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Biochemical changes in Bifidobacterium bifidum var. Pennsylvanicus after cell wall inhibition IV. Galactolipid composition
BIOCHIMICA
ET BIOPHYSICA
BIOCHEMICAL
ACTA
CHANGES
PENNSYLVANICUS IV. GALACTOLIPID
545
IN BIFIDOBACTERI~JM
AFTER
CELL
WALL
BIFiTDTJM
VAR.
INHIBITION
COMPOSITION
SUMMARY
A decrease of the lipid galactose content of cells and membranes was observed after growth of ~~~~~~cte~~~~ b~~~~~ var. ~~~~sy~v~~~c~swithout human milk. This change resulted both from a decrease of the amount of all glycolipid compounds and from a shift in the ratio of these compounds to the monog~aciosyl Iipids at the expense of the digalactosyl lipids. The shift was stronger after shortening of the lag phase of the cells. The alterations could be explained by a more limited disposal of UDPgalactose for glycolipid synthesis as a consequence of an increased cell wall polysaccharide synthesis.
INTRODUCTION
In a previous paper* we reported a considerable decrease of the galactose content in lipid extracts of whole cells, membrane and cytoplasmic preparations of Si$~~~~c~ey~~~~i~~~~~ var. ~en~s~~va~~cu~after inhibition of cell wall synthesis. Inhibition was effected by depriving the cells of human milk, which contains N-acetylglucosamine derivatives necessary for normal growth and cell wall mucopeptide synthesis2-4. Preliminary results suggested that the decrease in lipid galactose was correlated with an overall decrease of the galactolipids, which were identified as mono-, di-, and trigalactosyldiglyceride, mono-, and diacyl monogalactosyldiglyceride and acyl digalactosyldiglyceride l, In this paper we report the changes occurring in the galactolipid content and in the galactolipid composition after cell wall inhibition. MATERIALS
AND METHODS
Cells were inoculated at 23” and 37” and grown for 16 or 40 h with or without human milk’. The cells were harvested and washed as describedl. Membrane preparaBiochina.Biophys. A&z, 231
(197’)
545-549
tionswere obtained as before’~. Lipids were extracted and fractionated on si!icic acid columns according to described procedures;. Galactoiipids vvere present in the acetone column
fraction.
was established
Their
identification
after heating
was described
for 24 h at
previouslyl.
Membrane
70”.
Dry weight of cehs
fractions
were dried in WKGO
above P,&.
A modification of the procedure of ROUGEAS AND BATTY was used for rhe assay of the various galactolipids in acetone column fractions. The Silica gel G (Mercl;, Germany) was purified thoroughly with a slight’iy modified washing &-ocedare according to BROEKHCYSE~ in order to ob’cain acceptable blanks. Thin-layer chronzatograms of the acetone fractions (r-1.5 mg of total gala~to~~~~d~ were developed in ch_;oroforr~l-met~lanol-cone.
ammonia
(70 : zo : 2, by vol.). The separated
galartolipid
bands were located by a brief exposure to iodine vapour. The iodine wa,s removed by leading over SO, gas and by heating the plate for a short period, The bands were scrapedinto centrifuge tubes, as were areas of comparable size from the blank zone of the plate. To the adsorbent in the centrifuge tubes were added I ml 2% aqueous phenol and 4 ml cont. H&33,, the latter addition being rapid to ensure maximum heating of the mixture. The contents -were throughly mixed on a Vortex mixer, allowed to stand at room temperature for rs min and centrifuged at TQOOOg for I; min. The extinction of the supernatants was measured at 480 nm against a water blank. Standards of 20 pg and 50 ,t~ggallactose gave extinctions and 0.528 & 0.01, respectively. RESULTS
AND
0.223
&
0.01
(S.D.)
