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Release of a lipopolysaccharide-protein complex from Escherichia coli a by warm-water treatment
72
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26466 R E L E A S E OF A L I P O P O L Y S A C C H A R I D E P R O T E I N COMPLEX FROM ESCHERICHIA
C O L I A BY WARM-WATER TREATMENT
DEXTER ROGERS* Department of Biochemistry and t3iophysics Oregon 5"tate ~:niz,ersity, Corvallis, (}reg. 9733~ ( ('.5._4.)
(Received June 12th, 197oI (Revised manuscript received ()ctober 1st, i 07 o)
SUMMARY
Warm-water treatment of Escherichia coli A in o.I M Tris (pH 7.3), in the absence of added Mg z+, released protein and lipopolysaccharide simultaneously. After precipitation witti (NH4)2SO a and brief treatment with sodium deoxycholate, the solubilized material appeared as a single component when analyzed by analytical centrifugation. Chromatographic analysis with DEAE-cellulose indicated the coincidental distribution of protein and lipopolysaccharide components (e.g. heptose, 3deoxyoctulosonate and ester groups). Bound /5 hydroxymyristic acid was present, but colitose (3,6-dideoxy-L-galactose) was apparently not present in the lipopolysaccharide from this galactose-negative culture. Tile complex was precipitated with divalent cations and hydrolyzed with 14 mM HC1 to yield a lipid. About one-half of the complex was accounted for in terms of a full complement of amino acids. The complex was dissociated by chromatography in the presence of EDTA or sodium deoxycholate, and evidence was obtained for three subfractions containing varying proportions of protein and lipopolysaccharide. The procedure used for its release and its composition suggested that the lipopolysaccharide-protein complex might be derived from tile outermost laver of the cell wall. This complex showed no affinity for galactose.
INTRODUCTION When Escherichia coli A is suspended in o.I M Tris (pH 7.3) and stressed at 4 ~ °, a discrete amount of protein is released at a constant rate 1. This warm-water treatment releases protein without loss in viability of the treated cells or loss of cytoplasnlic material, and this release is prevented by the presence of divalent cations. The rate of release is related inversely to the osmolarity of the suspending medium. In IO mM solution, the release is faster than that in ioo mM solution, and there is evidence for a second sequence of release, which includes a sugar-binding protein 2. The purpose of this report is to identify the material released by warm-water treatment in o.i M Tris, in the absence of Mg 2+, and to relate it to the structure and transport function of the cell surface. * Present address: Department of Chemistry, Paterson State College, Wayne, N.J. o747o, U.S.A. Biochim. Biophys. Acla, 23o (1971) 72 81
RELEASE OF LIPOPOLYSACCHARIDE-PROTEIN COMPLEX
73
MATERIALS AND METHODS
The conditions for growing cultures of E. coli A in a lactic acid-inorganic salts medium have been described 3. This strain lacks both galactokinase (EC 2.7.1.6 ) and galactose-I-phosphate uridyltransferase (EC 2.7.7.io), and, under the conditions of growth, galactose should not have been available for the synthesis of a complete lipopolysaccharide. Presumably, this is a rough strain, a Re-type mutant 4, whose inner core is limiteeI t o a glucose residue attached to the heptose backbone. Except for preliminary experiments, when only small volumes of culture were needed, the culture was grown in a IOO-1fermentor. The cell paste, which weighed about 25 g on a dry basis, was suspended in 3 1 of o.i M Tris (pH 7.3) for I h at 23 ° before centrifuging. The cells were resuspended in 600 ml of o.I M Tris and placed in a 60 ° water bath. The temperature of the suspension rose to 48° within 4 min, and this temperature was maintained for 4 min longer. After centrifuging, the warm-water extract was dialyzed against a saturated solution of (NH4)2SOa, which had been adjusted to pH 7.3 and buffered with o.I M Tris. The precipitated material was centrifuged, redissolved and dialyzed overnight at 4 ° against several changes of o.I M Tris. The clarified extract, which contained about 35 ° mg of protein, was used for experimentation. In certain experiments, the clarified extract was dialyzed for 2 days at 4 ° against 4 or 5 changes of o.I M Tris containing either o.15 % sodium deoxycholate or 5 mM EDTA. As it will be shown, the extracted material is a lipopolysaccharide-protein complex plus a minor amount of protein 1 that is washed from the cells at o °, even in the presence of Mg2+. The total clarified extract was chromatographed on a 4 cm × 50 cm column of DEAE-cellulose 2. The column was equilibrated at 4 ° with the buffer used in the final preparation of the extract, i.e.o.I M Tris (pH 7.3) and, where indicated, o.15 % sodium deoxycholate or 5 mM EDTA. The column was eluted with a gradient prepared with a constant volume mixer 5. 2 1, or more, of I M NaC1 in o.i M Tris (pH 7.3) were mixed with 0. 5 1 of o.i M Tris. Io-ml fractions were collected and assayed for protein using (a) the Folin-Denis reagent 6, (b) the biuret reagent 7 or (c) turbidity measurement with trichloroacetic acid 2. The protein standard was a solution of recrystallized bovine serum albumin, which had been standardized spectrophotometrically s. The effluent fractions were also assayed for lipopolysaccharide components: (a) heptose by a modified cysteine-H2SO a reaction", 10, (b) 3-deoxyoctulosonate by a modified periodate-thiobarbiturate reaction 10.