Fatty acid Content in Whole-cell Hydrolysates and Phospholipid and Phospholipid Fractions of Pseudomonads: a Taxonomic Evaluation

Sytem. Appl. Microbiol. 19,528-540 (1996) © Gustav Fischer Verlag

Fatty acid Content in Whole-cell Hydrolysates and Phospholipid Fractions of Pseudomonads: a Taxonomic Evaluation MARC VANCANNEYTt, SABINE WITT2 , WOLF-RAINER ABRAHAM 2 , KAREL KERSTERSt, and HERB L. FREDRICKSONh 1

2

Laboratorium voor Microbiologie, Universiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium Gesellschaft fur Biotechnologische Forschung mbH, Abteilung Mikrobiologie, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Received April 11, 1996

Summary In a comparative taxonomic study, fatty acid contents of whole-cell hydrolysates and phospholipid fractions of strains belonging to the pseudomonads were analyzed. Major fatty acids in all taxa were 16:0 and various isomers of 16: 1 and 18: 1. Fatty acid patterns of phospholipids were easily differentiated from whole-cell profiles by their absence of hydroxylated fatty acids. The latter constituents proved to be diagnostic markers for differentiation of the main phylogenetic lineages within the pseudomonads (rRNA groups). Based on the presence or absence of three 3-hydroxy fatty acids, 10: 0 30H, 12: 0 30H, and 14: 0 30H, the majority of the strains could be allocated to one of the four rRNA groups (I-IV) studied. In general, rRNA group I organisms were characterized by the presence of 10:0 30H and 12:0 30H, rRNA group II strains contained 14: 0 30H, group III strains contained 10: 0 30H and group IV strains 12: 0 30H. A numerical analysis of the whole-cell fatty acid data yielded separate entities for all four rRNA groups. When using the fatty acid contents of phospholipids, no clear differentiation between rRNA group II and III taxa and rRNA group I organisms was obtained. Both approaches, however, yielded an analogous subgrouping of rRNA group I organisms which reflected their phylogenetic relationships.

Key words: Fatty acid analysis - Whole-cell hydrolysates - Phospholipids - Pseudomonads - Taxonomy

Introduction

As evidenced by a number of reviews whole-cell fatty acid analyses have found considerable use in the identification and classification of bacteria (Welch, 1991; Suzuki et al., 1993; Jantzen and Bryn, 1994). Also for the pseudomonads, it was shown that each of the five major rRNA subgroups, delineated by Palleroni et al. (1973), had a characteristic fatty acid profile (Moss et al., 1972; Ikemoto et al., 1978; Oyaizu and Komagata, 1983; Stead, 1992). Since Palleroni's work, many reclassifications have been proposed. Members of rRNA groups II to V have been reclassified in a number of different genera (Kersters et al., * Present address: Dr. Herb L. Fredrickson, Environmental Laboratory, Environmental Processes and Effects Division, Waterway Experimental Station, 3909 Halls Ferry Forad, Vicksburg, MS 39180-6199, USA

1996; Willems et al., 1992). Only taxa belonging or related to rRNA group I are considered as genuine Pseudomonas species. For a few validly described Pseudomonas species the phylogenetic position remains to be verified (Kersters et al., 1996). Most fatty acids occur in the cytoplasmic membrane and are constituents of polar lipids and glycolipids (Kates, 1964; Ratledge and Wilkinson, 1988). Quite a number of bacterial taxonomic studies already investigated the fatty acid composition of the polar lipid fractions (Fredrickson et al., 1986; Skerratt et al., 1991; Nunes et al., 1992; Kohring et al., 1994). These data showed that relationships among taxa based on polar lipid fatty acid contents paralleled their phylogenetic relatedness. We report here on a chemotaxonomic approach where the whole-cell fatty acid compositions and the fatty acid

Comparison of whole-cell and phospholipid fatty acid composition of pseudomonads contents of the phospholipids of 159 pseudomonad strains were compared. Reference strains of rRNA groups I to IV were included with special emphasis on the genuine Pseudomonas species. Materials and Methods Bacterial strains, growth conditions and harvesting ofcells. All of the strains which were studied are listed in Table 1. For wholecell fatty acid analysis, bacterial strains were precultured for 2 days on nutrient agar slants containing 1 g Lab Lemco (catalog no. L29; Oxoid, Ltd., Basingstoke, United Kingdom), 2 g yeast extract (catalog no. L21; Oxoid), 5 g bacteriological peptone (catalog no. L37; Oxoid), 5 g NaCl (catalog no.6404; Merck, Darmstadt, Germany) and 20 g agar (catalog no. U1; Oxoid) per liter distilled water. The precultures were streaked onto plates containing 30 g Trypticase soy broth (catalog no. 11768; BBL, Becton Dickinson Microbiology Systems, Cockeysville, Maryland, USA) supplemented with 15 g Bacto Agar (catalo? no. 0140-01; Difco Laboratories, Detroit, Michigan, USA) per lIter distilled water, and the plates were incubated for 24 h at 28°C. A loopful of cells from the overlap area of the second and third series of streaks was harvested. For phospholipid fatty acid analysis, cells were grown on a complex medium containing 20g tryptone (catalog no. 0123-173; Difco), 5g yeast extract (catalog no.0127-17-9; Difco), 5g glucose (catalog no. 16301; Riedel-de Haen AG, Seelze, Germany), 5 g NaCI (catalog no. 6404; Merck) and 15 g Bacto Agar (catalog no. 0140-01; Difco) per liter distilled water. After growing for at least one day a single colony was transferred to a 5 ml tube containing identical but liquid medium and shaken at 30°C overnight. Three ml of this overnight culture were inoculated into a 21 Fernbach flask, containing 11 of the above-mentioned medium. The culture was shaken for 24 h at 30°C. For harvesring, the liquid culture was centrifuged, washed twice in 0.01 M phosphate buffer and resuspended in 20 ml of buffer. A purity check of the liquid culture was performed on agar plates. Whole-cell fatty acid analysis. Cells were saponified (15% (w/v) NaOH, 30 min, 100°C), methylated to fatty acid methyl esters (FAMEs) (methanolic HCI, 10 min, 80°C) and extracted (hexane/methyl-tert-butyl ether [1: 1, v/v]) as described in detail by Osterhout et al. (1991). Fatty acid methyl esters were analyzed on a Hewlett-Packard (HP) 5890A gas chromatograph (Avondale, Pennsylvania, USA). Separation of fatty acid methyl esters was achieved with a fused-silica capillary column (25 m by 0.2 mm) with cross-linked 5% phenylmethyl silicone (film thickness 0.33 f.l.m; HP Ultra 2). The computer-controlled parameters were the same as those described by Osterhout et al. (1991). The instrument was equipped with a flame ionisation detector and an autosampler (HP 7673). Hz was serving as carrier gas. Phospholipid fatty acid analysis. Lipids were extracted from 2 g wet cells using a modified Bligh-Dyer procedure (Bligh and Dyer, 1959) as described previously (Fredrickson et al., 1986) and slightly modified in the present study by using dichloromethane (DCM) instead of chloroform. For the fractionation a chromatographic column (B & J EnviroPrep Silica Column; Baxter Diagnostic Inc., Burdick & Jackson Division, Muskegon, Michigan, USA) was used. The phospholipid fraction was dried under nitrogen, dissolved in DCM-MeOH (1: 1, v/v) and saponified overnight (1 M KOHlMeOH [1 :4, v/v], 40°C). A hexane extraction was performed to remove all residues. DCM, buffer and 6M HCI were added, the DCM phase was removed and dried. Methylation was performed in a mixture of MeOH, DCM and concentrated HCI (10: 1: 1, v/v/v) at 100°C for 90 min. After cooling, FAMEs were recovered in DCM, dried under nitrogen and 35

