Biochemical changes in Bifidobacterium bifidum var. pennsylvanicus after cell wall inhibition

BIOCHIMICA

ET BIOPHYSICA

267

ACTA

nnA 55715

BIOCHEMICAL

CHANGES

PENNSYLVANICUS

II. FATTY J. H.

ACID

AFTER

IN BIFIDOBACTERIUM CELL

WALL

BIFIDUM

VAR.

INHIBITION

COMPOSITION

VEERKAMP

Department of Biochemistry, lands) (Received

January soth,

University of Nijmegen,

Geert Grooteplein 21, Nijmegen

(The Nether-

1970)

SUMMARY I. Analyses of fatty acid composition of Bijidobacterium biJid%m var. pennsylvanicus were made on different lipid fractions isolated from cells grown with or without human milk. 2. No large differences were found in the fatty acid pattern between total, membrane and cytoplasmic lipids and glyco- and phospholipids. 3. Major constituents are the normal even-numbered saturated and monoenoic acids. The percentages of lactobacillic acid and branched fatty acids were low. 4. Cell wall inhibition by lack of human milk caused a shortening of the chain length of the fatty acids in all lipid fractions. The results were compared with reported lipid changes in L-forms of bacteria.

INTRODUCTION

Inhibition of cell wall synthesis by omission of human milk from the medium causes remarkable changes in the lipid composition of Bijdobacterium bijdum var. pennsylvanicusl. A considerable decrease in lipid-galactose content and a partial substitution of a poly-glycerol triester phospholipid by triacyl-bis-(glycerophosphoryl)glycerol were established. Considerable changes in morphology and osmotic properties of protoplasts prepared from cells grown on the deficient medium were also noticedz. Attempts to correlate differences in permeability with variations in fatty acid composition have been made for membranes of erythrocyte9. Investigation of glucose catabolism has shown+ that a classification of B. bifdum var. pennsylvanicus under the genus Lactobacillus is not justified Since fatty acid analysis by gas chromatography has previously been used for taxonomic studies of bacterial species+?, a study of the fatty acid composition of B. bijidum var. pennsylvanicus would seem to be of value from a taxonomic point of view. This paper presents the results of Biochim. Biophys. Acta, 210 (1970) 267-275

J. H. VEERKAMP

268 a study on the fatty acid composition inhibited

cells of B. biJidwn

of different lipid fractions

from normal and

var. pennsylvanicus.

MATERIALSAND METHODS Preparation and analysis of lipid fractions Cultivation of the organism and cell, membrane were obtained as previously

describedl.

and cytoplasmic

preparations

Cells were washed with 0.2 M sodium acetate

buffer (pH 5.0). Apart from the chemicals, solvents and reference compounds

previous-

ly mentioned*, reference fatty acids were obtained from Applied Science Laboratories, State College, Pa., U.S.A., extraction1

was applied

with or without

human

and from Hormel Institute,

to cells of cell preparations milk. The cytoplasmic

St. Paul, Minn., U.S.A. Lipid

from 6- or 24-l cultures,

fraction

and fresh growth

grown medium

were extracted according to BLIGH AND DYER *. Silicic acid fractionation of 100o300 mg lipids was carried out on 2.0 cm x 25 cm columns by elution with-in this order1600 ml chloroform,

700 ml acetone,

800 ml methanol

and 700 ml chloroform-

methanol-water (IO:IO:I, by vol.). Column fractions were identified by thin-layer chromatographyl. Glycolipids and Compound 15 were further purified by preparative thin-layer

chromatography

in chloroform-methanol-ammonia

Free fatty acids were separated extraction Preparation

(70 : 20 : 2, by vol.).

from esterified fatty acids of cytoplasmic

lipids by

with I M NaOH. of fatty

acid methyl esters and gas-liquid

chromatography

Fatty acid methyl esters were prepared by methanolysis of 0.1-5 mg lipid in 0.5 ml hexane with I ml BF,-methanol (100/b, w/v) at 100’ for 15 min8. Hydrolysis in I M methanolic

KOH solution

were used for comparison. Four gas chromatographic acids. Columns of 0.16 inchx6 Gas-Chrom

and subsequent

esterification

with diazomethane

systems were used to identify the bacterial ft of 2% silicone rubber SE 30 on 100-120

Q and of 109/o Apiezon

L on 60-80 mesh Chromosorb

fatty mesh

W were operated

at 200’. A column of 0.16 inchx6 ft of 15% diethyleneglycol succinate on 60-80 mesh Gas-Chrom P was used at 168”. These columns were used in a Packard model 7821 gas chromatograph.