~1 and (c) ester groups by an alkaline hydroxylamine reaction ~2. These results were normalized to give the relative distribution of material in the chromatographic effluent. Kinetic analysis of the rate of protein release during warm-water treatment of E. coli A and phosphatase assays on the chromatographic effluent were performed as described previously 1. The affinity of the lipopolysaccharide-protein complex for galactose was tested by equilibrium dialysis ~3. A portion of the clarified extract was dialyzed for 24 h at 4 ° against 20 mM D-Ei-~*Clgalactose in o.I M Tris (pH 7.3). Samples of the dialyzed material and its dialyzate were measured for radioactivity with a Packard model 3003 Tri-Carb scintillation spectrometer. Analytical centrifugation was performed with a Spinco model E ultracentrifuge. A portion of the clarified extract was dialyzed for 4 h at 4 ° against o.15 % sodium Biochim. Biophys. Acta, 230 (I97 I) 7 2 - 8 I
74
1). R O G E t { S
deoxycholate in o.I M Tris (pH 7.3). The dialyzed sample and its dialvzate were placed in opposite compartments of a double-sectored cell and centrifuged for 48 rain on speed at 52ooo rev./nfin. The sedimentation constant, s, was calculated ~4 and corrected for viscosity' and density, but not for concentration dependence. The following analyses were performed on portions of the same clarified extract that was analyzed bv sedimentation velocity (Fig. 5)- The dry wt. of the lipopolysaccharide-protein complex in solution was deternfined after exhaustive dialysis (3 days at 4 °) against 2oo volumes of o.I M Tris (pH 7.3). 5-ml portions of the dialyzed extract and the dialyzate were separately dried to constant weight at lO5 °. The difference in weight of the dried samples, 27. 5 rag, was considered to be the dry wt. of the Iipopolysaccharid~protein complex in 5 nil. The corresponding amount of protein was determined with the biuret reagent. A 5-nil sanlple contained 13.0 mg of protein. The protein content of the complex was therefore 49.5 %. A solution containing 7.36 mg of the complex ( 3.64 mg of protein) and ()% trichloroacetic acid was mixed thoroughly and centrifuged. The residue was suspended in constant boiling HC1, evacuated and flushed with purified nitrogen IO times, sealed in an ampoule under nitrogen and hydrolyzed for 2o h at IIO °, The hydrolyzate was analyzed for anlino acids with the method of SPACKMAX et al. ~ using a Beckman model I2o-B amino acid analyzer. A solution containing 7.36 mg of the complex ( 3.72 mg of lipopolysaccharide, by difference) and 14 mM HCI was treated in a boiling-water bath for 3o nlin and centrifuged. The residue was suspended in 5 ml of 5 M HC1 and refluxed overnight. The hydrolyzed material was extracted with three 5-ml portions of ether. After the ether was evaporated, the fatty acids were esterified by boiling for 2 rain with 3 ml of methanol containing BF a. The boiled nfixture was transferred to a separatory funnel containing 2o ml of water. It was extracted 3 times with Io-ml portions of petroleum ether. The combined extracts were washed with water and dried ower anhydrous Na,~S()4. After evaporation, the residue was dissolved in 2 ml of chloroform. Comparable w)luines of buffer, with and without I.O mg of I)L-/!]-hydroxymyristic acid, were also treated in the same manner. Io-/~l samples of the esters were analyzed with an 1" and M model 4o2 chromatograph fitted with a 6 ft • o.I0 in open tubular capillary containing Diatoport S (8o IOO nlesh) coated with diethvleneglycolsuccinate (6°o, w/w). The operating temperature range was I3O I 8 O ; the temperature was programmed to increase at a rate of 3°/min. The combined gas-ttow rates were He, 4 o lb/ineh"-, 7 ° inl/min; air, 20 lb/inch 'a, 28o ml/min; H,,, io lb/inch e, 35 ml/min. All solvents were redistilled and none revealed material in the analytical range. RESULTS
The effectiveness of warm-water treatment depends on the uniform application of pressure by the expanding cell membrane against the weakened and stressed cell wall. Since this pressure is related inversely to the activity of the impernleable solute in the suspending medium, it was reasoned that a differential effect could be achieved by controlling cell turgor with appropriate choices of solute concentration. By this means, the wall layers might be broken sequentially and material released selectiveh as the necessary disrupting pressures were attained. Preliminary experiments 1 proBiochtm. Biophy,~. Acta, 23 ° (~97 l) 72 81
RELEASE OF LIPOPOLYSACCHARIDE--PROTEIN COMPLEX
75
duced the expected results. By decreasing the osmotic pressure of the medium, a differential release of protein was obtained without loss in viability or apparent release of cytoplasmic components. To correlate this effect, the rate of protein release was compared with the average protoplast volume obtained by MARQUISis for Bacillus megaterium. This comparison showed that, within limits, the rate of protein release and the average protoptast volume were both logarithmic functions of the reciprocal of osmolarity (Fig. i). However, about one order of magnitude separated these functions. When two species are compared, some difference in their cell membranes is to be expected, but the noted difference could also be due to the inherent extensibility of the cell wall, which would reduce the effectiveness of warm-water treatment.