System. Appl. Microbiol. Vol. 19/4

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dissolved in 1 ml octane for analysis on a gas chromatograph (GC) and on a GC/mass spectrometer (MS). Dimethyldisulfide (DMDS) adducts were formed as described by Nichols et al. (1986) to determine the position of double bonds in monounsaturated fatty acids. Silylation was performed (Tri-Sil; Pierce, Rockford, USA) to determine the position of hydroxyl groups by GC/MS measurements. Capillary gas chromatographic analyses were performed on a HP 5890 Series II GC equipped with a capillary column which was differentiated from the column described above for whole-cell fatty acid analyses by its dimensions (50m; film thickness 0.11 11m). The oven program was also slightly different: 150°C for 2 min, 150°C to 280 °C at 4°C/min followed by an isothermal periode of 11 min. The GC/MS analyses were performed on a similar GC as described above (He was used as carrier gas). The GC was connected to a HP 5989A quadropole MS. The electron impact ion source was maintained at 200°C while the quadropole temperature was 100°C. The electron energy was 70 eV. Data analysis. Whole-cell fatty acid methyl ester profiles were identified by using the Microbial Identification System software (MIS version 3.8) obtained from Microbial ID, Inc. (Newark, Delaware, USA) and a calibration mixture of known standards (Hewlett-Packard). Phospholipid fatty acid methyl esters were identified by gas chromatograph-mass spectrometry analyses using the FAME adducts described earlier in this section for the determination of the hydroxy and double bond positions. Cis-trans isomers were differentiated by comparison with retention time of standards. The fatty acid data were subjected to hierarchical cluster analysis as peak square units. Normalization and log 10 transformation of the data was performed in the Pirouette software (lnfometrix, Seattle, USA). The dendrograms were generated by the program using Euclidean distances and the incremental clustering method (Manual for Pirouette, version 1.2; Infometrix).

Results and Discussion Cluster analysis A numerical comparison was performed on the fatty acid profiles of respectively whole-cell FAMEs and those obtained from the phospholipids. Both analyses are presented as a simplified dendrogram in Fig. 1. Analysis of whole-cell fatty acid fingerprints revealed major groups which corresponded well with the groupings based on DNA-rRNA hybridization techniques (Palleroni et aI., 1973; De Vos and De Ley, 1983; Willems et aI., 1992). Seven groups were delineated (Cl to C7). Four of them (Cl, C2, C3 and C7) grouped rRNA group I organisms, while rRNA group II, III and IV organisms formed separate entities and grouped in the clusters C4, C5 and C6, respectively. The considerable homogeneity observed within the rRNA groups II, III and IV may possibly be due to the restricted number of taxa investigated. One species, Pseudomonas stanieri which belongs to rRNA group I (Willems et aI., 1992), occupied an aberrant position and grouped with rRNA group III organisms in cluster C5. P. pertucinogena, for which the phylogenetic position in the gamma group of the Proteobacteria was uncertain (Kersters et aI., 1996), was closely related to rRNA group I organisms. The strains of rRNA group I investigated in the present study represented 29 different and validly described Pseu-

530

M. Vancanneyt

Table 1. List of strains studied Whole-cell FAMEsc

rRNA group' Species and strain numbers b

Phospholipid FAMEsC

Cluster

Subcluster

Cluster

Subcluster

C3

c

P4

c'

C1

a

PI

a'

C7 C7

g g

P7 ND

g'

rRNA group I

P. aeruginosa LMG 1242T (CCEB 481 T ), LMG 1272 (NCPPB 2195), LMG 5031 (NCPPB 1224), LMG 5033 (NCPPB 2652), LMG 5827 (NCPPB 1966), LMG 6395 (DSM 1117), LMG 9009 (NCIB 10421)