A Model 226 Perkin-Elmer

gas chromatograph

equipped

with a Golay Carbowax 1540 column (0.02 inch x 200 ft) at 160’ was also employed. All analyses were made with a flame ionization detector and nitrogen or helium as the carrier gas. Identification of fatty acids The fatty acids were identified by comparison of their relative retention volumes with those of standard methyl esters of saturated, unsaturated, iso- and of unsaturated fatty acids anteiso fatty acids on the four columns lo. Hydrogenation was carried out with 10% palladium-carbon in methanol under a hydrogen pressure of 3 atm at room temperature for 2 h. The methyl esters of unsaturated fatty acids were investigated by thin-layer chromatography on 30% AgNO,-impregnated plates according to MORRIS et al.ll. The positions of the double bonds of the individual fatty acids were established by oxidative cleavage according to the method of Von Rudloff as modified by B&him.

Biophys.

Ada,

210 (1970)

267-275

FATTY ACIDSOF B. bifidum

269

SCHEUERBRANDTAND BLOCH12. The oxidation time was extended to 4 h, and the mono- and dicarboxylic acids were extracted together with ether. The methyl esters of these acids were identified and determined by gas chromatography on SE 30 and diethyleneglycol succinate columns. The lower monocarboxylic acids were fractionated at 95”, the dicarboxylic acids at 168”. The separation of different positional isomers of monoethenoid fatty acids was achieved by Golay column gas chromatographyl3. The presence of the cyclopropane acids was established by their disappearance from the saturated fatty acid fraction after bromination14 or heating with z M methanolic HC115. Incorporation of [Me-lJC]methionine into fatty acids was assayed by the method of CRONAN’~. analysis of fatty acid comfiosition Fatty acid content of the fresh sterilized growth medium was determined with methyl pentadecanoate as internal standard. The relative composition of a fatty acid mixture was determined by measuring the area under the peaks by multiplication of peak height with their width at half height. Results for standards agreed with the stated composition data with a relative error of less than 4% for major components ( >IO% of total mixture) and less than 9% for minor components (
Quantitative

RESULTS Identijcation

of fatty

acids

Normal and branched saturated, unsaturated and cyclopropane fatty acids were detected in lipids of B. bijdum var. pennsylvanicus (Table I). The fatty acid methyl esters were characterized by their gas-liquid chromatographic retention volumes on the four polar and apolar columns before and after hydrogenation. Their identity was also established after separation into saturated and unsaturated components by the mercuric acetate adduct technique17 and thin-layer chromatographyle. Argentation thin-layer chromatography of the isolated methyl octadecenoate fraction indicated the presence of both cis-vaccenate and oleate and the absence of transoctadecenoates. Golay column gas chromatography and analysis of the mono- and dicarboxylic acids resulting from oxidative cleavage established that the octadecenoate fraction is a mixture of the g,ro-cis and 11,12-cis isomers. The identity of the cyclopropane fatty acids was established by comparison with fatty acid methyl ester samples prepared from Escherichia coli and Lactobacillus casei, which contained a large amount of cis-g,ro-methylene hexadecanoic acid and cis-rr,rz-methylene octadecanoic acid, respectivelylg. These components could be eliminated from the gas chromatographic profile pattern of the isolated saturated fatty acid fraction by brominationle and also by destruction with acid”. l4C transmethylation from [M&*C]methionine to fatty acids was observed to occur in cells oi late logarithmic phase which indicates a synthesis of cyclopropane fatty acids%20. The positions of the methylene groups were not established because of the small amounts of these fatty acids present in the lipid fractions. Biochim.

Biophys.

Acta,

210

(1970)

267-275

J. H. VEERKAMP

270 TABLE FATTY GROWN

I ACID WITH

COMPOSITION

OF

LIPIDS

ORWITHOUTHUMAN

ISOLATED

FROM

CELLS

OF

B. bi$dum

VAR.