~0
1.0
~/
B.megaterium
/
protoplosts
/ •8
o
g
~" o
7 _ /
D /
E. coli A
/ 0"1
I
I0
100
-2 ~"
oi
I/0smolarity
Fig. 1. Corre]ation b e t w e e n rate of release of p r o t e i n d u r i n g w a r m - w a t e r t r e a t m e n t of E. cull A a n d a v e r a g e p r o t o p l a s t v o l u m e of B. rnegaterium as f u n c t i o n s of osmolarity. T h e p r o t o p l a s t v o l u m e s were d e t e r m i n e d b y MARQUIS (see ref, 16).
As expected, the impermeable sucrose was more suitable in effecting osmotic shock than the partially permeable Tris buffer. However, at high concentrations, Tris contributed to the dissociation of the surface structure (cf. ref. 17). The release of lipopolysaccharide was not complete, although a discrete proportion was obtained by this warm-water treatment. In all of the following experiments, warm-water extracts were prepared with o.I M Tris (pH 7.3) because only a single sequence of protein release was obtained under these conditions 1. A warm-water extract was analyzed with the cysteine-H zSO 4 reaction and the periodate-thiobarbiturate reaction. The spectrum of the cysteineH2SO 4 reaction products, which was recorded after 24 h at 23 °, revealed peaks that corresponded to heptose (~max505-510 rim9), 3-deoxyoctulosonate (~max 385-39 ° nml°) and hexose, possibly glucose (~max 41°-415 nm9) - The secondary peak at about 590-600 nm was probably due to 3-deoxyoctulosonate and glucose9,10. The spectrum of the periodate-thiobarbiturate reaction products revealed peaks that corresponded to 3-deoxyoctulosonate (~m~ 548 rim1°' 1~) and a substance like that present in irradiated mouse skin which is not extracted with organic solvents (~max 450 nmlS). When the periodate treatment was performed at 55 °, instead of 23 °, the peak at 548 nm was intensified 3-fold without shifting its ~mx. This increase may have been due to hydrolysis of certain bound forms of 3-deoxyoctulosonate. It seems unlikely that colitose (3,6-dideoxy-L-galactose), whose reaction product absorbs at 532 nm ~s, after periodate treatment, was present. Colitose was not expected because, in the absence of galactose, the synthesis of the polysaccharide chain should have been Biochim. Biophys. Acta, 23o (1971) 72-81
7()
D. ROGERS
interrupted before colitose could be incorporated. Nevertlleless, a minor amount of colitose has been recovered from the lipopolysaccharide of a galactose-negative culture ~:'. Heptose-containing material was precipitated completely with trichloroacetic acid and with I0 mM Mg 2~, Ca 2= and Zn 2~. Its reactivity as heptose was enhanced three-fold by hydrolysis in 14 mM HC1 for I h at I00 °. Dilute acid hydrolysis als,~ liberated lipid, which was presumably lipid A 2°. During warm-water treatment, the rate of release of both protein and heptosecontaining material corresponded exactly (Fig. 2). In addition, Mg~- stabilized both protein and heptose-containing material against warm-water treatment. o I'C cL e :z:
dO8
OI A
0"8
o o
m
a.O.6
o 0"6
o
~t
+MgCl 2
o M i n u t e s at 4 8 *
80 Effluent
160 Frocliorts
240
Fig. 2. Release of protein and heptose-containing material (luring w a r m - w a t e r t r e a t m e n t of heptose; . - - . w i t h o u t Mg('l,z: . . . . . . . , w i t h MgC12. E . coli A. The concentration of MgC12 was o.ot M. ~,, protein; • ,
Fig. 3. C h r o m a t o g r a p h y of a w a r m - w a t e r e x t r a c t of E. coli A o n DEAl'; cellulose. The effluent was analvzed for protein ( 2 ) , heptose (O) and 3-deoxyoctulosonate (@).