P. agarici LMG 2110 (NCPPB 1996), LMG 2111 (NCPPB 1999), LMG 2112 T (NCPPB 2289 T ), LMG 2113 (NCPPB 2290), LMG 2115 (NCPPB 2471)

P. alcaligenes LMG 1224T (NCTC 10367T ), LMG 2854 (NCTC 8769), LMG 6353 (CCUG 1315) LMG 6354 (CCUG 1316), LMG 6355 (CCUG 15505)

P. amygdali LMG 2123 T (NCTC 10367T ) LMG 2125 (NCPPB 2610)

C1 C1

ND P2

P. asplenii LMG 2137T (NCPPB 1947T ) LMG 5147 (NRRL B-829)

C2 C2

b b

P6 PI

b"

C2

b

P3

b'

Cl

a

PI

a'

C2

b

P6

b"

C2

b

P3

b'

C1

a

PI

a'

C2 C2

b b

P6 ND

b"

P. caricapapayae LMG 2152 T (NCPPB 1873 T ), LMG 2153 (NCPPB 4086), LMG 5051 (PDDCC 4086), LMG 5375 (PDDCC 7496)

P. chlororaphis LMG 1245 (CCEB 518), LMG 5004T (DSM 50083T ), LMG 5832t1 (NCPPB 1800), LMG 6220T (DSM 50083 T )

P. cichorii LMG 1248 (NCPPB 906), LMG 2162 T (NCPPB 943 T ), LMG 2163 (NCPPB 950), LMG 2164 (NCPPB 1511), LMG 2165 (NCPPB 1039), LMG 5052 (PDDCC 691)

P. coronafaciens LMG 13190T (ATCC 19607T ), LMG 2330 (NCPPB 1898), LMG 5030 (NIAS 1017), LMG 5060 (PDDCC 3113), LMG 5061 (PDDCC 4421)

P. corrugata LMG 1276 (NCPPB 2455), LMG 2172 T (NCPPB 2445 T ), LMG 2173 (NCPPB 2447), LMG 5036 (NCPPB 2451), LMG 5038 (NCPPB 2458)

P. ficuserectae LMG 5694T (PDDCC 7848 T ), LMG 5695 (PDDCC 7849) LMG 5696 (PDDCC 7850)

P. f/uorescens LMG 1799 (NCIB 8194) LMG 2189 (CCEB 488), LMG 5848 (NCPPB 3126), LMG 5849 (NCPPB 3141), LMG 6812 (W263 R. Vantomme), LMG 7207 (A2 R. Vantomme), LMG 7216 (All R. Vantomme)

C2 Cl

P6 a

PI

a'

Comparison of whole-cell and phospholipid fatty acid composition of pseudomonads

531

Table 1. Continued rRNA group' Species and strain numbers b

Whole-cell FAMEsc

Phospholipid FAMEsc

Cluster

Subcluster

Cluster

Subcluster

Cl

a

PI

a'

C2 Cl

a

PI PI

a' a'

Cl C2

a

PI

a'

Cl

a

PI

a'

Cl

a

PI

a'

C2

b

P6

b"

Cl

a

PI

a'

Cl

a

PI

a'

Cl

a

PI

a'

P. {luorescens by. I LMG 1794T (MMCA 40T ), LMG 5825 (CCEB 004), LMG 5916 (CIP 73.25), LMG 5829 (NCPPB 263), LMG 5830 (NCPPB 316) P. {luorescens by. III LMG 1244 (ATCC 17571), LMG 5822 (CCEB 295), LMG 5938 (CCUG 1320) LMG 5831 (NCPPB 1796)

P. {luorescens by. IV LMG 5168 (NCPPB 1803) LMG 5939 (CCUG 1255)

P6

P. {luorescens by. V LMG 5167 (NCPPB 1804), LMG 5833 (NCPPB 1805), LMG 5940 (CCUG 1320) P. fragi LMG 2191 T (CCEB 387T ), LMG 5919 (CIP 60.46), LMG 5920 (CIP 60.47)

P. fuscovaginae LMG 2158 T (PDDCC 5940T ), LMG 2192 (PDDCC 5939), LMG 5097 (PDDCC 5941), LMG 5742 (HMB 264) P. marginalis Py. alfalfae LMG 2214 (PDDCC 5708), LMG 5039 (NCPPB 2645), LMG 5040 (NCPPB 2646) P. marginalis py. marginalis LMG 1243 (ATCC 17815), LMG 2210T (NCPPB 667T ), LMG 2211 (NCPPB 668), LMG 5170 (NCPPB 1795), LMG 5177 (NCPPB 1676) P. marginalis py. pastinacae LMG 2238 (NCPPB 806), LMG 5042 (NCPPB 804), LMG 5043 (NCPPB 805), LMG 5044 (NCPPB 807)

P. meliae LMG 2220 T (NCPPB 3033 T )

PI

C2

P. mendocina LMG 1223 T (ATCC 25411 T ), LMG 5941 (CCUG 5916), LMG 6396 (H609 Hansen)

C7

g

P7

g'

C2

b

P3

b'

C7

g

P7

g'

C7

g

P7

g'

Cl C2

a

PI PI

a' a'

P. mucidolens LMG 2223 T (NCTC 8068 T )

P. oleovorans LMG 2229 T (MMCA 4T ) P. pseudoalcaligenes LMG 1225 T (ATCC 17440T ), LMG 5516 (CIP 60.76), LMG 5517 (CIP 61.21), LMG 6036 (CCUG 15237), LMG 6037 (CCUG 15284) P. putida LMG 2171 (259 Voets), LMG 2232 (CCEB 380), LMG 2259 (CCEB 520), LMG 9070 (P28-139 Emerson), LMG 12644 (DSM 1868) LMG 2258 (101 Delft)