DISCUSSIQX
The lipid content
of inhibited
which can be explained ‘TABLE
of
cells was higher than that of normal cells (Table I),
by the nearly absence
of the cell wall mucopeptide
layer and
I
ZIPID
COXTEKT
GROWN
WITII
AND
COMPOSITIOX
OR WITROUT
NUMASi
OF CELLS
.4ND
MEMBRANES
OF B.
biJ%hPn
VAR. jW?Z’JtSyh&c7As
MILK
were inoculated a.t 23’ and harvested after 16 h growth at 37”. The values are expressed as means with standard errors. Membrane preparations were prepared after lysozyme treatment of cells as described before1 and dried in vactlo above P,O,. The number of pfeparaticns, all ana!gzed in duplicate are given between parentheses.
Cells
the increased number of internal membranes in these cells?. The galactose content of the lipids decreased however remarkably. Data of the lipid content of the membrane fractions give a more useful comparison. The membrane lipid proved to be rather low in comparison to membranes of other gram-positive bacteria@. Snip for membranes of 3ac~~~~~ ~e~a~e~~~~ a similar value was reported lo. The relatively i low lipid content Biochirn. Biaphys. Acta,
231
(rgpj
545-549
GALACTOLIPID COMPOSITION OF
of the preparations ribonucleoprotein
B. bijdzw
may be caused
547
by the presence
and polysaccharide8.
Another
of the remarkable
amount
of
cause may be the fact that membrane
preparations were dried in vacua above P,O,. Membrane lipid did not change very much after cell wall inhibition, only the galactose content of the lipids decreased to about 50%. The decrease of the galactose content of the lipids was not due to the acidity 5.0-5.~
of the medium
of inhibited
cells at the time of harvesting
(pH 6.0-6.4
for the medium of normal cells). By column fractionation it was found that in normal cells 30-45%
lipid was accounted
by the phospholipids,
45-60%
by the glycolipids
veTsus
of the total and 1o-2o~~
by the neutral lipids. Alterations in the galactose content of the lipids were reflected in the distribution of the lipid fractions in cells in which cell wall synthesis was inhibited. indicated
The glycolipid fraction amounted to 15-30% an overall decrease of the galactolipids. The
of the total lipids, which polar fraction, containing
mainly phospholipids, was increased (60-70~/~) but the neutral fraction remained constant (IO-ZO~/~). Because the phosphorus content in total lipid extracts did not a phosphorus-free polar lipid may be change significantly after cell wall inhibitionl, eluted from the column together present
with the phospholipids.
time concerning the nature The decrease of the galactose
the glycolipid
fraction
galactolipids
at the
decrease of
from a shift between
glycolipids
could result
and/or from a decline of the total number the individual
No data are available
of this polar component. content of the lipids and the relative of glycolipid
the individual
molecules.
in normal cells of the late logarithmic
Determination
of
phase (16 h growth)
after inoculation at 23O showed high proportions of digalactosyldiglyceride and monogalactosyldiglyceride (Table II). The amount of acylmonogalactosyldiglyceride is less TABLE
II
GALA~TOLIPID GROWNWITN
COMPOSITION
0F
ORWITHOUTHUMAN
CELLS
bijidunz v_4~.pe?znsylvanicus
0FB.
INOCULATED
AT
23’
AND
MILK
Cells were harvested after 16 h of growth at 37”. The composition was determined by quantitative thin-layer chromatography of the total glycolipid fraction. The values are expressed as percent of total lipid galactose in the first two columns. Means with standard errors for duplicate determinations in three extracts are shown. The composition is given in percentages of total galactolipid molecules in the third and fourth column.
