FEMS Microbiology Letters 13 (1982) 87-91
Published by Elsevier Biomedical Press
87
Cellular fatty acid composition of strains of three species
of
Sphingobacterium gen.
nov. and
Cytophagajohnsonae
Eiko Y a b u u c h i and C. W a y n e Moss *
Kansui Medical Unit~ersio', Department ~[ Microbiology, Moriguchi, Osaka 570, Japan, and * Cetlters for Disease Control, Analytical
Bacteriology Branch, Bacteriology Dicision, Atlanta, GA 30333. U.S.A.
Received 28 September 1981
Accepted 30 September 1981
1. I N T R O D U C T I O N
In a recent report, Holmes et al. [1] proposed
the name Flavobacterium multivorum for a group
of nonfermentative, nonsporeforming, G r a m negative, rod-shaped bacteria which were previously designated group IIK-2 [2,3]. The cellular
lipids of IIK-2 and related organisms are characterized by the presence of sphingophospholipids
in which the major long chain base is a branchedchain 17-carbon sphinganin [4]. On the basis of
this unique compound, and data from numerical
analysis of 92 phenotypic characters, Yabuuchi et
al. [5] placed the organisms in a new genus and
proposed new names Sphingobacterium gen. nov.
and Sphingobacterium versatilis sp. nov. S. versatilis KM2138 ( = A T C C 3 3 3 0 0 ) was designated as
the nomenclatural type for the species. Three biovars of S. versatilis [5] are now regarded to compose 3 separate species. Biovar 1 includes KM2138
and is S. versatilis. Biovar 2 corresponds with F.
multivorum and the species is transferred to the
genus Sphingobacterium. Sphingobacterium multivorum comb. nov. is proposed with KM2812
(NCTCl1343) as type strain. The name Sphingobacterium mizutae sp. nov. is proposed for the
biovar 3 organism and strain KM1203 ( =
ATCC33302) is designated as nomenclatural type
for the species.
Chemical data such as cellular fatty acids have
provided valuable additional information for the
recognition of genus or species of various bacteria
[6-11]. The most abundant acid in organisms previously designated IIK-2 is a saturated, branchedchain 15-carbon acid (i-15:0) with the methyl
branch at the iso (penultimate) carbon atom or a
saturated, branched-chain 15-carbon hydroxy acid
(i-2-OH-15:0) with the hydroxy group at the 2carbon atom [8]. Also present in relatively small
amounts is a saturated, branched-chain 15-carbon
hydroxy acid with the hydroxyl group at the 3carbon atom (i-3-OH-15:0). In most studies, determination of these acids (as methyl esters) has
been done with gas-liquid chromatography (GLC)
using glass columns (3-m length × 2 or 4-mm i.d.)
packed with 3% OV-101 as stationary phase [12].
However, with these columns it is difficult to
identify the branched-chain hydroxy acids since
i-2-OH-15:0 co-elutes with a monounsaturated,
straight-chain 16-carbon acid (16: 1) and i-3-OH15:0 co-elutes with palmitic acid (16:0). In the
present study, we use a fused-silica capillary column for G L C analysis of the cellular fatty acids of
the three species of Sphingobacterium and strains
of Cytophaga johnsonae. The increased resolution
of components on the capillary column permitted
accurate quantitative comparison of the fatty acids
of these organisms.
0378-1097/82/0000-0000/$02.75 ~ 1982 Federation of European Microbiological Societies
88
2. MATERIALS A N D METHODS
Four strains of S. versatilis, five strains of S.