A warm-water extract was chromatographed on DEAE cellulose with tile result shown in Fig. 3. The distribution of protein, heptose and 3 deoxyoctulosonate corresponded closely and indicated one major peak and two minor peaks. The first minor peak came off the column at about the break-through point. This peak may have consisted of material aggregated by the presence of o.I mM Mg('l., that was present in the buffer used in this experiment only. In the absence of added MgC12, only one minor peak is observed. Assays for ester groups, which are not shown in this figure, also indicated a close correspondence. A similar distribution of these components was observed with material that was precipitated with Mg2., Ca 2~ and Zn 2~, except for the absence of material usually appearing as a shoulder to the major peak (Fractions
Io5 125). Also expected in the warm-water extract were tllose components a that can be washed from the cells at o °, even in the presence of Mg2-. Among these components is a phosphatase, whose distribution in the DEAE-cellulose effluent is shown in Fig. 4This activity was located in the shoulder to the major peak (Fractions lO5-125). Analysis of the warm-water extract for homogeneity was performed by analytical centrifugation. This analysis revealed both a nlajor component and a minor component, whose Sobs were 2.3 S and 4.9 S, respectively. A similar analysis in the presence of IO mM MgC12 revealed four peaks, whose Sobs were 3.45 S, 5.7 S, 15.9 S and 24.5 S. When the warm-water extract was treated briefly with o.15 Oo sodium B i o c k i m . B i o p h y s . A c l a . 23o (197 t) 72-81
77
RELEASE OF LIPOPOLYSACCHARIDE--PROTEIN COMPLEX
deoxycholate before analytical centrifugation, only a single component was revealed (Fig. 5). The Sobs and s20, w were 3.25 S and 5.35 S, respectively. After treatment for 2 days with either o.15 % sodium deoxycholate (Fig. 6) or 5 mM EDTA (Fig. 7), the lipopolysaccharide-protein complex was dissociated by chromatography on DEAE-cellulose into three subfractions, which contained varying proportions of protein and lipopolysaccharide. However, complete resolution of these subfractions was not obtained. I.C
i
ii
_
il
i
.~° 0"4
i
i i
-6 ¢c0. 2
::~
./" ,,, 0 . . . . .
J'
0
'\
" .........
80 Effluent
160 Fractions
240
Fig. 4- Distribution of p h o s p h a t a s e activity in t h e effluent from DEAE-cellulose. The p h o s p h a t a s e substrate was glucose 6-phosphate (see ref. i). The d o t t e d line represents the distribution of protein in the effluent. Fig. 5. Analytical centrifugation of a w a r m - w a t e r e x t r a c t of E. coli A t h a t was precipitated with (NH4)~SO 4 and dialyzed briefly against o.15 % sodium deoxycholate (4 h at 4 °). The buffer was o.i M Tris (pH 7.3). The dialyzed sample and its dialyzate were placed in opposite c o m p a r t m e n t s of a double-sectored cell and centrifuged for 48 min on speed at 52000 rev./min at 5 °. Sedimentation is toward the left. i.o I
1.0
2"
0`8
=0-8
=°0`6 <
~0`6
~0`4
.~0"4
0.2
¢0-2
it ¢ =
=
o
S I
a/
eo
o/
120 tr ffluont
o
160 Fractions
200
eO
120
Eftluen!