PENNSYLVANICUS

MILK

The percentage of each fatty acid is the mean of the percentages for duplicate analyses of the lipid extracts from fifteen experiments with and seven experiments without human milk. Standard errors and in parentheses the range of the percentages are listed behind the means. The fatty acid methyl esters are designated by the number of carbon atoms, followed by the number of double bonds, with the prefix anteiso- and iso- indicating the type of branching and cycle- standing for cyclopropane. The retention volumes were determined on the 15% diethyleneglycol succinate column at 168”. Retention volume for palmitate was arbitrarily set on 1.00. + denotes presence in an amount less than 0.5%. Fatty

acid

12:o

iso-14:o 14:o anteiso-15 : 0 15:o iso-16:o 16:o 16:r anteiso-17 : 0 17:o cycle-I 7 : 0 iso-r8:o 18:o 18:r ant&so-19:o 18:~ 19:o cycle-19:o

Relative retention

With

human

milk

Without

human

milk

vol.

0.33 0.49 0.55 0.68

+

0.73 0.88

+ +

I.I &

0.2 (0.5-2.5)

3.5 & 0.3 (2.0-4.6) +

22.9 f 4.9 f + +

1.00

1.15 1.27 1.38 1.56 1.68 I.89 2.14 2.45 2.66 2.83 2.96

0.9 (17-28) 0.3 (3.1-8.9)

1.5 1.8 10.1 1.0 + + 23.9 9.2 + +

* + + 5

0.2 0.4 (0.5-3.8) 1.0 (8.6-12) 0.2 (0.5-1.5)

7.9 42.9 + 1.5 + I.2

+ I.3 (2.8-14) zt 2.5 (34-56)

* 2.4 (15-33) f 0.9 (5.9-12)

+

2.7 & 1.0 (0.5-16) 22.6 & I.3 (17-29) 38.4 & I.5 (29-46) 2.2 &

0.3 (0.5-4.0)

+ 2.8 f

0.5 (0.5-6.1)

& 0.4 (0.5-3.6) f

0.1 (0.5-1.5)

Fatty acid conzpositiox of total lipids The composition of the fatty acid methyl esters from lipids of B. bijidwn var. pennsylvanicus grown with and without human milk is shown in Table I. The growth medium contained in both cases about 75 mg of fatty acids per 1, present largely in polyoxyethylene sorbitan mono-oleate (Tween-80). Since defatted human milk was used, no significant differences existed in the fatty acid composition of both media. The media contained 3.4% of myristic, 2% of pentadecanoic, 3.8% of palmitic, 11% of palmitoleic, 78% of oleic, 1% of linoleic and traces of heptadecanoic and nonadecanoic acids. The analysis procedure was checked on standard mixtures and also by repeated determinations with a single lipid extract using different times and temperatures for the methylation with BF,-methanol. The relative standard error of the mean for major components was less than 3% and for minor components (< 5%) less than 10% (eleven determinations). Major fatty acid constituents are the normal C14, C,, and C,, saturated and the C,, and C,, monoenoic acids. Low amounts of cyclopropane and branched fatty acids were found. The separation of n- and isooctadecanoic acid was not always satisfactory, resulting in high standard errors for these fatty acids. A relatively large range of percentages was obtained for all fatty acids in consequence of differences in the growth phase at which cells were harvested. Lipids were isolated from cells of late logarithmic or early stationary phase (pH of medium 5.2-4.9 for normal cells, B&him.

Biophys.

Acta.

210

(1970)

26;-275

FATTY ACIDS OF

B. bijdum

271

5.8-6.3 for cells grown without human milk). Differences in growth phase and conditions have a marked effect on the bacterial fatty acid composition*. Cell wall inhibition by cultivation without human milk caused a significant decrease of octadecanoic acid and increase of myristic and palmitoleic acid. These changes in fatty acid composition are not due to the lower acidity of the medium, as will be demonstrated below. The ratio of the two isomers of octadecenoic acid appeared to be about the same in both cell types. In normal cells 12% of the octadecenoic fraction was cis-vaccenic acid, in inhibited cells 15%. Fatty acid composition of diferent lipid fractions Analyses of lipids isolated from membrane preparations and the cytoplasmic fractions showed no large differences in the fatty acid composition of these two cell fractions, only the isooctadecanoic and lactobacillic acid content was somewhat higher in the cytoplasmic lipids (Table II). Cell wall inhibition was accompanied by TABLE FATTY PLASMIC HUMAN

II ACID

COMPOSITION

FRACTION

OF

OF

LIPIDS

CELLS

OF

ISOLATED

FROM

B. bi$dum

VAR.