532

M. Vancanneyt

Table 1. Continued rRNA group' Species and strain numbers b

Whole-cell FAMEsc

Phospholipid FAMEsc

Cluster

Subcluster

Cluster

Subcluster

C1

a

PI

a'

C1

a

PI

a'

C7

g

P7

g'

P. putida by. A LMG 2257T (ATCC 12633T ), LMG 5834t1 (NCPPB 1806), LMG 5835 (NCPPB 1807)

P. putida by. B LMG 1246t1 (ATCC 17430)

P. resinovorans LMG 2274T (ATCC 14235T )

P. stanieri LMG 6791 (CCUG 16025) LMG 6846 (CCUG 16023), LMG 6847T (CCUG 16021 T )

C5 C5

ND P3

P. stutzeri LMG 1228 LMG 2839 LMG 6397 LMG 2243

(NCTC 10475), LMG 2332 (MMCA 39), LMG 2334 (CCEB 522), (P18/27 Pane), LMG 5838 (NCPPB 1972), LMG 6394 (ATCC 11607), (H610 Hansen), LMG 11199T (ATCC 17588) (ATCC 14405)

C7 C7

g g

P7 P3

g'

C1

a

PI

a'

C2 C2

b b

PI

P3

b'

Cl

a

PI

a'

C1

a

PI

a'

C2

b

P3

b'

C4

d

P2

d'

C4 C4

d d

PI

C5

e

P3

P. synxantha LMG 2335 T (NCIB 8178 T )

P. syringae LMG 1247T (NCPPB 281 T ), LMG 12648 (DSM 6693) LMG 12643 (DSM 1856)

P. taetrolens LMG 2336T (CCEB 381 T )

P. tolaasii LMG 2339 (NCPPB 741), LMG 2342T (NCPPB 2192T ), LMG 2345 (NCPPB 2325), LMG 2346 (NCPPB 2412), LMG 2829 (NCPPB 1116)

P. viridi{lava LMG 12647 (DSM 6694), LMG 2352T (NCPPB 635 T ), LMG 2353 (NCPPB 1080), LMG 5101 (PDDCC 3884) rRNA group II

Bu. cepacia LMG 1222T (ATCC 25416T ), LMG 6889 (NCPPB 1962)

R. solanacearum LMG 2299T (NCPPB 325T ) LMG 2301 (NCPPB 446)

P3

rRNA group III

A. delafieldii LMG 1792t1 (ATCC 17506), LMG 5943 T (CCUG 1779T )

e'

Comparison of whole-cell and phospholipid fatty acid composition of pseudomonads

533

Table 1. Continued rRNA group' Species and strain numbers b

Whole-cell FAMEs'

Phospholipid FAMEs'

Cluster

Subcluster

Cluster

Subcluster

C5 C5

e e

ND P3

e'

C5 C5

e e

PI P3

e'

C5

e

P3

e'

C5

e

P3

e'

C5

e

P2

C5 C5

e e

P2 ND

C5 C5

e e

P6 P3

e'

P5

f'

A. facilis

LMG 2193 T (ATCC 11228 T ) LMG 6599 (CCUG 15920)

C. acidovorans LMG 1226T (ATCC 15668 T ) LMG 5932 (CCUG 727)

C. terrigena LMG 1253 T (NCIB 8193 T ), LMG 5520 (CCUG 12940)

C. testosteroni LMG 1787 (ATCC 17407), LMG 1800T (NCTC 10698 T )

H. (lava LMG 2185 T (DSM 619T )

H. pal/eronii LMG 2366 T tl (ATCC 17724T ) LMG 6348 (RH 2) H. pseudo(lava LMG 5945T (CCUG 13799T ) LMG 7584 (DSM 1084) rRNA group IV Br. diminuta

LMG 2089 T (ATCC 11568 T ), LMG 2337 (CIP 63.38)

C6

Ungrouped species

P. pertucinogena LMG 1874T (ATCC 190T ) LMG 1875 (ATCC 6627)

P3 P2

• Groupings of Pal/eroni et al. (1973) b Abbreviations: A., Acidovorax; Br., Brevundimonas; Bu., Burkholderia; c., Comamonas; H., Hydrogenophaga; P., Pseudomonas; R., Ralstonia T, Type strain The origin of the strains is indicated between brackets. Abbreviations: ATCC, American Type Culture Collection, Rockville, Maryland, USA; CCEB, Culture Collection of Entomogenous Bacteria, Department of Insect Pathology, Institute of Entomology, Prague, Czech Republic; CCUG, Culture Collection University of Goteborg, Department of Clinical Bacteriology, Goteborg, Sweden; CIP, Collection Bacterienne de L'Institut Pasteur, Paris, France; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany; HMB, Collection of H. Maraite, Louvain-la-Neuve, Belgium; LMG, Laboratorium voor Microbiologie Gent Culture Collection, Gent, Belgium; MMCA, Medical Microbiology Culture Collection, Aarhus, Denmark; NCIB, The National Collection of Industrial Bacteria, Aberdeen, Scotland, U.K.; NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, U.K.; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, U.K.; NIAS, National Institute of Agro-Environmental Sciences, Ministry of Agriculture, Forestry and Fishery, Ibaraki, Japan; NRRL, Agricultural Research Service Culture Collection, Northern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois, USA; PDDCC, Plant Diseases Division Culture Collection, Auckland, New Zealand; RH, Rudolph Hugh Collection, George Washington University, Washington D.C., USA , Clusters as indicated in Fig. 1 Subclusters are used for calculation of mean fatty acid profiles in Table 2 and 3 d U, ungrouped

tr

-

P. aeruginosa mean c (7)

tr 1.1(0.2) 1.2(0.2)

1.6 4.2(0.4) 1.4(0.6) 1.3(1.3)

-

delafieldii fadlis acidovorans terrigena testosteroni H. flava H. palleronii H. pseudoflava mean e (15)