,
,
160 Fractions
,
200
Fig. 6, Effect of sodium deoxycholate on t h e c h r o m a t o g r a p h y of a w a r m - w a t e r e x t r a c t of E. coli A on DEAE-cellulose. The w a r m - w a t e r e x t r a c t was dialyzed against o.1 M Tris (pH 7.3) containing o.i5 % sodium deoxycholate (2 days at 4 °). The effluent was analyzed for protein (A), heptose ( • ) and 3-deoxyoctulosonate (O). Fig. 7. Effect of E D T A on the c h r o m a t o g r a p h y of a w a r m - w a t e r e x t r a c t of E. call A on D E A E cellulose. The w a r m - w a t e r e x t r a c t was dialyzed against o.I M Tris (pH 7-3) containing 5 mM E D T A (2 days at 4 o). The effluent was analyzed for protein (A), heptose ( • ) , 3-deoxyoctulosonate (©) and ester groups (V). Biochim. Biophys. Acta, 23o (1971) 72-8i
78
D . R()GI~R~
TABLE
I
ANALYSIS
FOR
The material
Peak
THE
FATTY
Retention time (rain)
I 2
ACID
was hydrolyzed,
2.8 5.0
COMPONENTS
OF
THE
esterified and analyzed
In *
LIPOPOLYSACCHARIDE
Relative peak height .Sample
o.I 3 O.211
Control
I]. 5 I9. 5
-PROTEIN
COMPLEX
l)y g a s c h r o m a t o g r a p h y .
O 4.O
t,¢ "
Pcrtcnta~,e ,,f total /arty acids
<~:O
O.I 5
,~" t J
lO :O
0 3 I
I
3
0.8
0. 31
I "5
4 .5 {) 7
8. 5 9.9 I o.8 1-.o
o.39 o-45 o.49 (>.M
)o.o 42.{>
o 7.o
12 : o
().44
7 -'5
I 1.1}
5.0
14 : o
o.4~
4
2.o
11
t
N
1 3.2
{).{)O
().O
i).O
()
0
14.()
o.{){)
) o'5
1{} II 12 13
15. 7 17.2 10. I 28. 3
o.71 o.79 (1.87 1.29
I().(}
7 .o IO.O
15"5
I{},O
43.O ,q.o
{) 9,o
* Stearic
TABLE
a c i d { 18 :o}
O
Tentative identification
3
{).7 I
l{): 1
0.79
fl-{)H 14:o 18 : i
o.S 7 1.2()
4 nl
31 ~)
J .oo.
I1
ANALYSIS
FOR
The complex
A miuo acid
AMINO
ACID
was hydrolyzed
COMPONENTS
OF
and analyzed
THE
LIPOPOLYSACCHARIDE--PROTE1N
with an automatic
mg residue (3.64 mg protein) *
Lys
i . 7 (}
o . 2 2{)
o . 51 I . 1~
o.o7o
3.23
o-372
Thr
1.o 3
o. 10 5
i .5 °
,). 13 l o. 4 1 3 o.272 o. 14 °
Ser * * Glu*" Pro Gly *"
Val * * Met 1 le
Leu Tyr Phe Cy~ NH a Trp
amino
l~moles residue (3.64 mg protein) "
His A r g *" Asp''
.\la
I0:o
COMPLEX
acid analyzer.
O. l 8 4
3.z°
2.4o 2.45 3-o9
o. 22o
2 .o{) 11.5q ) .63 2.43 o.95 ~.oo . . *** ""*
o.2o 7 o.o77 o. 1 8 5 I).275 o. i `55 o. 147 .
Total
.
.
. .., """ 3.239
• 7.3 {) n a g c o m p l e x = 3.{)4 m g p r o t e i n ( b i u r e t ) . indicated amino acids, in the form of polypeptide (biuret), are also found in the lipopolysaccharide moiety of the complex (see ref. 2 U. "** } ' r e s e n t , b u t n o t d e t e r m i n e d ; tryptophan w a s n o t e d i n t h e s p e c t r u m a t 2t}z n m . " * The
Biochim. Biophys. Acta, 2 3 0 ( I 9 7 I) 7 2 - 8 I
RELEASE OF LIPOPOLYSACCHARIDE-PROTEIN COMPLEX
79
Another characteristic component of lipopolysaccharide is/~-hydroxymyristic acid, which was detected by means of gas chromatography after hydrolysis and esterification (Table I). Peak 12 corresponded to the observed appearance of /5hydroxymyristic acid. The relative distribution of fatty acids reported in this paper and by ELBEIN AND HEATH19 and by BURTON AND CARTER20 all differ quite strikingly from each other. The biuret assay of the lipopolysaccharide-protein complex had indicated that protein was 49.5 % of the dry weight. An amino acid analysis of the hydrolyzed complex confirmed this estimate (Table II). A full complement of amino acids was obtained and the recovered residue weights accounted for 45 % of the dry wt. of the complex and 9° % of the biuret assay value. This account did not include the contributions of tryptophan, half cysteine and NHs. A numerical analysis 22 of the ratio of hydrophilic-to-apolar amino acid residues and the ratio of charged-to-small amino acid residues revealed that the protein component of the lipopolysaccharide-protein complex resembled more closely membranous lipoprotein than soluble lipoprotein and oligomeric protein than monomeric protein. All attempts failed to show any affinity between the lipopolysaccharide-protein complex and galactose. DISCUSSION