multivorum, five strains of S. mizutae and three
strains of C. johnsonae, including the type strain
for each species, were used. Each of the 17 strains
was inoculated on a plate of agar medium composed of Bacto-peptone (1%), Bacto-yeast extract
(0.5%), and Bacto-agar (1.5%). After 20h incubation at 30°C, the cells were harvested, saponified,
and the resulting fatty acids were methylated as
described previously [12]. The methyl esters were
analyzed with a series 5880A gas chromatograph
(Hewlett-Packard Co., Avondale Division, Pennsylvania, U.S.A.) equipped with a flame ionization
detector and a 25-m × 0.2-mm fused-silica capillary
column with OV-1 as stationary phase. The injection temperature was maintained at 250°C, and
the detector was maintained at 300°C. The column
conditions were as follows: 190°C for 5 min, then
temperature program to 250°C at 6 ° C / m i n with a
final 8-min hold at 250°C. Fatty acids were identified by retention time comparison of their methyltrifluoroacetyl derivatives with those of known
standards and further confirmed by both electron
impact (EI) and chemical ionization (CI) mass
spectrometry. Quantitation of both methyl esters
and acetylated methyl esters was accomplished
with a series 3350 microprocessor unit (HewlettPackard) connected to the 5880A gas chromatograph.
3. RESULTS A N D DISCUSSION
Chromatograms of the cellular fatty acids (as
methyl-trifluoroacetyl esters) of the type or reference strain of each of the three species of
Sphingobacterium are shown in Fig. 1. The sharpness of the peaks in the chromatogram and the
Table 1
C e l l u l a r f a t t y a c i d c o m p c , s i t i o n o f 14 s t r a i n s of three s p e c i e s o f Sphingobacterium a n d 3 s t r a i n s o f ()'tophaga iohn.~om~e
S. cersatihs
Fatty
S. mtdticorunt
acids ~
2134 b
2138 d
2148
2087
2532
4
5
4
5
2812 d
2813
14:0
2 c
.
16 : 1
24
24
23
22
29
28
27
13
29
5
-
5
6
7
II
9
1
2
8
18
23
9
27
17
-
-
i-15:l
.
i-15:0
23
a-15:0
.
i-16:0
.
2
926.
15:0
16:0
17:1
2
2289
.
.
.
.
.
.
25
.
25
.
.
.
26
.
.
.
.
i-17:1
-
i-17:0
.
a-17:l
-
a-17:0
.
2-OH-14:0
3-OH-14:0
1
.
2-OH-16:0
-
-
I
-
-
3-OH-16:0
3
3
3
2
2
3
30
4
28
3
28
4
26
4
19
7
20
6
4
3
4
3
I
3
i - 2 O H - 15 : 0
i-3OH-15:0
i-3OH-17:0
" Number
.
6
.
.
3
.
.
.
.
I
.
I
.
.
1
.
1
1
3
.
3
9
35
3
21
6
4
7
2
10
1
b e f o r e the colon indicates the n u m b e r o f c a r b o n a t o m s , a n d t h e n u m b e r after r e f e r s to n u m b e r o f d o u b l e b o n d s : i i n d i c a t c s
a methyl branch at the iso carbon atom, a n d a indicates that the m e t h y l b r a n c h is at t h e anteiso c a r b o n :
h y d r o x y l g r o u p at t h e 2 o r 3 carbon from the c a r b o x v l e n d o f t h e c h a i n .
2 or 3OH
indicates an
89
organism from Sphingobacterium and Flavobacterium both of which contain relatively large
amounts (more than 16%) of this acid. The relative
large amount (13-29%) of 16:1 acid in Sphingobacterium distinguishes these organisms from
Flavobacterium species which contain relatively
small amounts (less than 6%) of 16 : 1 [9]. Thus, the
fatty acid data provide additional support for
establishing Sphingobacterium as a new genus.
baseline resolution of components illustrate the
excellent separating efficiency of the capillary column. The quantitative fatty acid data for each of
the 17 strains are presented in Table 1. The values
listed in the table are averages from two separate
analyses of the organism processed through the
entire procedure of regrowth, saponification, and
G L C analysis.
In general, the fatty acid composition of the
three Sphingobacterium species was similar. Each
strain of Sphingobacterium contained i-15:0, i-2OH-15:0, and 16:1 as major peaks while most
strains contained 14 : 0, i-3-OH- 15 : 0, 3-OH- 16 : 0,
and i-3-OH-17:0 as minor components. The relative amount of i-2-OH-15:0 in the 14 strains of
Sphingobacterium ranged from 19 to 35%, but in
C. johnsonae this acid was present in only small
amounts (1-2%). The small amount of i-2-OH15:0 in C. johnsonae clearly distinguishes this
ACKNOWLEDGEMENTS
Coordination of this study was made possible
through the support of the Japan Association for
the Promotion of Science. The authors acknowledge the excellent technical support of Mary Ann
Lambert.