Br. diminuta mean f (2)

A. A. C. C. C.

-

-

-

Bu. cepacia R. solanacearum mean d (4)

-

-

-

tr

-

2.6(3.6) 4.3(2.9)

-

3.6(0.3) 4.4(0.6) 3.5(0.3) 7.8(2.1) 8.0(1.2) 4.4

-

-

6.5(1.3)

5.8(0.2) 4.7(0.2) 4.3(0.9) 5.2(0.8) 4.5(0.3) 7.1(1.0) 6.2 5.4(0.7) 4.5(0.6) 5.3(1.3)

-

-

tr tr

-

-

-

tr

-

5.4(1.0)e 8.1(0.9) 6.7(0.7) 5.7(1.0) 6.9(0.4) 6.3(1.6) 7.3(1.2) 6.4 5.8 5.2(0.6) 6.3(1.3)

_d

P. asplenii P. caricapapayae P. cichorii P. coronafadens P. ficuserectae P. fuscovaginae P. mucidolens P. syringae P. viridiflava mean b (33)

P. agarid P. chlororaphis P. corrugata P. fluorescens P. fragi P. marginalis P. putida P. synxantha P. taetrolens P. tolaasii mean a (62)b

Species/ subduster b

10:0 30H

8:0 30H

-

2.3(1.7)

-

-

3.2(0.1) 3.7(0.1) 3.0(0.3) 3.8(0.4) 3.5(0.4)

-

4.4(0.4)

1.9(0.3) 5.8(0.4) 5.6(0.5) 5.7(0.3) 5.3(0.4) 2.4(0.6) 2.9 6.4(0.8) 6.9(0.4) 5.1(1.7)

2.5(0.4) 2.1(0.2) 5.4(1.4) 3.6(1.1) 4.7(0.2) 4.0(0.5) 3.3(1.6) 3.3 10.0 3.1(0.8) 3.7(1.5)

12:0

-

-

-

-

-

-

-

7.3(1.9)

8.0(0.1) 3.6(0.4) 3.7(0.7) 3.9(0.3) 3.8(0.3) 8.5(1.4) 6.1 4.6(0.6) 4.2(0.4) 5.0(2.0)

6.7(1.3) 8.1(1.2) 5.4(0.9) 6.9(1.3) 6.3(0.6) 7.2(1.1) 8.6(2.4) 5.6 1.9 7.2(0.7) 7.1(1.7)

12:0 20H

2.3(0.4)

-

-

-

-

-

-

6.6(1.2)

5.9(0.2) 4.9(0.5) 5.2(0.9) 5.4(0.5) 5.2(0.4) 6.7(1.1) 5.6 6.2(0.7) 6.3(0.5) 5.8(0.9)

5.8(1.2) 7.5(0.7) 6.1(0.9) 6.4(0.7) 6.5(0.3) 6.7(0.9) 7.1(1.1) 6.1 6.1 6.1(0.4) 6.5(0.9)

12:0 30H

1.3(0.1)

3.6(0.1) 3.2(0.1) tr 3.9(0.1) tr 3.5 tr 3.5(0.3) 2.3(1.6)

4.9(0.1) 4.8(0.1) 4.9(0.1)

tr

tr

-

tr tr tr tr tr tr

tr

-

tr tr tr tr tr tr

tr

tr

-

1.3(0.6) tr

14:0

-

-

-

-

12.3(1.0) 9.3(2.1) 10.8(2.2)

-

-

-

-

-

-

-

-

tr

-

-

-

-

tr

tr

-

14:0 30H

Table 2. Fatty acid content (mean percentage of total) of whole-cell hydrolysates of pseudomonads'

29.9(1.5)

26.8(2.0) 27.8(3.4) 32.9(2.9) 29.9(0.2) 24.6(1.4) 24.4 26.9(1.1) 20.5(3.2) 26.9(4.0)

19.8(1.2) 19.6(1.8) 19.7(1.3)

21.8(2.4)

26.5(0.7) 24.0(0.9) 25.6(1.9) 24.9(1.2) 24.1(1.2) 25.3(1.7) 26.3 24.0(1.7) 22.1(0.6) 24.4(1.8)

33.4(1.1) 27.3(1.5) 23.9(0.5) 26.6(1.3) 26.5(1.9) 27.5(1.7) 24.6(1.5) 25.9 28.6 30.5(2.1) 27.2(2.9)

16:0

1.6(0.1)

41.4(0.6) 42.2(1.1) 41.5(2.3) 39.8(2.4) 33.1(0.5) 49.6 41.7(1.1) 50.9(0.6) 42.0(5.3)

21.4(0.6) 30.7(0.1) 26.1 (5.4)

17.3(2.7)

36.9(1.3) 36.8(1.2) 36.0(1.3) 38.6(1.7) 36.2(1.2) 35.0(1.1) 31.1 35.8(2.2) 34.9(1.3) 35.9(1.9)

29.4(1.4) 26.5(7.0) 28.2(2.1) 30.3(2.1) 27.0(3.9) 30.4(2.8) 25.6(2.9) 23.4 29.0 23.7(1.8) 28.2(3.6)

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tr

tr tr tr tr 1.1(0.7) tr

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22.1(1.1) 18.7(4.0) 20.4(3.1)

32.6(3.3)