Galactolipid
Diacylmonogalactosyldiglyceride Monogalactosyldiglyceride (+acylmonogalactosyldiglyceride) Monogalactosylmonoglyceride Acyldigalactosyldiglyceride Digalactosyldiglyceride Trigalactosyldiglyceride
Galactolipad composition 0/Oof lipid gala&se (= 100)
0h of total galactolipid molecules (= 100)
+ Human milk
+ Human milk
3.5 f 22.8 1.1 8.6 52.6 11.4
& * f & *
- HWYLaVl milk
- Huvnan milk
0.29
9.5 f
1.34
5.6
13.8
0.64 0.40 0.57 0.50 0.15
31.2 *
1.41
36.9
45.2 3.3 3.5 28.8 5.8
2.3 i: 1.37 4.9 & 0.86 39.8 f 3.42 12.1 f 0.83
I.8 6.9 42.4 6.1
than 1% of lipid galactose. After cell wall inhibition a remarkable shift to monogalactosyldiglyceride and its acylated derivatives was observed. The total monogalactosyl lipids were increased from 44 to 62%. This increase was reflected in a drop of the total digalactosyl lipids which was about the same. Biochim. Biophys. Ada,
231
(1971)545-549
~noc~~atio~ of celis at 37” izstead of at 23” gave a. shortening of the lag phase from 8 to 4 II and a slightly lower pH value after a6 h growth. The proportions of the various gaiactolipids in normal cells remained the same (Table III). Inhibitedcellshowever showed a m&e distinct shift to total manogalactosyl lipids from 41 to 7ic/;i with a concomitant decrease cf total digalactosyl lipids. After 40 h growth (pH 3: harvest 5.5) the amount of trigalactosyldiglyceride of inhibited cells ixreased from 6 to x.jC;& at the cost of the total monoga!actosyl lipids (from Sz to 57%) and the total digaiactosyl lipids (from 32 to z~*/~o)_ TABI.E
III
GALACTOLIPID GXOU7N
WITH
COMPOSITIOX OR WITHOUT
OF
CELLS
HUMAN
B. ~.~~d~~~VAR.
OF
~~~Z~zSy~~U~~Z~C~~S INOCULATED
AT
3~~ ASD
EYJfLi<
Cells were harvested after 16 h of growth at 37>. The composition was determined by quantitative thin-layer chromatography of the total glycolipid fraction. The values are expressed as percent of total lipid galactose in the first two columns. Means with standard errors for duplicate determinations in three extracts are shown. The composition is given in percentages of total gaiactolipid molecules in the third and fourth column. ..~___ _._.. Galactolipid Galactolipid compositl:o9z .I__ - -..-._.._.__ 91, of lipid galaclose 74 of total glycol$Ad wkoleccdes (= 100) (= IOO)
Diacylmonogala,ctos~Idiglyceride Monogalactos yldiglyceride (+ acylmonogalactosyldigl~ceride~ Zlonogalactosyllnonoglyceride ~c~lldigalactosyld~glyceride Digalactosyldiglyceride Trigalactosyldiglyceride
..-.~.. Nwnm
+ Human
-_ Irlwm,an
$ N~tlman
-
mikk
milk
m itk
w&2 _I__.-
1.5 = 0.01
12.3 :t 1.36
2.5
15.7
22.6 i 0.44 0.9 = 0.45 12.4 = 1.28
37.7 rt 0.87 6.0 i- 0.81 9.9 r. 0.23
47.8 7.2 6.1
jZ.1
27.0
37-r 1.5 10.0 42.5 5.7
+
0.72
10.5 + 0.73 ~--
&
1.27
7.2 I: 0.96 ..- --.~
..-.
i7.2
5.3
The decrease of the galactose content of the lipids after cell wall inhibit&, anpeared to be caused not only by a decrease of the mean number of the galactose molecules/galactolipid molecule but also by a decrease of the proportion of gaiactolipids of total lipids (Table IV). The galactolipid content of the total cell decreases still after cell wall inhibition in spite of the near absence of the mucopeptide. The
GXLACTOLIPXU
CO3TENT
OF CELLS
ANU
MEMIBRASES
OF ./it. bij%&m YAR.
~en?Z.~~~~~~z~~l,~S
Cellswereiaoc~~lated at 23’ and harvested after IS h of growth at 37” with or without human milk. Values were calculated out the data of Tables I and II.
{Lmoles galactose/,umole galactolipid pmoles galactolipid/mg lipid ,umoles galactolipid/mg dry weight _____-~.---
1.62
1.44
1.62
I.44
0.28
0.18
0.27
0.16
0.017
o.or4 -~.