MEMBRANE

PREPARATIONS

PENNsYLVANICUS

GROWN

AND WITH

THE OR

CYTO-

WITHOUT

MILK

The percentage of each fatty acid is the mean of duplicate analyses of lipid extracts from the number of experiments listed in parentheses at the top of each column. The standard error is given for every mean. Notation of the fatty acids is as in Table I. + denotes presence in an amount less than 0.5%. Fatty aced

12:o

iso-r4:o 14:o anteiso-15 : 0 16:o 16:r anteiso-17:o iso-18:o 18:o 18:1

18:2 cycle-1g:o

Without human milk

With human milk Membrane

+

(15)

CytoPlasmic fraction(g)

+

+

1.0 5 0.3 2.8 &

+ 28.1 3.8 + + 24.6 36.8 2.3 1.3

0.2

4 1.3 & 0.2

+ 1.0 5 1.0 & 0.2 + 0.2

Cytoplasmic fraction(T)

Membrane(ro)

+ 2.4 + 24.4 2.4 + 4.4 23.3

zt 0.7 5 1.0

39.9

&

1.0 *

l

0.2

* 1.6 zt 0.7 I.6

2.1 & 0.3 2.1 f 0.4

0.2

I0.g

+

0.8 25.6 7.9 + + 7.6 41.9 2.8 1.4

* 0.2 k 2.2 * 0.5

I.5 &

0.2

I.0 *

0.3

10.3 + 22.8 4.9 + + 7.5 45.1 2.7 3.3

I.0

=k 1.4 i 2.3 * 0.8 -I- 0.3

& 0.8 k 1.2 * 0.7

f f zk zt

1.5 0.9 0.5 1.3

the same alterations of fatty acid composition in lipids of both cell fractions as were observed in lipids from total cells. The effect of differences in acidity of the medium was investigated by adjusting the pH of three 15-h cultures of normal cells to 6.8. The cultures were incubated for an additional hour leading to a final pH of 6.1. The membranes of the cells were isolated and the lipids extracted. The membrane lipids had the following mean fatty acid composition: myristic acid, 3.8% ; palmitic acid, 22.3%, palmitoleic acid, 5.9% ; stearic acid, 24.8% ; octadecenoic acid, 34.5% ; linoleic acid, 3.2% ; and lactobacillic acid, 2.17/o. These results do not deviate significantly from the results for membrane preparations from normal cells harvested at pH 4.9-52 (Table II). So the differences in fatty acid composition after cell wall inhibition are not due to lower acidity of the medium. Biochim. Biophys.

Ada,

210

(1970)

267-275

J. H. VEERKAXIP

272 TABLE

III

FATTY

ACID

COMPOSITION

OF

LIPID

FRACTIONS

FROM

CELLS

B. bifidum

OF

PEXiYSYLVASICUS

“AR.

GROWN WITH OR WITHOUT HUMANMILK The percenta.ge

of each fatty acid is given as the mean of duplicate analyses of the lipid fractions. The number of experiments is given in parentheses at the top of each column. Fatty acid notation is as in Table I. + denotes presence in an amount less than 0.50;. F&y

With human milk I (IS) II (9) III

acid

12:o

+

I-

iso-14:o 14:o ant&o-r5 :0

2.3 3.3

-

16:o 16:1 anteiso-I 7 : 0 iso-IS:0 18:0 18:1

23.5

3.3

4.6

27.8

6.0 +

t

3.0 20.X

32.0

38.0

4.1 23.9

7.2

I.5 2.5

31.9 I.3 3.5

of the fatty

27.8 2.0

3.4

III.

I.‘,

Z-j.0

25.2

~-

II.0

34.5 6.8

-t

i-

8.4

9.3

1.0 r3.8

I.2 4.6

44.6

39.8

3s.3

2.5

2.2 I.2

1.0 1.1

of lipid fractions

is given in Table

3.2 9.1

IO.2

+

I.8

(3)

1.j

4m

T

acid composition

acid column chromatography

17.3

4.5

Il’

2.2

I.8

_

2.1 25.0

2.6

A survey

:

28.0

+

25.9

cycle-1g:o

3.8 0.7

I.5 6.6 I.7

(3)

.1_

0.9 c 13.3

0.8

I.5 3.6

f

10.9 +

IVztkout human milk II (3) III 1 (3)