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domonas species. Within this rRNA group, three major genomic subgroups were recognized by Palleroni et al. (1973). Differentiation was made among a saprophytic fluorescent subgroup, a second subgroup of fluorescent phytopathogenic species, and a third subgroup containing the "alcaligenes" group, the "stutzeri" group and P. aeruginasa. The latter species was genomically more close to the "alcaligenes" group, but phenotypically more similar to the saprophytic pseudomonads (Palleroni, 1984). Although we investigated a lot more species than those presented in the latter work, it is remarkable to see the correlation between the DNA homology groupings of Palleroni (1984) and the whole-cell fatty acid clustering from the present study. Cluster C1 grouped strains of the saprophytic fluorescent species P. {luorescens, P. putida, P. chlororaphis, but also strains of the species P. agarici, P. amygdali, P. corrugata, P. fragi, P. marginalis, P. synxantha, P. taetrolens and P. tolaasii. Cluster C2 mainly grouped strains of phytopathogenic species, P. asplenii, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. fuscovaginae, P. meliae, P. syringae and P. viridi{lava. Cluster C7 grouped both members of the "alcaligenes" group (P. alcaligenes and P. pseudoalcaligenes), of the "stutzeri" group (P. stutzeri and P. mendocina) and the type strain of the species P. pertucinogena, P. oleovorans and P. resinovorans. P. aeruginosa clustered as a separate entity in cluster C3 close to the fluorescent pseudomonads. Stead (1992) also studied many rRNA group I taxa on the basis of cellular fatty acid analysis but did not differentiate among species of cluster C1 (P. agarici, P. chlororaphis, P. (luorescens, P. marginalis, P. putida and P. tolaasii), cluster C2 (P. asplenii, P. caricapapayae, P. cichorii, P. ficuserectae, P. fuscovaginae, P. syringae and P. viridi{lava) and cluster C3 (P. aeruginosa). The latter author did describe separate subgroups for members of cluster C7 (P. alcaligenes, P. pseudoalcaligenes and P. stutzeri), and for two species of cluster C1, P. corrugata and P. amygdali, respectively. Data from our study did not differentiate the latter two specIes. 5ubgrouping of rRNA group I organisms was also investigated using 165 rRNA sequence analyses (Moore et aI., 1996). Within the genus Pseudomonas two distinct intrageneric divisions were designated. The" P. aeruginosa (intrageneric) cluster" included the following species investigated in the present study: P. aeruginosa, P. alcaligenes, P. mendocina, P. oleovorans, P. pseudoalcaligenes, P. resinovorans and P. stutzeri. A second cluster, "the P. {luorescens (intrageneric) cluster" included the following species: P. {luorescens, P. agarici, P. amygdali, P. asplenii, P. chlororaphis, P. cichorii, P. coronafaciens, P. ficuserectae, P. marginalis, P. putida, P. syringae, P. tolaasii and P. viridi{lava. Except for the position of P. aeruginosa, fatty acid

536

M. Vancanneyt

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Fig. 1. Simplified dendrogram showing the relationships of pseudomonads strains based on numerical analysis of whole-cell fatty acids and phospholipid fany acids. The groups are indicated CI to C7 and PI to P7, respectively. Strains are indicated in Table 1. Subclusters were delineated (a to g and a' to g', respectively) which group strains that were used for calculation of mean fany acid compositions.

Comparison of whole-cell and phospholipid fatty acid composition of pseudomonads

data corroborated with this genomic division. Fatty acid analysis did not reveal all described genomic sublineages (Moore et aI., 1996). Within "the P. {luorescens cluster", no distinct fatty acid clusters were observed for P. asplenii and P. putida, and for P. agarici, respectively. On the other hand, a subgrouping of various phytopathogenic species was reflected in both approaches. Numerical analysis of the phospholipid fatty acid data also revealed seven groups (PI to P7). When comparing this phospholipid fatty acid clustering with the whole-cell fatty acid clustering, both similarities and discrepancies were observed. Similarities were the more or less analogous grouping of rRNA group I organisms and the separate position of rRNA group IV strains. Indeed, most of the strains of rRNA group I which were delineated in the whole-cell FAME clusters C1, C3 and C7 were found in the phospholipid clusters PI, P4 and P7, respectively. Discrepancies were the subdivision of the phytopathogenic species of cluster C2 into the clusters P3 and P6. Other major differences were observed for representatives of rRNA groups II and III, which did not form separate entities using phospholipid fatty acid data and clustered among rRNA group I organisms. Fatty acid content of the major groupings The major fatty acid content of the whole-cell hydrolysates and phospholipid fractions are presented in Table 2 and 3, respectively. Delineation of groups for calculation of mean fatty acid contents was largely based on the whole-cell fatty acid clustering because the latter grouping was more consistent with available genomic information (see above). However, mean values were not calculated using all strains of the respective clusters C1 to C7 and PI to P7 because of the aberrant or unexpected position of some strains in the numerical analyses (Fig. 1). A subset of strains was selected for calculation of mean fatty acid compositions, indicated as subclusters a to g for whole-cell hydrolysates and a' to g' for phospholipid fractions, respectively (Fig. 1; Table 1). Both tables (Table 2 and 3) indicated that 16: 0 and isomers of 16: 1 and 18: 1 were major fatty acid components of all taxa studied. Whole-cell fatty acid patterns were easily differentiated from phospholipid fatty acid profiles by the presence of significant amounts of hydroxylated fatty acids. Wollenweber and Rietschel (1990) demonstrated that hydroxy fatty acids predominate within the lipopolysaccharides and that they are generally missing in other cell-wall lipids of Gram-negative bacteria. As indicated in literature, they also might be used as diagnostic markers for characterization of the major phylogenetic groups within the pseudomonads (Moss et aI., 1972; Ikemoto et aI., 1978; Oyaizu and Komagata, 1983; Stead, 1992). Data from the present study and literature data demonstrated that, based on the presence or absence of three 3hydroxy fatty acids, 10:0 30H, 12:0 30H, and 14:0 30H, the majority of the taxa could be allocated to one of the four rRNA groups I to IV. Characteristic for the rRNA group I organisms was the presence of both 10:0 30H and 12: 0 30H and the absence of significant amounts of

