FEMS MicrobiologyLetters 59 (1989) 101-106 Published by Elsevier
101
FEM 03545
The presence of ll-methyloctadec-ll-enoic acid in the extractable lipids of Pseudomonas vesicularis A. Barnes, L. G a l b r a i t h a n d S.G. W i l k i n s o n School of Chemistry, Universityof Hull, Hull, U.K. Received 20 December 1988 Accepted 6 January 1989 Key words: Pseudomonas vesicularis; Lipid; Fatty acid
1. S U M M A R Y The polar lipids of Pseudomonas vesicularis do not contain a methyleneoctadecanoic acid but instead the isomeric l l - m e t h y l o c t a d e c - l l - e n o i c acid. The presence of the latter acid distinguishes the lipids of P. vesicularis from those of P. diminuta, but links them with the lipids of Rhizobium and Caulobacter species.
2. I N T R O D U C T I O N The species Pseudomonas vesicularis and P. diminuta form r R N A group IV of the organisms currently included in the genus Pseudomonas [1]. The placement of this group is unsatisfactory, and a phylogenetic location within subgroup a-2 of the purple bacteria has been diagnosed [2]. Among the distinctive features of the organisms are a lipid A based on 2,3-diamino-2,3-dideoxy-D-glucose rather than 2-amino-2-deoxy-D-glucose [3,4] and a unique combination of glycolipids [5]. During a study [5] of the polar lipids of P. vesicularis, an unusual
Correspondence to: A. Barnes, School of Chemistry, University of Hull, Hull, U.K.
fatty acid was detected. The identification of this component as l l - m e t h y l o c t a d e c - l l - e n o i c acid is now reported.
3. M A T E R I A L S A N D M E T H O D S 3.1. Organisms and growth conditions Cultures of three strains of P. vesicularis ( N C T C 10900, N C T C 11166, and N C T C 11167) were grown for 48 h at 3 0 ° C on nutrient agar CM3 (Oxoid). The cells were harvested and washed three times with water, then freeze-dried. 3.2. Extraction and fractionation of lipids Cells were stirred for 2 h at room temperature with c h l o r o f o r m / m e t h a n o l (2:1, v / v ; 60 m l / g cells), the suspension was filtered (No. 4 porosity glass sinter), and the insoluble residue was washed with further solvent. The combined filtrate and washings were dried by rotary evaporation, the lipids were dissolved in chloroform (1 vol.) and fractionated by the addition of acetone (4 vol.). Subsequent studies were based mainly on the soluble (glycolipid-enriched) fraction. 3.3. Preparation and analysis of fatty esters Fatty acids were released by mild alkaline methanolysis of the lipids [6]. The methyl esters
0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies
102
produced were fractionated by thin-layer chromatography on Silica gel G (Merck) containing 5% ( w / w ) A g N O 3 with dichloromethane as the solvent. The separated fractions were detected by using 2',7'-dichlorofluorescein and recovered by elution with diethyl ether. Methyl esters were converted into 3-picolinyl esters [7,8] by the method of Wait and Hudson [9]. Monoenoic esters were hydrogenated for 2 h at room temperature and atmospheric pressure, using 5% ( w / w ) palladium on charcoal as the catalyst and methanol as the solvent. Oxidation of the double bond in the branched, monoenoic ester from P. vesicular&, followed by H P L C of phenacyl esters of the products, was done by the method of Longmuir et al. [10]. Methyl cis-vaccenate and heptanoic acid were used as standards for the identification of products. G L C of methyl and 3-picolinyl esters was carried out with a fused-silica capillary column (30 m) of BP1 (Scientific Glass Engineering) in a Mega 5160 chromatograph (Carlo Erba). Mass spectra (from combined G L C - M S or by direct insertion) were obtained by using a Finnigan 1020B mass spectrometer.