(II)

_. I.0

+

IO.5

Is:2

IV

$

+ 3.0 +

I

1j:o

(13)

4~ 9.1

separated

The first fraction

3.9 0.6 31.4

2.0 +

by silicic

contained

the

neutral lipids, the second fraction the glycolipids and the last two the phospholipids. The different lipid fractions showed much similarity in their fatty acid patterns. The neutral lipids contained than the other fractions.

relatively

less palmitic

The percentage

acid and more octadecanoic

of oleic acid of the octadecenoic

acids

acid fraction

was also somewhat higher than in total lipids (90 veyszbs 85%). No large differences were found between the glycolipids and the two phospholipid fractions. The percentage of unsaturated fatty acids appeared to be somewhat lower in the more polar phospholipids differences a decrease TABLE FATTY

(Fraction

between

IV). All four lipid fractions

normal

of octadecanoic

and inhibited

demonstrated

cells as observed

acid and an increase

of myristic

the same marked

in total

lipid extracts,

and palmitoleic

acid.

IV ACID

COMPOSITION

OF

DIFBEREPjT

LIPIDS

OF

B. bijidum

VAR.

PENNSYLVAKICUS

The percentage of each fatty acid is given as the mean of duplicate analyses of two preparations of each lipid. Abbreviations : MGD, monogalactosyldiglyceride ; DGD, digalactosyldiglyceride : TGD, trigalactosyldiglyceride; PGP, polyglycerolphospholipid (Compound 15)‘. Fatty acid notation is as in Table I. + denotes presence in an amount less than o.so/“. Fatty

Diacyl-MGD

acid

iso-

12:o

:0

14:o anteiso-15:o 16:o 16:1 anteiso-17 : 0 iso-18:o 18:o 18:1 18:2 cycle-19 : 0 Biochim.

Biophys.

-

I.1

c

1

2.5 -1

3.2 1.0

16.0 4.2

23.7

t

4.3

+

2.8 16.5 44.’ I.1 i

Acta,

MGD

i 23.0 42.6 2.0 5.1

‘3 210

(1970)

26>-27j

Acyl-DGD

DGD

+

-t

0.6

2.9 _30.4 4.6 + 17.6 18.9 19.1 I.4 1.0

-

3.0 I.5 18.7 5.4 + + 20.7 43.4 2.2 3.4

TGD

PGP

f

c

2.2 3.6

I.3 19.1 5.6 c 1.0 18.7 41.0 3.3 5.3

7.0 I.2

.<2 .7 6.7 I.3 18.7 21.7 I.2 3.7

FATTY ACIDS OF

B. bi$dum

273

The fatty acid composition of the five quantitatively most important galactolipids and of an isolated polyglycerolphospholipid (Compound 15)’ were also determined (Table IV). The galactolipids, except for the monoacyldigalactosylglyceride, show a remarkable similarity in their fatty acid pattern. The monoacyldigalactosyldiglyceride contained more saturated acids, especially palmitic and isooctadecanoic acid. The polyglycerol phospholipid resembled in its fatty acid composition the second phospholipid fraction (Fraction IV). The free fatty acids of the cytoplasmic phase had the same distribution as the esterified fatty acids. DISCUSSION

B. bifidum var. pennsylvanicus resembles in its fatty acid composition that of the lactobacilli4+~20~21 and streptococci4~23 in so far as it has high proportions of normal even-numbered saturated and unsaturated fatty acids and low amounts of odd-numbered branched-chain acids. In most other Gram-positive families of the Eubacteriales the branched fatty acids are the major and the straight-chain acids Comparison with the lactobacilli data, however, also the minor components 41a3p24. shows distinctive differences. The lactobacilli also have less octadecanoic acid and much more lactobacillic acid when grown in the medium with the same fatty acid contenP. Because other bifidobacteriaz6 give a similar fatty acid pattern as B. bij&m var. pennsylvanicus, the fatty acid composition may be another argument in support of a taxonomical differentiation between bifidobacteria and lactobacilli. The small amount of octadecadienoic acid found originated from the medium, because true bacteria do not synthesize poly-unsaturated long-chain fatty acids. Only for Bacillus licheniformisz7 and Mycobacterium phlei28 has a synthesis of this fatty acid been reported. Cell wall inhibition appears to have a marked effect on the fatty acid composition of all lipid fractions from B. bijdum var. pennsylvanicus. Membrane fatty acid composition resembled that of whole cells. No important differences were observed between the glycolipid and phospholipid fractions. The effect was not explicable in terms of the pH at which the cells were harvested. The substitution of stearic acid by especially myristic and palmitoleic acids increases the ratio of unsaturated/saturated fatty acids and the total C,,/C,, ratio (Table V). This shortenTABLE