537

14: 0 30H. The rRNA group II strains only contained 14:0 30H, group III organisms only contained 10:0 30H and group IV organisms only contained 12: 0 30H. Exceptions were P. stanieri, belonging to the P. {luorescens rRNA branch (Willems et aI., 1992), which lacked 12: 0 30H (Table 2) and Hydrogenophaga palleronii, an organism of rRNA group III, did not contain the fatty acid 10: 0 30H. The latter statement was confirmed by Willems et al. (1989) who demonstrated that also H. taeniospiralis lacked this component. In general, results from the present study correlated well with data from literature. Only two differences were observed with the study of Oyaizu and Komagata (1983). At first, the latter authors mentioned 14:0 30H as a diagnostic peak for Brevundimonas species (rRNA group IV). Data from this study and those of a more extended polyphasic work on these taxa (Segers et aI., 1994) did not confirm the presence of this feature. Secondly, Oyaizu and Komagata (1983) indicated for Acidovorax avenae, a species which was not included in our work, the following four fatty acids as diagnostic markers: 10:0 30H, 12:0 30H, 14:0 30H and 14: 1 30H. Stead et al. (1992) and Willems et al. (1989) also studied A. avenae strains and their data lacked the latter three fatty acids. The present study, in which two other Acidovorax species were studied, corroborated with data from the latter authors. Except for the characteristic and discriminating 3hydroxy fatty acids, also other qualitative differences in the whole-cell fatty acid pattern were observed among the major phylogenetic groups. Two hydroxy fatty acids, 16: 1 20H and 18: 1 20H, were characteristic for Burkholderia and Ralstonia species (rRNA group II). Only Brevundimonas diminuta strains, representing rRNA group IV, showed an appreciable amount of 19: 0 cyclo <.o8c. The latter fatty acid, however, was not detected in Br. vesicularis (Segers et aI., 1994). Besides the presence of unique fatty acids, also quantitative information could be used to differentiate the four rRNA groups. Major quantitative differences were observed among the dominant fatty acids 16:0 and isomers of 16:1 and 18:1 (indicated as SF 7 in Table 2). For instance, rRNA group IV organisms showed the lowest amount of 16: 1 and the highest amount of 18:1 (SF 7). When comparing the whole-cell fatty acid content with the phospholipid fatty acid content a complete absence of hydroxylated fatty acids in the phospholipid fraction was detected (Table 3). Also the shorter chain fatty acid 12: 0 was not found. A few fatty acids, 16: 1 <.o7t, several isomers of 19: 1 and 20: 1 <.o9c, were unique for the phospholipid fraction. From Table 3, it is demonstrated that 16: 1 <.o7t was characteristic for most of the rRNA group I organisms, the isomers of 19: 1 characterized rRNA group II organisms and 20: 1 <.o9c did occur in taxa of rRNA groups II, III and IV. At this moment it is not clear why these features were not detected in the whole-cell hydrolysates. Only growth conditions and extraction procedures differentiated both fatty acid analyses, two parameters which may influence more the quantitative content rather than the qualitative fatty acid composition (Suzuki et aI., 1993). An overall comparison of the fatty acid composi-

27.5 (9.4)

tr

tr

tr

tr

c' (7)

d' (2)

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28.1 (0.7)

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1.6 (0.6)

41.1 (2.4)

tr

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28.7 (5.4)

33.8 (2.2)

8.2 (3.8)

-

tr

tr

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9.1 (5.4)

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tr

tr

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tr

tr

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1.4 (0.5)

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tr

tr

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1.6 (1.4)

tr

1.3 (1.0)

20.7 (3.8)

4.0 (1.9)

1.0 (0.7)

2.7

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1.3 (0.3)

tr

tr

tr

2.0 (0.6)

2.5 (2.0)

1.6 (0.7)

cyclo w7c

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tr

tr

tr

5.2 (1.1)

1.1 (0.5)

tr

1.2 (1.1)

5.3 (1.7)

w9c

18:1

36.9 (5.6)

48.1 (3.1)

16.3 (4.5)

2.2 (0.5)

35.0 (6.7)

17.5 (4.4)

20.8 (0.9)

2.8 (3.3)

1.0 (0.9)

tr

tr

3.0 (2.0)

1.1 (1.2)

tr (2.8)

2.1 (1.9)

w7t

w7c/w9t 11.2 (4.8)

18:1

18:1

2.1 (0.3) tr

-

tr

1.6 (0.1)

tr

tr

tr

tr

3.9 (3.5)

tr

tr tr

tr

tr

tr (1.2)

1.4 (0.6)

C

19:1 c

1.7 (1.8)

1.8

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tr

B

19:1"

tr

A

19:1"

20:1

tr

4.8 (2.7)

19.0 (0.1)

7.1 (4.2)

tr

1.8 (1.7)

tr

5.9 (0.1)

3.1 (0.8)

1.6 (0.5)

tr

tr

cyclo w8c w9c

19:0

• Only the fatty acids counting for more than 1.0% (mean amount) were indicated. Subcluster d' contained also a considerable amount of two other isomers of 19: 1 (3.0% and 3.4% respectively) b Subclusters are delineated in Fig. 1 and their respective strains are indicated in Table 1. Total number of strains are indicated between brackets c The double bond position indicated by the capital letters is unknown d tr, Trace amount « 1.0%) e Standard deviation f -, Not detected

g' (21)

1.8 (1.8)

30.9 (1.3)

tr

b" (13)

(2)

36.3 (3.2)

tr

b' (17)

37.3 (5.4)

34.2

1.1 (0.8)d

40.3 (4.3)

16:0

a' (66)

subclusterb

14:0

Table 3. Fatty acid composition (mean percentage of total) of phospholipid fractions of pseudomonads'

'<

....