0.022 -__---
o.or5
_ _--
amount of gakactolipid molecules/mg membrane decreased about 33% together with the decrease of the mean nuniber of galactose rnoleculesig~ac~o~~~~d molecule. The most probable e~F~ana~on for the decrease of the galactose content of the
GALACTOLIPID
COMPOSITION
OF B.
bijdum
519
lipids seems to be a competition for UDP-galactose. After cell wall inhibition an outer electron-dense layer was always seen in electronmicrographs of these cells before and after treatment with lysozyme*. This layer presumably consists of cell wall polysaccharide material composed of glucose, galactose and rhamnosell. Increase of polysaccharide synthesis could be a compensation for the lack of a rigid peptidoglycan layer and could compete with glycolipid synthesis for UDP-galactose. ACKNOWLEDGEMENT
The authors are indebted to Mr. G. A. M. Rutten for excellent technical assistance. REFERENCES F. A. EXTERKATE AND J. H. VEERKAMP, Biochim.Bio$hys. Acta, 176 (1969) 65. P. GYBRGY, R. F. NORRIS AND C. S. ROSE, Arch.Biochem.Biophys., 24 (1954) 193.
G. BASCHANG, Fovtschv. Chew.. Ovg. Naturstoffe, 20 (1962) 200. M. C. GLICK, T. SALL, F. ZILLIKEN AND S. MUDD, Biochim. Biophys. Acta, 37 (1960) 361. J. H. VEERKAMP, Biochim. Biophys. Acta, 210 (1970) 267. P. G. ROUGHAN AND R.D. BATT, Anal.Biochem., 22 (1968) 74. R. M. BROEKHUYSE, Biochim.Biophys. Acta, 152 (1968) 307. F. A.EXTERKATE,G.F.J.VRENSENAND J.H.VEERKAMP, Biochim.Biophys. Acta, 219 (1970) 141. 9 M. R. J. SALTON, Ann. Rev. Micvobiol., 21 (1967) 417. IO S. MIZUSHIMA, M. ISHARA AND K. KITAHARA, J. Biochem. Tokyo, 59 (1966) 374. R. LAMBERT AND Y. SAITO, Arch.Biocheun.Biophys., 112 (1965) 120. II J. H. VEERKAMP, Biochim. Biophys. Acta, 231 (1971)545-549
ET BIOPHYSICA
BIOCHEMICAL
ACTA
CHANGES
PENNSYLVANICUS IV. GALACTOLIPID
545
IN BIFIDOBACTERI~JM
AFTER
CELL
WALL
BIFiTDTJM
VAR.
INHIBITION
COMPOSITION
SUMMARY
A decrease of the lipid galactose content of cells and membranes was observed after growth of ~~~~~~cte~~~~ b~~~~~ var. ~~~~sy~v~~~c~swithout human milk. This change resulted both from a decrease of the amount of all glycolipid compounds and from a shift in the ratio of these compounds to the monog~aciosyl Iipids at the expense of the digalactosyl lipids. The shift was stronger after shortening of the lag phase of the cells. The alterations could be explained by a more limited disposal of UDPgalactose for glycolipid synthesis as a consequence of an increased cell wall polysaccharide synthesis.
INTRODUCTION
In a previous paper* we reported a considerable decrease of the galactose content in lipid extracts of whole cells, membrane and cytoplasmic preparations of Si$~~~~c~ey~~~~i~~~~~ var. ~en~s~~va~~cu~after inhibition of cell wall synthesis. Inhibition was effected by depriving the cells of human milk, which contains N-acetylglucosamine derivatives necessary for normal growth and cell wall mucopeptide synthesis2-4. Preliminary results suggested that the decrease in lipid galactose was correlated with an overall decrease of the galactolipids, which were identified as mono-, di-, and trigalactosyldiglyceride, mono-, and diacyl monogalactosyldiglyceride and acyl digalactosyldiglyceride l, In this paper we report the changes occurring in the galactolipid content and in the galactolipid composition after cell wall inhibition. MATERIALS
AND METHODS
Cells were inoculated at 23” and 37” and grown for 16 or 40 h with or without human milk’. The cells were harvested and washed as describedl. Membrane preparaBiochina.Biophys. A&z, 231
(197’)
545-549
tionswere obtained as before’~. Lipids were extracted and fractionated on si!icic acid columns according to described procedures;. Galactoiipids vvere present in the acetone column
fraction.
was established
Their
identification
after heating
was described
for 24 h at
previouslyl.