Current conceptions of the cell wall of E. coli and other gram-negative bacteria consider a multilayered, asymmetric structure zz-26. The outer, L-layer of the cell wall contains lipopolysaccharide and lipoprotein, and by electron microscopy it appears as two visible layers separated by a transparent layer. The intermediate, G-layer contains globular protein and mucopeptide, and it appears as a transparent layer and a visible layer. Since the L-layer can be removed with phenol or detergents, it may be only loosely connected to the G-layer. These treatments, however, fragment the L-layer. The isolated lipopolysaccharide contains less than I0 % protein, which may be attached covalently to the lipopolysaccharide~1. In cell envelopes, in addition to hydrophobic bonding, lipids and proteins may be bound by polar bonds involving Mg 2÷ (see ref. 27). The L-layer is also stabilized by divalent cations because osmotic shock with EDTA releases about one-half of the lipopolysaccharide 28. Much of this release is prevented with Mg2+. The lipopolysaccharide-protein complex that is isolated by warm-water treatment may be derived from the outer, L-layer of the cell wall because of the similarity in composition and the mode of isolation. The complexity of this material, which is evident from chromatography in the presence of sodimn deoxycholate or EDTA, may reflect the composition and structule of this layer in its original state. Warm-water treatment in 0.I M Tris, in the absence of added Mg2+, may prove to be a specific probe of the outer, L-layer of the cell wall and discriminate it from the intermediate, G-layer. Among the proteins that are released by osmotic shock are several nutrient binding proteins 29-31. Since the isolated lipopolysaccharide-protein complex lacks any affinity for galactose, nutlient binding proteins as a class may be located beneath the lipopolysaccharide-protein complex. This possibility is substantiated by the inability of antiserum for the leucine-binding protein to inhibit uptake by the intact Biochim. Biophys. Acta, 23 ° (1971) 72-8I
~O
I). R O G E R S
cellaL It is possible that warm-water treatment might be modified to probe deeper into the cell wall. When warm-water treatment is performed in I0 mM Tris, instead of I00 mM Tris, a second sequence of protein release occurs 1. Among the pr-teins released under these conditions is a binding protein for galactoseL Further work may be able to establish the location of binding proteins among the globular proteins at the outerface of the G-layer or within the periplasmic space. The process of nutrient transport into a cell has been recognized as a surface phenomenon and the cell membrane, the osmotic barrier, is considered to hold at least some of the transport components a3. Certain components have already been isolated from membrane fragments a4, 35. Still other components mav be located outside of the cell membrane because thev can be released by osmotic shock 29 a~ a pr.cess that apparently does not damage the cell membrane or release cytoplasmic material 3°,a7. In fact, a treated cell can regain its lost function when the binding protein is reinstalled in the cell 29. For a multicomponent transport system, the anisotr~q~ic distribution of its components would contribute to the vectored movement ~)f the transported nutrient 38. Therefore, the transport components should be located experimentally within the cell surface in order to fully understand the process. ACKNOWLEDGEMENTS
This study was made while the author was a Special Fellow of the U.S. Public Health Service, National Institutes of Health and was supported by a Public Health Service Fellowship 5 I:3 AM-II 083. I am grateful for the warm hospitality that was provided by the late Vernon H. Cheldelin. I thank Robert R. Becker for many useful discussions and Robert L. Howard for performing the amino acid analysis. REFERENCES I D. ROGERS, Science, I 5 9 (1968) 531. 2 I). ROGERS, J. Bacteriol., 88 (1964) 279. 3 D. ROGERS .aND S . - H . YO, J. Bacteriol., 8 4 ( I 9 0 2 ) 8 7 7 . 4 0 . L/3DERITZ .aND O. WESTPHAL, Angew. Chem. Intern. Ed., 5 (1966) I 9 8 . 5 R . S. ALM, t{. J . P . WILLIAMS AND A. TISELIUS, Acta Chem. Scan&, 6 (1952) 8 20. 6 0 . H . LOWRY, N. J. ROSEBROUGH, A. [~. FARR AND R . J. RANDALL, ./. Biol. Chem., I03 (It#3l) 265 . 7 A. G. GO~NALL, C. J . BaRDAWILL AND M. 3I. DAVID, .[. Biol. Chem., 177 (1949) 751 • 8 B . J . ADKINS .aND J. F. FOSTER, Biochemistry, 5 ( r 9 6 6 ) 2579. q Z. DlSCHE, J. Biol. Chem., 2 o 4 (1953) 983. i o M. J . OSBORN, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 4991~ A. WEISSBACH AND J . HURWlTZ, J . Biol. Chem., 2 3 4 (1959) 705 . 1 2 R . F . GOODU, N. F. LEBLANC AND C. M. WRIGHT, Anal. Chem., 27 (1955) 125J. 13 R . M. ROSENBERG ANI) I. M. KLOTZ, in P. ALFXANDER AND R . J . BLOCK, A Laboratory 3lamtal of Analytical Methods of Protein Chemistry, Vol. 2, Pergamon Press, New York, 196o, p. 1 3 t . 14 H . K . SCHACHMAN, in S. P . COLOXVICK AND N. O. I~_APL.aN, Methods in E,zymologl,, Vol. 4, Academic Press, New York, I 9 5 7 , p. 32. I 5 D. H . SPACKMAN, \ ¥ . H . STEIN AND S. ~'~OORE, Anal. Che~l., 3o ( t 9 5 8 ) 1 I9(1. I 0 R. E. I~.IARQUIS, Arch. BiochenL Biophys., i i 8 (1967) 323 . 17 H . C. NEU, D. F . ASHMAN AND T. D. PRICE, J. Bacteriol., 93 (1967) 130o18 I-~. M. \¥ILBUR, F. BERNHEIM AND (). ~ r . SHAPIRO, Arch. Biochem. Biophys., 24 (1949) 3o5. ~9 A. D. ELBEIN AND E. C. HEATH, J . Biol. Chem., 2 4 0 (1965) 1919. 20 A. J . BURTON AND H . E. CARTER, Biochemistry, 3 (1964) 4 T M 21 A. ~OV~OTNY, A. ~ . NO\VOTNY AND M. P. BRIGHAM, Bacteriol. Proc., p. ~)ti, ~Of~8. 22 1?. T. HATCH AND A. L. BRUCE, Nature, 218 (1968) IlOU. 23 S. DE PETRIS, J. Ultrastruct. Res., 19 (1967) 45.
Biochim. Biophys. Acta, 2 3 0 (~97 l) 7 a 81
RELEASE OF LIPOPOLYSACCHARIDE-PROTEIN COMPLEX 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38
8I
H. H. MARTIN, J. Theoret. Biol., 5 (1963) i. R. G. E. MURRAY, P. STEED AND H. E. ELSON, Can. J. 2~Iicrobiol., I i (1965) 547. R. E. BURGE AND J. C. DRAPER, J. Mol. Biol., 28 (1967) 173. C. W. F. McCLARE, Nature, 216 (1967) 766. L. LEIVE, Biochem. Biophys. Res. Commun., 21 (1965) 290. Y. ANRAKU, J. Biol. Chem., 243 (1968) 3128. J. R. PIPERNO AND D. L. OXENDER, J. Biol. Chem., 241 (1966) 5732. A. 13. PARDEE, J. Biol. Chem., 241 (1966) 5886. P. K. NAKANE, G. E. •ICHOALDS AND D. L. OXENDER, Science, 161 (1968) 182. P. MITCHELL AND J. MOYLE, Syrup. Soc. Gen. Microbiol., 6 (1956) 15o. W. KUNDIG, F. D. KUNDIG, B. ANDERSON AND S. ROSEMAN,J. Biol. Chem., 241 (1966) 3243 . H. R. KAEACK AND E. R. STADTMAN, Proe. Natl. Aead. Sci. U.S., 55 (1966) 920. H. C. NEU AND L. A. I-IEPPEL, Biochem. Biophys. Res. Commun., 17 (1964) 215. L. A. HEPPEL, Science, 156 (1967) 1451. P. MITCHELL, in A. KLEINZELLER AND A. KOTYK, Membrane Transport and Metabolism, Academic Press, New York, 1961, p, 22.
Biochim. Biophys. Aeta, 23o (1971) 72-81
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26466 R E L E A S E OF A L I P O P O L Y S A C C H A R I D E P R O T E I N COMPLEX FROM ESCHERICHIA
C O L I A BY WARM-WATER TREATMENT
DEXTER ROGERS* Department of Biochemistry and t3iophysics Oregon 5"tate ~:niz,ersity, Corvallis, (}reg. 9733~ ( ('.5._4.)