V

CHANGESOF FATTY ACID PATTERN OF BACTERIALLIPIDS BY CELL WALL INHIBITION Percentages of total fatty acids are given for each fraction. Data for Streptococcus pyogelzes, normal and L-forms, were taken from PANOS et al. 33. For a good comparison the figures for streptococci grown in L-form medium were used. Fatty acid fraction r4:o + 16:1 + 18 :2 16:o + 18:1 18:o + cycle-rg:o Unsaturated Total C,, Total C,, cis-Vaccenic acid Oleic acid

B. bijidzcm oav. pennsylvanicus

Streptococcus pyogenes

Normally grown

Inhibited

coccus

L-form

IO 61 25 45 28 66 6 32

19

67 9 54 33 52 7 36

25 59 6 47 46 41 8

24 61 8 5.5 33 56 7 28

I7 Biochim. Biophys.

Acta, 210

(1970)

267-275

“74

J.H.VEERKAMP

ing of the chain length of the fatty

acids may affect the properties

their function in the cell and its membrane. fluenced the osmotic fragility of Mycoplasma methylgalactoside

accumulation

of the lipids and

Changes in the fatty acid pattern inlaidlawii B membranes28, the thio-

in E. c01i~~ and the penetration

of nonelectrolytes

into liposomes31. Alterations in the fatty acid composition of the membrane lipids resulted in characteristic morphological changes of Mycoplasma laidlawii B but did not appear to adversely affect the viability of the organism32. Lipid alterations during or after inhibition of cell wall synthesis studied in Gram-positive coccus

auyeus36 with their derived

Streptococcus increase

cells by comparing

$yoge?aes

stable

is associated

of octadecenoic

L-forms.

with

of the unsaturated/saturated

an increase

Streptococcus

fatty

pyogenes33-35

Permanent

a decrease

have been

and Staphylo-

cell wall inhibition

of the

C,,,K,,

ratio

acids ratio by a decrease of palmitic

and octadecadienoic

of

and an

acids. The lipids from L-forms

and of

Staphylococcus aureus IOO and Staphylococcus aureus H did not show significant differences in their fatty acid patterns. Altogether, the results for L-forms indicate only small changes

in the effective

chain length of the fatty

acids (Table V).

PANOS et a1.33 observed upon loss of cell wall a redistribution within the octadecenoic isomers, consisting of a relative decrease of cis-vaccenic acid in the stable L-form. This is in contrast with the small changes in the oleic acid/&-vaccenic acid ratio of B. bifidum var. pennsylvanicus

after cell wall inhibition.

A decrease in fatty acids containing a cyclopropane ring was observed in Gram-negative bacteria in an L-form of Proteus P 18 (ref. 37) and in E. coli B (ref. 38)

under

conditions

of partial

cell

wall

inhibition.

Staphylococcus aweus lack these acids. No change content was found in B. bijdum var. pennsylvanicus

Streptococcus

pyogenes

and

in the C,,-cyclopropane acid after growth without human

milk. Earlier’ we reported changes of lipid content and composition after cell wall inhibition, changes which also contrasted with observations on the L-forms34y38. Cell wall inhibition by lack of glucosamine derivatives, serving as cell wall precursors, and an L-form cell wall inhibition defect may cause various alterations of lipid composition and metabolism. These various changes may account for the differences in the alteration of fragility and osmotic properties which have been found after both types of cell wall inhibition. L-forms seem to be less fragile than the protoplasts from streptococci, whereas protoplasts derived from inhibited cells of B. bijidum var. pennsylvanicus

show a marked

decrease

of osmotic

stabilityz.