('. johns,mac
S. Dll2lllblC
1203 d
2055
2120
-
.
.
2789
2274
-
.
t
.
2500 d
2584
2586
4
5
I
I
1
5
17
15
18
29
15
27
35
12
3
I
3
IO
5
23
16
6
.
.
.
26
.
.
32
.
27
-
3
.
I
.
.
18
36
23
15
3
31
2
3
4
7
--
--
2
I
-
5
--
2
4
.
.
.
.
I
.
.
.
.
-
.
.
.
.
I
.
I
I
-
3
4
-
I
5
6
I
-
2
-
1
2
I
30
29
26
19
28
2
I
2
3
2
2
5
2
5
6
8
I1
I1
9
I
6
I
I
3
h Numbers
r e f e r to K M ( K a n s a i M e d i c a l U n i v e r s i t y ) s t r a i n n u m b e r .
c N u m b e r s r e f e r to p e r c e n t o f t o t a l f a t t y a c i d s , - i n d i c a t e s n o t d e t e c t e d o r p r e s e n t at Jess t h a n I ~ .
d Type strain for each species.
A.
Sphingobacterlum versatUis
hi
W
Z
o
o.
oE
t
t !
~
T
~ ~t
t
~:
~
MIN
B. Sphingobacterium multivorum
Y
C. Sphingobacterium mizufae
o
Fig. I. Gas chromatograms of trifluoroacetylated methyl ester of fatty acids from saponified whole cells of S p h i n g o b a c t e r i u m .
Chromatogram A = S. versatilis KM2138, type strain; B = S. m u l t i v o r u m KM926, reference strain: C - S. m i z u t a e KM 1203, type strain.
Analysis on a fused-silica, OV-I capillary column (25 m × 0.20 mm). See footnote a of Table 1 for peak designation. (Chromatograms
here are cut off at around 14 min, because there appeared no significant peaks afterward.)
91
REFERENCES
[1] Holmes, B., Owen, R.J. and Weaver, R.E. (1981) Int. J.
Syst. Bacteriol. 31, 21-34.
[2] Tatum, H.W, Ewing, W.H. and Weaver, R.E. (1974) in
Manual of Clinical Microbiology, 2nd ed. (E.H. Lennette,
E.H. Spaulding and P. Truant, Eds.), pp. 281-283,
American Society of Microbiology, Washington, DC.
[3] Hugh, R. and Gilardi, G.L. (1980) in Manual of Clinical
Microbiology, 3rd ed. (E.H. Lennette, A. Balows, W.J.
Hansler Jr. and J.P. Truant, Eds.), pp. 312-314, American
Society of Microbiology, Washington, DC.
[4] Yano, I., Yamamoto, A., Ohno, Y., Masui, M. and Yabuuchi, E. (1977) Proc. Japan. Confer. Biochem. Lipids (in
Japanese) 19, 19--22.
[5] Yabuuchi, E., Yano, I., Kaneko, T. and Ohyama, A.
(1981) in The Flavobacterium-CvtophagaGroup (H. Re-
[6]
[7]
[8]
[9]
[10]
[I I]
[12]
ichenbach and O.B. Weeks, Eds.), pp. 79-90, Verlag
Chemie, Weinheim.
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Dees, S.B. and Moss, C.W. (1978) J. Clin. Microbiol. 8,
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Dees, S.B., Moss, C.W., Weaver, R.E. and Hollis, D.
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Moss, C.W. and Dees, S.B. (1978) J. Clin. Microbiol. 8,
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Yabuuchi, E., Tanimura, E., Ohyama, A., Yano, I. and
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Yano, I., Ohno, Y., Masui, M., Kato, K., Yabuuchi, E. and
Ohyama, A. (1976) Lipids I 1,685-688.
Moss, C.W. and Dees, S.B. (1975) J. Chromatogr. 112,
595-604.