(l)

::l ::l

("l

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Comparison of whole-cell and phospholipid fatty acid composition of pseudomonads tion of the phospholipids showed no diagnostic peaks for each of the main phylogenetic groups (rRNA groups). Groupings were generally based on quantitative differences. The relative amount of the fatty acids was in some cases comparable to those found in the whole-cell fatty acid content. For instance, both data sets showed the lowest amount of 16: 1 and the highest amount of 18: 1 for rRNA group IV organisms and rRNA group III taxa showed a highest mean value of 16: 1. A significantly higher amount of cyclo fatty acids (17: 0 cyclo and 19: 0 cyclo) in the phospholipid fraction of rRNA group II strains compared to the whole-cell extracts could be explained by different cultivation conditions (Welch, 1991). As indicated above, organisms belonging to the genuine genus Pseudomonas (rRNA group I) consisted of some major fatty acid clusters which reflected rather well the phenotypic and genomic groupings. Whole-cell fatty acid data demonstrated some qualitative differences among these subgroups: e. g. the "alcaligenes-stutzeri" group (cluster C7) lacked significant amounts of 12: 0 20H and the "{luorescens" group (cluster C1) consisted of high relative amounts of 17: 0 cyclo. Also quantitative differences were observed among the various rRNA group I clusters which were comparable to those observed in the fatty acid content of phospholipids. Because of the limited number of strains studied within the genera Burkholderia and Ralstonia (rRNA group II) and Brevundimonas (rRNA group IV), no general statements about intrageneric relationships could be drawn. Among rRNA group III organisms, including taxa of the genera Acidovorax, Comamonas and Hydrogenophaga, only the latter genus was easily discriminated by the absence of 12:0. These data confirmed those of Willems et al. (1989), although the latter authors detected a small amount of 12: 0 in H. taeniospiralis.

Fatty acid content at the species level From the phospholipid fatty acid profiles, no mean fatty acid compositions for individual species were calculated because species-specific differentiating characteristics were not observed within the different clusters (a'-g ' ). From the whole-cell fatty acid composition, a mean fatty acid content was calculated for each species although species discrimination was not possible within all clusters a to g (Table 2). Within cluster a there was a large overlap between fatty acid profiles of the majority of the species. Only on a basis of smaller quantitative differences, some of the species were distinguished e. g. Pseudomonas agarici and P. synxantha. Also species of cluster b showed overlapping FAME contents and proved to be very similar. Cluster c only grouped P. aeruginosa strains which were characterized by a unique fatty acid content. Cluster d grouped Burkholderia cepacia and Ralstonia solanacearum which could be differentiated both qualitatively and quantitatively. Characteristic for Burkholderia cepacia was the presence of 16: 0 30H and for Ralstonia solanacearum small amounts of 15: 0,17: 0 and 17: 1 w6c were detected. Within cluster e, grouping the genera Acidovorax, Comamonas and Hydrogenophaga, species-specific profiles were ob-

539

served for Comamonas testosteroni which contained significant amounts of 16: 0 20H and 16: 120H and for Hydrogenophaga palleronii which lacked 10: 0 30H and contained 17: 0 cyclo. Except for the Acidovorax species, at least quantitative discrimination among species of these genera was found. Cluster f only grouped Brevundimonas diminuta. This species was distinguished from the second member of the genus Br. vesicularis as demonstrated by Segers et al. (1994). Among taxa of cluster g most species showed quantitatively unique FAME contents. Only four species were not classified into one of the above mentioned clusters a to g. Pseudomonas amygdali contained some very characteristic fatty acids 16: 0 30H and 19: 0 10 methyl. The position of P. meliae was different after numerical analysis of whole-cell and phospholipid fatty acid profiles (C2 and P1, respectively). The two investigated strains of P. pertucinogena occupied distinct positions on the dendrogram mainly due to quantitative differences in their fatty acid profiles (Fig. 1; Table 2). P. stanieri was not grouped because it showed the diagnostic fatty acids of rRNA group III although the species belongs to rRNA group I (Willems et aI., 1992).

Conclusions Numerical analysis of fatty acid contents of phospholipid fractions and whole-cell hydrolysates of pseudomonads yielded more or less analogous groupings which reflected their phylogenetic relationships. Whole-cell hydrolysates contained various hydroxy fatty acids which could be used as taxonomic markers. Also for characterization at the species level whole-cell fatty acid data proved to be more valuable than data from phospholipids. Acknowledgements. This work was carried out in the framework of contracts BIOT-CT91-0294 and BI02-CT94-3098 of the Commission of European Communities. We thank Michaela Blank for excellent technical assistence in preparing phospholipid fatty acid extracts.

References Bligh, E. G., Dyer, W.].: A rapid method for total lipid extraction

and purification. Can. J. Biochem. Physiol. 37, 911-917 (1959)

De Vos, P., De Ley, ].: Intra- and intergeneric similarities of Pseudomonas and Xanthomonas ribosomal ribonucleic acid cis-

trons. Int. J. Syst. Bacteriol. 33, 487-509 (1983)

Fredrickson, H. L., Cappenberg, T. E., de Leeuw, ].: Polar lipid

fatty acid compositions of Lake Vechten seston - an ecological application of lipid analysis. FEMS Microb. Ecol. 38,381-396 (1986) Ikemoto, 5., Kuraishi, H., Komagata, K., Azuma, R., Suto, T., Murooka, H.: Cellular fatty acid composition in Pseudomonas species. J. Gen. Appl. Microbiol. 24, 199-213 (1978) Jantzen, E., Bryn, K.: Analysis of cellular constituents from Gram-negative bacteria, pp.21-61. In: Chemical Methods in Prokaryotic Systematics (M. Goodfellow, A. G. O'Donnell, eds.), New York, John Wiley and Sons Ltd. 1994 Kates, M.: Bacterial lipids, Advances in Lipid Research 2, 17-90 (1964)

540

M. Vancanneyt

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Marc Vancanneyt, Laboratorium voor Microbiologie, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium