

Ident flcation of unknown strains on the basis of cellular fatty acid composition Strains of Nos. 7, 14, 10-B-A, 10-B-C, 26, and 33 were gram-negative polarly flagellated rods, showed nutritional requirement, and turned nutrient agar con- taining tyrosine to brown. Although identification of these strains was difficult on the basis of conventional routine tests, they have been identified as P, diminuta in the point of characteristic fatty acid composition of this species which demon- strated the presence of acids of 3-OH C12:o,cyclopropane C19;o,and a small amount of acid of iso-C16:o,and the absence of acid of 3-OH C10.0,as shown in Fig. 6. As mentioned above, characteristic cellular fatty acid composition of the bacteria, such as P. maltophilia, P. cepacia, P. putrefaciens, and P. diminuta, would be useful for rapid identification of the bacteria.

Similarity values based on fatty acid composition The strains tested showed high similarity values on the basis of fatty acid composition, and fell into distinct clusters, as shown in Fig. 7. These clusters correlated fairly well with the grouping mentioned above, and P. maltophilia and P, putrefaciens differed clearly from other members of the genus Pseudomonas.

Taxonomic consideration of the genus Pseudomonas IIZUKAand KoMAGATA(11) divided the genus Pseudomonas into the three groups as mentioned above. In their grouping, P. maltophilia was included in the chromogenic group, and P, putrefaciens, P. diminuta, and P. iners in the achromo- genic group. However, these species indicated characteristic cellular fatty acid compositions, and were different from the species in the groups. Therefore, 1978 Fatty Acid Composition in Pseudomonas Species 211

Fig. 6. Fatty acid pattern of Pseudomonas diminuta. A, Pseudomonas diminuta KS 0016; B, Pseudomonas diminuta No. 26; C, Pseudo- monas diminuta No. 33. these facts suggest the revision of their grouping of the genus Pseudomonas. STANIERet al. (17) reported an extensive taxonomic study of the genus Pseudo- monas, and recognized six groups; fluorescent group, acidovorans group, alcali- genes group, P. multivorans, P, stutzeri, and P. maltophilia. PALLERONIet al. (26) divided this genus into five groups on the basis of r-RNA-DNA hybridization. Moreover, YANG et al. (27) analyzed "extractable lipid" and "bound lipid" of Pseudomonas species, and reported a good agreement of lipid composition with Palleroni's grouping. The species of the fluorescent and achromogenic groups have been fairly well studied taxonomically, but those of the chromogenic group, only a little. Chemotaxonomic studies of the species in the chromogenic group may suggest a clear taxonomic relation within the genus and to Xanthomonas and related bacteria. 212 IKEMOTO, KURAISHI, KOMAGATA, AZUMA, SUTO, and MUROOKA VOL. 24

Fig. ?. Dendrogram of similarity values based on cellular fatty acids. 1978 Fatty Acid Composition in Pseudomonas Species 213

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