REFERENCES I F. A. EXTERKATE AND J. H. VEERKAMP, Biochim. Biophys. Acta. 176 (1969) 65. 2 F. A. EXTERKATE, J. H. VEERKAMP AND G. J. VRENSEN, Biochinz. Biophys. Acta, submitted for publication. 8 L. L. M. VAN DEENEN AND J. DE GIER, in CH. BISHOP AND D. M. SURGEON, The Red Cell, Academic Press, New York, 1964, Chapter 7. J. H. VEERKAMP, Arch. Biochem. Biophys., 129 (1969) 257. M. KATES, Advan. Lipid Res., z (1964) 17. J. E. STEINHAUER, R. L. FLENTGE AND R. V. LECHOWITZ, Appl. Microbial., 15 (1967) 876. 7 R. W. IFKOVITZ AND H. S. RAGHEB, AppI. Microbial., 16 (1968) 1406. E E. G. BLIGH AND W. J. DYER, Can. J. Biochem. Physiol., 37 (1958) grr. o W. R. MORRISON AND L. M. SMITH, J. Lipid Res., 5 (1964) 600. o A. T. JAMES, J. Chromatog., 2 (1959) 552. him. Biophys.

Acta,

210 (1970) 267-275

FATTY

ACIDS OF

B. bijdum

275

II I2 I3 I4 15 16 I7 18 I9

L. J. MORRIS, D. M. WHARRY AND E. W. HAMMOND, J. Chromatog., 31 (1967) 69. G. SCHEUERBRANDTAND K, BLOCH, J. Bid. Chem., 237 (rg6z) 1064. C. PANOS, J. Gas Ch~~matog., 3 (1965) 278. 3. L. BRIAN AND E. W. GARDNER, Appl. Microbial., 16 (rg68) ,549. K. J. THORNE AND E. KODICEK. B&him. Biojdys. Ada, 59 (1962) 306. J. E. CRONAN, Anal. Biochem., 21 (1967) 293. H. K. MANGOLD, J. Am. Oil Chemists’ Sot., 38 (1961) 708. H. K. MANCOLD AND R. KAMMERECK, Chem. Id., (1961) 1032. W. M. O’LEARY, in S. K. SHAPIRO AND F. SCHLENK, Transmethylafion and Methiokne Biosylathesis, University of Chicago Press, Chicago, 1965, p. 94. 20 K. HOFFMANN, Fatty Acid metabolism in ~~~c~ooygan~sms, John Wiley, New York, 1963, p. 48. 2I 1. H. VEERKAMP, H. W. WASSENBERG A;“~‘DF. A. EXTERKATE, Biochim. Biophys. Acta, submitted for publication. 22 P. MACLEOD AND 1. P. BROWN, .I. Bacteriol., 85 (1963) 1056. 23 K. Y. CHO AND M.-R. J. SALTON, Biochim. Biophys. Acta, 116 (1966) 73. 24 J. ASSELINEAU, The Bacterial Lipids, Herman, Paris, 1966, p. 204. 25 F. A. EXTERKATE, B. OTTEN AND J. H. VEERKAMP, Biochim. Biophys. Acta, submitted for publication. 26 J. ERWIN AND K. BLOCH, Science, 143 (1964) 1006. 27 A. J. FULCO, Biochim. Bio$hys. Acta, 187 (1969) 169. 28 C. ASSELINEAU, H. MONTROIZIER AND J. C. PROM& European J. Biochem., IO (1969) 580. 2g S. RAZIN, M. E. TOURTELLOTTE, R. N. MCEL~IANEY AND J. D. POLLACK, J. Bacteriol., gr (1966) 609. 30 H. U. SCHAIRER AND P. OVERATH, J. Mol. Biol., 44 (1969) 209. 31 J. DE GIER, J. G. MANDERSLOOT AND L. L. M. VAN DEENEN, Biochim. Biophys. Acta, 150 (1968) 666. 32 R. N. MCELHANEY AND M. E. TOURTELLOTTE, Science, 164 (1969) 433. 33 C. PA~OS, M. COHEN AND G. FAGAN, Biochemistry, 5 (1966) 1461. 34 M. COHEN AND C. PANOS, Biochemistry, 5 (1966) 2385. 35 C. PANOS, Ann. N.Y. Acad. Sci., 143 (1967) 152. 36 J. B. WARD AND H. R. PERKINS, Biochem. J., 106 (1968) 391. 37 J. A. I. NESBIT AND W. J. LENNARZ, J. Bacteriol., 8g (1965) 1020. 38 G. WEINBAUM AND C. PANOS, J. Bacterial., 92 (1966) 1576. B~och~m. B~ophys. Acta, 2Io (‘970) 267-275