Membrane
70”.
Dry weight of cehs
fractions
were dried in WKGO
above P,&.
A modification of the procedure of ROUGEAS AND BATTY was used for rhe assay of the various galactolipids in acetone column fractions. The Silica gel G (Mercl;, Germany) was purified thoroughly with a slight’iy modified washing &-ocedare according to BROEKHCYSE~ in order to ob’cain acceptable blanks. Thin-layer chronzatograms of the acetone fractions (r-1.5 mg of total gala~to~~~~d~ were developed in ch_;oroforr~l-met~lanol-cone.
ammonia
(70 : zo : 2, by vol.). The separated
galartolipid
bands were located by a brief exposure to iodine vapour. The iodine wa,s removed by leading over SO, gas and by heating the plate for a short period, The bands were scrapedinto centrifuge tubes, as were areas of comparable size from the blank zone of the plate. To the adsorbent in the centrifuge tubes were added I ml 2% aqueous phenol and 4 ml cont. H&33,, the latter addition being rapid to ensure maximum heating of the mixture. The contents -were throughly mixed on a Vortex mixer, allowed to stand at room temperature for rs min and centrifuged at TQOOOg for I; min. The extinction of the supernatants was measured at 480 nm against a water blank. Standards of 20 pg and 50 ,t~ggallactose gave extinctions and 0.528 & 0.01, respectively. RESULTS
AND
0.223
&
0.01
(S.D.)
DISCUSSIQX
The lipid content
of inhibited
which can be explained ‘TABLE
of
cells was higher than that of normal cells (Table I),
by the nearly absence
of the cell wall mucopeptide
layer and
I
ZIPID
COXTEKT
GROWN
WITII
AND
COMPOSITIOX
OR WITROUT
NUMASi
OF CELLS
.4ND
MEMBRANES
OF B.
biJ%hPn
VAR. jW?Z’JtSyh&c7As
MILK
were inoculated a.t 23’ and harvested after 16 h growth at 37”. The values are expressed as means with standard errors. Membrane preparations were prepared after lysozyme treatment of cells as described before1 and dried in vactlo above P,O,. The number of pfeparaticns, all ana!gzed in duplicate are given between parentheses.
Cells
the increased number of internal membranes in these cells?. The galactose content of the lipids decreased however remarkably. Data of the lipid content of the membrane fractions give a more useful comparison. The membrane lipid proved to be rather low in comparison to membranes of other gram-positive bacteria@. Snip for membranes of 3ac~~~~~ ~e~a~e~~~~ a similar value was reported lo. The relatively i low lipid content Biochirn. Biaphys. Acta,
231
(rgpj
545-549
GALACTOLIPID COMPOSITION OF
of the preparations ribonucleoprotein
B. bijdzw
may be caused
547
by the presence
and polysaccharide8.
Another
of the remarkable
amount
of
cause may be the fact that membrane
preparations were dried in vacua above P,O,. Membrane lipid did not change very much after cell wall inhibition, only the galactose content of the lipids decreased to about 50%. The decrease of the galactose content of the lipids was not due to the acidity 5.0-5.~
of the medium
of inhibited
cells at the time of harvesting
(pH 6.0-6.4
for the medium of normal cells). By column fractionation it was found that in normal cells 30-45%
lipid was accounted
by the phospholipids,
45-60%
by the glycolipids
veTsus
of the total and 1o-2o~~
by the neutral lipids. Alterations in the galactose content of the lipids were reflected in the distribution of the lipid fractions in cells in which cell wall synthesis was inhibited. indicated
The glycolipid fraction amounted to 15-30% an overall decrease of the galactolipids. The
of the total lipids, which polar fraction, containing
mainly phospholipids, was increased (60-70~/~) but the neutral fraction remained constant (IO-ZO~/~). Because the phosphorus content in total lipid extracts did not a phosphorus-free polar lipid may be change significantly after cell wall inhibitionl, eluted from the column together present
with the phospholipids.
time concerning the nature The decrease of the galactose
the glycolipid
fraction
galactolipids
at the
decrease of
from a shift between
glycolipids
could result
and/or from a decline of the total number the individual
No data are available
of this polar component. content of the lipids and the relative of glycolipid
the individual
molecules.
in normal cells of the late logarithmic
Determination
of
phase (16 h growth)
after inoculation at 23O showed high proportions of digalactosyldiglyceride and monogalactosyldiglyceride (Table II). The amount of acylmonogalactosyldiglyceride is less TABLE
II
GALA~TOLIPID GROWNWITN
COMPOSITION
0F
ORWITHOUTHUMAN
CELLS
bijidunz v_4~.pe?znsylvanicus
0FB.
INOCULATED
AT
23’
AND
MILK
Cells were harvested after 16 h of growth at 37”. The composition was determined by quantitative thin-layer chromatography of the total glycolipid fraction. The values are expressed as percent of total lipid galactose in the first two columns. Means with standard errors for duplicate determinations in three extracts are shown. The composition is given in percentages of total galactolipid molecules in the third and fourth column.
Galactolipid
Diacylmonogalactosyldiglyceride Monogalactosyldiglyceride (+acylmonogalactosyldiglyceride) Monogalactosylmonoglyceride Acyldigalactosyldiglyceride Digalactosyldiglyceride Trigalactosyldiglyceride
Galactolipad composition 0/Oof lipid gala&se (= 100)
0h of total galactolipid molecules (= 100)
+ Human milk
+ Human milk
3.5 f 22.8 1.1 8.6 52.6 11.4
& * f & *
- HWYLaVl milk
- Huvnan milk
0.29
9.5 f
1.34
5.6
13.8
0.64 0.40 0.57 0.50 0.15
31.2 *
1.41
36.9
45.2 3.3 3.5 28.8 5.8
2.3 i: 1.37 4.9 & 0.86 39.8 f 3.42 12.1 f 0.83
I.8 6.9 42.4 6.1
than 1% of lipid galactose. After cell wall inhibition a remarkable shift to monogalactosyldiglyceride and its acylated derivatives was observed. The total monogalactosyl lipids were increased from 44 to 62%. This increase was reflected in a drop of the total digalactosyl lipids which was about the same. Biochim. Biophys. Ada,
231
(1971)545-549
~noc~~atio~ of celis at 37” izstead of at 23” gave a. shortening of the lag phase from 8 to 4 II and a slightly lower pH value after a6 h growth. The proportions of the various gaiactolipids in normal cells remained the same (Table III). Inhibitedcellshowever showed a m&e distinct shift to total manogalactosyl lipids from 41 to 7ic/;i with a concomitant decrease cf total digalactosyl lipids. After 40 h growth (pH 3: harvest 5.5) the amount of trigalactosyldiglyceride of inhibited cells ixreased from 6 to x.jC;& at the cost of the total monoga!actosyl lipids (from Sz to 57%) and the total digaiactosyl lipids (from 32 to z~*/~o)_ TABI.E