4. RESULTS As the previous study [5] of the lipids in P.
vesicular& N C T C 10900 had shown that the unidentified fatty acid was concentrated into monoglycosyldiacylglycerols, the acetone-soluble (glycolipid-enriched) lipids were used as the source of this acid. Determination of the fatty acid composition (Table 1) for these lipids confirmed that the unidentified acid (compound X) was a major component for all three strains examined. On GLC, compound X as the methyl ester had a retention time very similar to that of methyl octadecanoate (18:0). However, previous results obtained by G L C with a polar column and by argentation thin-layer chromatography indicated that compound X was unsaturated [5]. This was confirmed by mild hydrogenation, which produced a change in retention time of the ester on the BP1 column from 11.42 min to 13.25 min, and by mass spectrometry, which showed that corn-
Table 1 Fatty acid composition of the acetone-soluble fraction of lipids from P. vesicularis Fatty acid
14:0 15:0 cis-16:l
16:0 cis-17:l ~
17:0 cis-18:1
18:0 Compound X Others
Strain N C TC 10900
N C TC 11166
NCTC 11167
0.5 5.9 4.7 23.3 4.8 3.8 39.2 Tr 17.8 Tr
0.6 4.9 7.4 13.0 10.4 4.6 36.9 Tr 21.9 0.3
0.4 3.8 4.3 15.9 5.3 6.1 37.6 1.7 23.5 1.4
a Two isomers present. Results are expressed as percentages of the total peak area on G L C of the methyl esters. Fatty acid shorthand: number before the colon, the total number of carbon atoms; number after the colon, the number of double bonds. Tr, trace.
pound X was a C19 monoenoic acid (molecular ion for the methyl ester at m / z 310, changed to m / z 312 by hydrogenation). Inspection of the mass spectra for the methyl and the 3-picolinyl esters of the hydrogenated acid identified it as ll-methyloctadecanoic acid. In the mass spectrum of the methyl ester (Fig. 1), ions diagnostic for a methyl branch at position 11 were present at m / z 213, 185, 181, and 163 [11,12], and there was no significant ion with m / z 199 which is prominent for n-alkaline esters [13]. Similarly, the mass spectrum of the 3-picolinyl ester contained diagnostic ions with m / z 290 and 262 but no peak at m / z 276 [7]. The position of the double bond in compound X could not be determined from the mass spectrum of the methyl ester, although the spectrum closely resembled that reported for methyl 11methyloctadec-ll-enoate [12]. The mass spectrum of the 3-picolinyl ester of compound X (Fig. 2) contained the molecular ion ( m / z 387) and the typical fragments with m / z 164, 151, 108 and 92 [7,8]. The gap between the signals with m / z 302 and 262 is consistent with fragmentation of an ester with the double bond between C-11 and C-12 and a methyl group attached at one of these positions. Further evidence for an 11-methyl 11-
103
4:3
1OO.O -
74
87
50..~
-
•
]
111
17
. -
t
221,
2~$
Fig. 1. Mass spectrum of the methyl ester of hydrogenated compound X.
1~3.0
92
-
164
55
JI
151
81
JI,,hl,.,,,,ll,l,.,ll ° ....
' ....
4e lOe.0
-
5~.o
-
I ....
' ....
~
I I],, I ....
21~
~,h,,,lh ...... 11, ,Z,.,1 ....... 1~, i ....
~
I ....
' ....
1~
I ....
' ....
128
,.,,I,I
I ....
,
,....... ,l~,~l,J,,~..=,. i ....
t4~
I ....
. ....
1~3
I ....
i ....
188
220
. , ,,.. I ....
I ....
2~1
,., I ....
,
' ....
2~
33~
344 :382
....
248
, ....
I .... 2 ~
, ....
I .... 298
i ....
I .... ~
316
' ....
I .... 329
' ....
I .... 348
i ....
I .... To9
i ....
I .... ~ee
~ ....
I .... 4ea
Fig. 2. Mass spectrum of the 3-picolinyl ester of compound X.
' ....
I .... 491R
I ' ~ "
104
enoic ester was obtained by oxidative cleavage of the double bond [10] and conversion of the monocarboxylic acid produced into its phenacyl ester. On HPLC, the latter ester had the retention time of phenacyl heptanoate, and the mass spectra of the reference ester and that derived from the bacterial lipid also matched (molecular ions at m / z 248, major fragment ions at m / z 178 and 113). Thus, compound X is an ll-methyloctadecll-enoic acid of undefined geometric configuration.
5. DISCUSSION The fatty acid identified here as 11-methyloctadec-11-enoic acid has not been described previously as a component of the lipids of P. vesicular& [14-17], possibly because of the similar properties on GLC of its methyl ester and that of octadecanoic acid. However, a 11-methyloctadecll-enoic acid is a typical component of the lipids of Rhizobiurn and Bradyrhizobium species [12,18, 19] and of Caulobacter species [20]. The possibility of different geometrical isomers cannot be discounted, as the fatty acid in Rhizobium is relatively resistant to hydrogenation and bromination [12], and the behaviour on argentation thin-layer chromatography of the methyl ester of the acid in Caulobacter [20] (described as the cis isomer, without rigorous proof) differed somewhat from that found [5] for P. vesicular&. Compound X may reasonably be considered as a product from ring-opening of the isomeric cyclopropane acid (lactobacillic acid), itself derived from cis-vaccenic acid. It is therefore interesting to note that the lack of lactobacillic acid is one feature that distinguishes the lipids of P. vesicular& from those of P. diminuta [21]. Conversely, P. vesicularis produces carotenoids and a heptosyldiacylglycerol not found in P. diminuta [5]. Unusual properties shared by the two species include a lipid A based on 2,3-diamino-2,3-dideoxy-D-glucose, the lack of phosphatidylethanolamine, and presence of unusual glycolipids incorporating glucuronic acid. Intriguingly, Caulobacter species also lack phosphatidylethanolamine [2224], produce glycolipids as major polar lipids
[24,25], and do not contain 2-amino-2-deoxy-Dglucose in their lipid A [26]. The recent observation [27] that slow-growing rhizobia also incorporate some 2,3-diamino-2,3-dideoxyglucose into lipid A is also noteworthy, although the similarity with P. vesicularis does not extend to their polar lipids [28].
REFERENCES [1] Palleroni, N.J. (1984) in Bergey's Manual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G., eds.), Vol. 1, pp. 141-199, Williams and Wilkins, Baltimore, MD. [2] Woese, C.R. (1987) Microbiol. Rev. 51, 221-271. [3] Wilkinson, S.G. and Taylor, D.P. (1978) J. Gen. Microbiol. 109, 367-370. [4] Kasai, N., Arata, S., Mashimo, J., Akiyama, Y., Tanaka, C., Egawa, K. and Tanaka, S. (1987) Biochem. Biophys. Res. Commun. 142, 972-978. [5] Wilkinson, S.G. and Galbraith, L. (1979) Biochim. Biophys. Acta 575, 244-254. [6] Wilkinson, S.G. (1968) Biochim. Biophys. Acta 164, 148-156. [7] Harvey, D.J. (1982) Biomed. Mass Spectrom. 9, 33-38. [8] Christie, W.W., Brechany, E.Y. and Holman, R.T. (1987) Lipids 22, 224-228. [9] Wait, R. and Hudson, M.J. (1985) Lett. Appl. Microbiol. 1, 95-99. [10] Longmuir, K.J., Rossi, M.E. and Resele-Tiden, C. (1987) Anal. Biochem. 167, 213-221. [11] Ryhage, R. and Stenhagen, E. (1960) Arkiv Kemi 15. 291-315. [12] Gerson, T., Patel, J.J. and Nixon, L.N. (1975) Lipids 10, 134-139. [13] McCloskey, J.A. (1970) Top. Lipid Chem. 1, 369-440. [14] Kaltenbach, C.M., Moss, C.W. and Weaver, R.E. (1975) J. Clin. Microbiol. 1, 339-344. [15] Kiprianova, E.A., Andreev, L.V. and Boiko, O.I. (1980) Mikrobiol. Zh. (Kiev) 42, 11-16. [16] Chen, M.M.G. and Chen, P.H. (1981) Proc. Natl. Counc., Repub. China 5A, 202-212. [17] Oyaizu, H. and Komagata, K. (1983) J. Gen. Appl. Microbiol. 29, 17-40. [18] Gerson, T. and Patel, J.J. (1975) Appl. Microbiol. 30, 193 198. [19] MacKenzie, S.L., Lapp, M.S. and Child, J.J. (1979) Can. J. Microbiol. 25, 68-74. [20] Andreev, L.V., Akimov, V.N. and Nikitin, D.I. (1986) Folia Microbiol. (Prague) 31, 144-153. [21] Moss, C.W. (1978) in Glucose Nonfermenting Gramnegative Bacteria in Clinical Microbiology (Gilardi G.L., Ed.), pp. 171-201, CRC Press, West Palm Beach. [22] Contreras, I., Shapiro, L. and Henry, S. (1978) J. Bacteriol. 135, 1130-1136.
105 [23] Jones, D.E. and Smith, J.D. (1979) Can. J. Biochem. 57, 424-428. [24] De Siervo, A.J. and Homola, A.D. (1980) J. Bacteriol. 143, 1215-1222. [25] De Siervo, A.J. (1985) J. Bacteriol. 164, 684-688. [26] Agabian, N. and Unger, B. (1978) J. Bacteriol. 133, 987-994.
[27] Weckesser, J. and Mayer, H. (1988) FEMS Microbiol. Rev. 54, 143-154. [28] Wilkinson, S.G. (1988) in Microbial Lipids, Vol. 1 (Ratledge, C. and Wilkinson, S.G., eds.), pp. 299-488, Academic, London.
101
FEM 03545
The presence of ll-methyloctadec-ll-enoic acid in the extractable lipids of Pseudomonas vesicularis A. Barnes, L. G a l b r a i t h a n d S.G. W i l k i n s o n School of Chemistry, Universityof Hull, Hull, U.K. Received 20 December 1988 Accepted 6 January 1989 Key words: Pseudomonas vesicularis; Lipid; Fatty acid
1. S U M M A R Y The polar lipids of Pseudomonas vesicularis do not contain a methyleneoctadecanoic acid but instead the isomeric l l - m e t h y l o c t a d e c - l l - e n o i c acid. The presence of the latter acid distinguishes the lipids of P. vesicularis from those of P. diminuta, but links them with the lipids of Rhizobium and Caulobacter species.
2. I N T R O D U C T I O N The species Pseudomonas vesicularis and P. diminuta form r R N A group IV of the organisms currently included in the genus Pseudomonas [1]. The placement of this group is unsatisfactory, and a phylogenetic location within subgroup a-2 of the purple bacteria has been diagnosed [2]. Among the distinctive features of the organisms are a lipid A based on 2,3-diamino-2,3-dideoxy-D-glucose rather than 2-amino-2-deoxy-D-glucose [3,4] and a unique combination of glycolipids [5]. During a study [5] of the polar lipids of P. vesicularis, an unusual
Correspondence to: A. Barnes, School of Chemistry, University of Hull, Hull, U.K.
fatty acid was detected. The identification of this component as l l - m e t h y l o c t a d e c - l l - e n o i c acid is now reported.
3. M A T E R I A L S A N D M E T H O D S 3.1. Organisms and growth conditions Cultures of three strains of P. vesicularis ( N C T C 10900, N C T C 11166, and N C T C 11167) were grown for 48 h at 3 0 ° C on nutrient agar CM3 (Oxoid). The cells were harvested and washed three times with water, then freeze-dried. 3.2. Extraction and fractionation of lipids Cells were stirred for 2 h at room temperature with c h l o r o f o r m / m e t h a n o l (2:1, v / v ; 60 m l / g cells), the suspension was filtered (No. 4 porosity glass sinter), and the insoluble residue was washed with further solvent. The combined filtrate and washings were dried by rotary evaporation, the lipids were dissolved in chloroform (1 vol.) and fractionated by the addition of acetone (4 vol.). Subsequent studies were based mainly on the soluble (glycolipid-enriched) fraction. 3.3. Preparation and analysis of fatty esters Fatty acids were released by mild alkaline methanolysis of the lipids [6]. The methyl esters
0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies
102
produced were fractionated by thin-layer chromatography on Silica gel G (Merck) containing 5% ( w / w ) A g N O 3 with dichloromethane as the solvent. The separated fractions were detected by using 2',7'-dichlorofluorescein and recovered by elution with diethyl ether. Methyl esters were converted into 3-picolinyl esters [7,8] by the method of Wait and Hudson [9]. Monoenoic esters were hydrogenated for 2 h at room temperature and atmospheric pressure, using 5% ( w / w ) palladium on charcoal as the catalyst and methanol as the solvent. Oxidation of the double bond in the branched, monoenoic ester from P. vesicular&, followed by H P L C of phenacyl esters of the products, was done by the method of Longmuir et al. [10]. Methyl cis-vaccenate and heptanoic acid were used as standards for the identification of products. G L C of methyl and 3-picolinyl esters was carried out with a fused-silica capillary column (30 m) of BP1 (Scientific Glass Engineering) in a Mega 5160 chromatograph (Carlo Erba). Mass spectra (from combined G L C - M S or by direct insertion) were obtained by using a Finnigan 1020B mass spectrometer.
4. RESULTS As the previous study [5] of the lipids in P.
vesicular& N C T C 10900 had shown that the unidentified fatty acid was concentrated into monoglycosyldiacylglycerols, the acetone-soluble (glycolipid-enriched) lipids were used as the source of this acid. Determination of the fatty acid composition (Table 1) for these lipids confirmed that the unidentified acid (compound X) was a major component for all three strains examined. On GLC, compound X as the methyl ester had a retention time very similar to that of methyl octadecanoate (18:0). However, previous results obtained by G L C with a polar column and by argentation thin-layer chromatography indicated that compound X was unsaturated [5]. This was confirmed by mild hydrogenation, which produced a change in retention time of the ester on the BP1 column from 11.42 min to 13.25 min, and by mass spectrometry, which showed that corn-
Table 1 Fatty acid composition of the acetone-soluble fraction of lipids from P. vesicularis Fatty acid
14:0 15:0 cis-16:l
16:0 cis-17:l ~
17:0 cis-18:1
18:0 Compound X Others
Strain N C TC 10900
N C TC 11166
NCTC 11167
0.5 5.9 4.7 23.3 4.8 3.8 39.2 Tr 17.8 Tr
0.6 4.9 7.4 13.0 10.4 4.6 36.9 Tr 21.9 0.3
0.4 3.8 4.3 15.9 5.3 6.1 37.6 1.7 23.5 1.4
a Two isomers present. Results are expressed as percentages of the total peak area on G L C of the methyl esters. Fatty acid shorthand: number before the colon, the total number of carbon atoms; number after the colon, the number of double bonds. Tr, trace.
pound X was a C19 monoenoic acid (molecular ion for the methyl ester at m / z 310, changed to m / z 312 by hydrogenation). Inspection of the mass spectra for the methyl and the 3-picolinyl esters of the hydrogenated acid identified it as ll-methyloctadecanoic acid. In the mass spectrum of the methyl ester (Fig. 1), ions diagnostic for a methyl branch at position 11 were present at m / z 213, 185, 181, and 163 [11,12], and there was no significant ion with m / z 199 which is prominent for n-alkaline esters [13]. Similarly, the mass spectrum of the 3-picolinyl ester contained diagnostic ions with m / z 290 and 262 but no peak at m / z 276 [7]. The position of the double bond in compound X could not be determined from the mass spectrum of the methyl ester, although the spectrum closely resembled that reported for methyl 11methyloctadec-ll-enoate [12]. The mass spectrum of the 3-picolinyl ester of compound X (Fig. 2) contained the molecular ion ( m / z 387) and the typical fragments with m / z 164, 151, 108 and 92 [7,8]. The gap between the signals with m / z 302 and 262 is consistent with fragmentation of an ester with the double bond between C-11 and C-12 and a methyl group attached at one of these positions. Further evidence for an 11-methyl 11-
103
4:3
1OO.O -
74
87
50..~
-
•
]
111
17
. -
t
221,
2~$
Fig. 1. Mass spectrum of the methyl ester of hydrogenated compound X.
1~3.0
92
-
164
55
JI
151
81
JI,,hl,.,,,,ll,l,.,ll ° ....
' ....
4e lOe.0
-
5~.o
-
I ....
' ....
~
I I],, I ....
21~
~,h,,,lh ...... 11, ,Z,.,1 ....... 1~, i ....
~
I ....
' ....
1~
I ....
' ....
128
,.,,I,I
I ....
,
,....... ,l~,~l,J,,~..=,. i ....
t4~
I ....
. ....
1~3
I ....
i ....
188
220
. , ,,.. I ....
I ....
2~1
,., I ....
,
' ....
2~
33~
344 :382
....
248
, ....
I .... 2 ~
, ....
I .... 298
i ....
I .... ~
316
' ....
I .... 329
' ....
I .... 348
i ....
I .... To9
i ....
I .... ~ee
~ ....
I .... 4ea
Fig. 2. Mass spectrum of the 3-picolinyl ester of compound X.
' ....
I .... 491R
I ' ~ "
104
enoic ester was obtained by oxidative cleavage of the double bond [10] and conversion of the monocarboxylic acid produced into its phenacyl ester. On HPLC, the latter ester had the retention time of phenacyl heptanoate, and the mass spectra of the reference ester and that derived from the bacterial lipid also matched (molecular ions at m / z 248, major fragment ions at m / z 178 and 113). Thus, compound X is an ll-methyloctadecll-enoic acid of undefined geometric configuration.
5. DISCUSSION The fatty acid identified here as 11-methyloctadec-11-enoic acid has not been described previously as a component of the lipids of P. vesicular& [14-17], possibly because of the similar properties on GLC of its methyl ester and that of octadecanoic acid. However, a 11-methyloctadecll-enoic acid is a typical component of the lipids of Rhizobiurn and Bradyrhizobium species [12,18, 19] and of Caulobacter species [20]. The possibility of different geometrical isomers cannot be discounted, as the fatty acid in Rhizobium is relatively resistant to hydrogenation and bromination [12], and the behaviour on argentation thin-layer chromatography of the methyl ester of the acid in Caulobacter [20] (described as the cis isomer, without rigorous proof) differed somewhat from that found [5] for P. vesicular&. Compound X may reasonably be considered as a product from ring-opening of the isomeric cyclopropane acid (lactobacillic acid), itself derived from cis-vaccenic acid. It is therefore interesting to note that the lack of lactobacillic acid is one feature that distinguishes the lipids of P. vesicular& from those of P. diminuta [21]. Conversely, P. vesicularis produces carotenoids and a heptosyldiacylglycerol not found in P. diminuta [5]. Unusual properties shared by the two species include a lipid A based on 2,3-diamino-2,3-dideoxy-D-glucose, the lack of phosphatidylethanolamine, and presence of unusual glycolipids incorporating glucuronic acid. Intriguingly, Caulobacter species also lack phosphatidylethanolamine [2224], produce glycolipids as major polar lipids
[24,25], and do not contain 2-amino-2-deoxy-Dglucose in their lipid A [26]. The recent observation [27] that slow-growing rhizobia also incorporate some 2,3-diamino-2,3-dideoxyglucose into lipid A is also noteworthy, although the similarity with P. vesicularis does not extend to their polar lipids [28].