III
GALACTOLIPID GXOU7N
WITH
COMPOSITIOX OR WITHOUT
OF
CELLS
HUMAN
B. ~.~~d~~~VAR.
OF
~~~Z~zSy~~U~~Z~C~~S INOCULATED
AT
3~~ ASD
EYJfLi<
Cells were harvested after 16 h of growth at 37>. The composition was determined by quantitative thin-layer chromatography of the total glycolipid fraction. The values are expressed as percent of total lipid galactose in the first two columns. Means with standard errors for duplicate determinations in three extracts are shown. The composition is given in percentages of total gaiactolipid molecules in the third and fourth column. ..~___ _._.. Galactolipid Galactolipid compositl:o9z .I__ - -..-._.._.__ 91, of lipid galaclose 74 of total glycol$Ad wkoleccdes (= 100) (= IOO)
Diacylmonogala,ctos~Idiglyceride Monogalactos yldiglyceride (+ acylmonogalactosyldigl~ceride~ Zlonogalactosyllnonoglyceride ~c~lldigalactosyld~glyceride Digalactosyldiglyceride Trigalactosyldiglyceride
..-.~.. Nwnm
+ Human
-_ Irlwm,an
$ N~tlman
-
mikk
milk
m itk
w&2 _I__.-
1.5 = 0.01
12.3 :t 1.36
2.5
15.7
22.6 i 0.44 0.9 = 0.45 12.4 = 1.28
37.7 rt 0.87 6.0 i- 0.81 9.9 r. 0.23
47.8 7.2 6.1
jZ.1
27.0
37-r 1.5 10.0 42.5 5.7
+
0.72
10.5 + 0.73 ~--
&
1.27
7.2 I: 0.96 ..- --.~
..-.
i7.2
5.3
The decrease of the galactose content of the lipids after cell wall inhibit&, anpeared to be caused not only by a decrease of the mean number of the galactose molecules/galactolipid molecule but also by a decrease of the proportion of gaiactolipids of total lipids (Table IV). The galactolipid content of the total cell decreases still after cell wall inhibition in spite of the near absence of the mucopeptide. The
GXLACTOLIPXU
CO3TENT
OF CELLS
ANU
MEMIBRASES
OF ./it. bij%&m YAR.
~en?Z.~~~~~~z~~l,~S
Cellswereiaoc~~lated at 23’ and harvested after IS h of growth at 37” with or without human milk. Values were calculated out the data of Tables I and II.
{Lmoles galactose/,umole galactolipid pmoles galactolipid/mg lipid ,umoles galactolipid/mg dry weight _____-~.---
1.62
1.44
1.62
I.44
0.28
0.18
0.27
0.16
0.017
o.or4 -~.
0.022 -__---
o.or5
_ _--
amount of gakactolipid molecules/mg membrane decreased about 33% together with the decrease of the mean nuniber of galactose rnoleculesig~ac~o~~~~d molecule. The most probable e~F~ana~on for the decrease of the galactose content of the
GALACTOLIPID
COMPOSITION
OF B.
bijdum
519
lipids seems to be a competition for UDP-galactose. After cell wall inhibition an outer electron-dense layer was always seen in electronmicrographs of these cells before and after treatment with lysozyme*. This layer presumably consists of cell wall polysaccharide material composed of glucose, galactose and rhamnosell. Increase of polysaccharide synthesis could be a compensation for the lack of a rigid peptidoglycan layer and could compete with glycolipid synthesis for UDP-galactose. ACKNOWLEDGEMENT
The authors are indebted to Mr. G. A. M. Rutten for excellent technical assistance. REFERENCES F. A. EXTERKATE AND J. H. VEERKAMP, Biochim.Bio$hys. Acta, 176 (1969) 65. P. GYBRGY, R. F. NORRIS AND C. S. ROSE, Arch.Biochem.Biophys., 24 (1954) 193.
G. BASCHANG, Fovtschv. Chew.. Ovg. Naturstoffe, 20 (1962) 200. M. C. GLICK, T. SALL, F. ZILLIKEN AND S. MUDD, Biochim. Biophys. Acta, 37 (1960) 361. J. H. VEERKAMP, Biochim. Biophys. Acta, 210 (1970) 267. P. G. ROUGHAN AND R.D. BATT, Anal.Biochem., 22 (1968) 74. R. M. BROEKHUYSE, Biochim.Biophys. Acta, 152 (1968) 307. F. A.EXTERKATE,G.F.J.VRENSENAND J.H.VEERKAMP, Biochim.Biophys. Acta, 219 (1970) 141. 9 M. R. J. SALTON, Ann. Rev. Micvobiol., 21 (1967) 417. IO S. MIZUSHIMA, M. ISHARA AND K. KITAHARA, J. Biochem. Tokyo, 59 (1966) 374. R. LAMBERT AND Y. SAITO, Arch.Biocheun.Biophys., 112 (1965) 120. II J. H. VEERKAMP, Biochim. Biophys. Acta, 231 (1971)545-549