REFERENCES [1] Palleroni, N.J. (1984) in Bergey's Manual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G., eds.), Vol. 1, pp. 141-199, Williams and Wilkins, Baltimore, MD. [2] Woese, C.R. (1987) Microbiol. Rev. 51, 221-271. [3] Wilkinson, S.G. and Taylor, D.P. (1978) J. Gen. Microbiol. 109, 367-370. [4] Kasai, N., Arata, S., Mashimo, J., Akiyama, Y., Tanaka, C., Egawa, K. and Tanaka, S. (1987) Biochem. Biophys. Res. Commun. 142, 972-978. [5] Wilkinson, S.G. and Galbraith, L. (1979) Biochim. Biophys. Acta 575, 244-254. [6] Wilkinson, S.G. (1968) Biochim. Biophys. Acta 164, 148-156. [7] Harvey, D.J. (1982) Biomed. Mass Spectrom. 9, 33-38. [8] Christie, W.W., Brechany, E.Y. and Holman, R.T. (1987) Lipids 22, 224-228. [9] Wait, R. and Hudson, M.J. (1985) Lett. Appl. Microbiol. 1, 95-99. [10] Longmuir, K.J., Rossi, M.E. and Resele-Tiden, C. (1987) Anal. Biochem. 167, 213-221. [11] Ryhage, R. and Stenhagen, E. (1960) Arkiv Kemi 15. 291-315. [12] Gerson, T., Patel, J.J. and Nixon, L.N. (1975) Lipids 10, 134-139. [13] McCloskey, J.A. (1970) Top. Lipid Chem. 1, 369-440. [14] Kaltenbach, C.M., Moss, C.W. and Weaver, R.E. (1975) J. Clin. Microbiol. 1, 339-344. [15] Kiprianova, E.A., Andreev, L.V. and Boiko, O.I. (1980) Mikrobiol. Zh. (Kiev) 42, 11-16. [16] Chen, M.M.G. and Chen, P.H. (1981) Proc. Natl. Counc., Repub. China 5A, 202-212. [17] Oyaizu, H. and Komagata, K. (1983) J. Gen. Appl. Microbiol. 29, 17-40. [18] Gerson, T. and Patel, J.J. (1975) Appl. Microbiol. 30, 193 198. [19] MacKenzie, S.L., Lapp, M.S. and Child, J.J. (1979) Can. J. Microbiol. 25, 68-74. [20] Andreev, L.V., Akimov, V.N. and Nikitin, D.I. (1986) Folia Microbiol. (Prague) 31, 144-153. [21] Moss, C.W. (1978) in Glucose Nonfermenting Gramnegative Bacteria in Clinical Microbiology (Gilardi G.L., Ed.), pp. 171-201, CRC Press, West Palm Beach. [22] Contreras, I., Shapiro, L. and Henry, S. (1978) J. Bacteriol. 135, 1130-1136.
105 [23] Jones, D.E. and Smith, J.D. (1979) Can. J. Biochem. 57, 424-428. [24] De Siervo, A.J. and Homola, A.D. (1980) J. Bacteriol. 143, 1215-1222. [25] De Siervo, A.J. (1985) J. Bacteriol. 164, 684-688. [26] Agabian, N. and Unger, B. (1978) J. Bacteriol. 133, 987-994.
[27] Weckesser, J. and Mayer, H. (1988) FEMS Microbiol. Rev. 54, 143-154. [28] Wilkinson, S.G. (1988) in Microbial Lipids, Vol. 1 (Ratledge, C. and Wilkinson, S.G., eds.), pp. 299-488, Academic, London.