(Received June 12th, 197oI (Revised manuscript received ()ctober 1st, i 07 o)
SUMMARY
Warm-water treatment of Escherichia coli A in o.I M Tris (pH 7.3), in the absence of added Mg z+, released protein and lipopolysaccharide simultaneously. After precipitation witti (NH4)2SO a and brief treatment with sodium deoxycholate, the solubilized material appeared as a single component when analyzed by analytical centrifugation. Chromatographic analysis with DEAE-cellulose indicated the coincidental distribution of protein and lipopolysaccharide components (e.g. heptose, 3deoxyoctulosonate and ester groups). Bound /5 hydroxymyristic acid was present, but colitose (3,6-dideoxy-L-galactose) was apparently not present in the lipopolysaccharide from this galactose-negative culture. Tile complex was precipitated with divalent cations and hydrolyzed with 14 mM HC1 to yield a lipid. About one-half of the complex was accounted for in terms of a full complement of amino acids. The complex was dissociated by chromatography in the presence of EDTA or sodium deoxycholate, and evidence was obtained for three subfractions containing varying proportions of protein and lipopolysaccharide. The procedure used for its release and its composition suggested that the lipopolysaccharide-protein complex might be derived from tile outermost laver of the cell wall. This complex showed no affinity for galactose.
INTRODUCTION When Escherichia coli A is suspended in o.I M Tris (pH 7.3) and stressed at 4 ~ °, a discrete amount of protein is released at a constant rate 1. This warm-water treatment releases protein without loss in viability of the treated cells or loss of cytoplasnlic material, and this release is prevented by the presence of divalent cations. The rate of release is related inversely to the osmolarity of the suspending medium. In IO mM solution, the release is faster than that in ioo mM solution, and there is evidence for a second sequence of release, which includes a sugar-binding protein 2. The purpose of this report is to identify the material released by warm-water treatment in o.i M Tris, in the absence of Mg 2+, and to relate it to the structure and transport function of the cell surface. * Present address: Department of Chemistry, Paterson State College, Wayne, N.J. o747o, U.S.A. Biochim. Biophys. Acla, 23o (1971) 72 81
RELEASE OF LIPOPOLYSACCHARIDE-PROTEIN COMPLEX
73
MATERIALS AND METHODS
The conditions for growing cultures of E. coli A in a lactic acid-inorganic salts medium have been described 3. This strain lacks both galactokinase (EC 2.7.1.6 ) and galactose-I-phosphate uridyltransferase (EC 2.7.7.io), and, under the conditions of growth, galactose should not have been available for the synthesis of a complete lipopolysaccharide. Presumably, this is a rough strain, a Re-type mutant 4, whose inner core is limiteeI t o a glucose residue attached to the heptose backbone. Except for preliminary experiments, when only small volumes of culture were needed, the culture was grown in a IOO-1fermentor. The cell paste, which weighed about 25 g on a dry basis, was suspended in 3 1 of o.i M Tris (pH 7.3) for I h at 23 ° before centrifuging. The cells were resuspended in 600 ml of o.I M Tris and placed in a 60 ° water bath. The temperature of the suspension rose to 48° within 4 min, and this temperature was maintained for 4 min longer. After centrifuging, the warm-water extract was dialyzed against a saturated solution of (NH4)2SOa, which had been adjusted to pH 7.3 and buffered with o.I M Tris. The precipitated material was centrifuged, redissolved and dialyzed overnight at 4 ° against several changes of o.I M Tris. The clarified extract, which contained about 35 ° mg of protein, was used for experimentation. In certain experiments, the clarified extract was dialyzed for 2 days at 4 ° against 4 or 5 changes of o.I M Tris containing either o.15 % sodium deoxycholate or 5 mM EDTA. As it will be shown, the extracted material is a lipopolysaccharide-protein complex plus a minor amount of protein 1 that is washed from the cells at o °, even in the presence of Mg2+. The total clarified extract was chromatographed on a 4 cm × 50 cm column of DEAE-cellulose 2. The column was equilibrated at 4 ° with the buffer used in the final preparation of the extract, i.e.o.I M Tris (pH 7.3) and, where indicated, o.15 % sodium deoxycholate or 5 mM EDTA. The column was eluted with a gradient prepared with a constant volume mixer 5. 2 1, or more, of I M NaC1 in o.i M Tris (pH 7.3) were mixed with 0. 5 1 of o.i M Tris. Io-ml fractions were collected and assayed for protein using (a) the Folin-Denis reagent 6, (b) the biuret reagent 7 or (c) turbidity measurement with trichloroacetic acid 2. The protein standard was a solution of recrystallized bovine serum albumin, which had been standardized spectrophotometrically s. The effluent fractions were also assayed for lipopolysaccharide components: (a) heptose by a modified cysteine-H2SO a reaction", 10, (b) 3-deoxyoctulosonate by a modified periodate-thiobarbiturate reaction 10.~1 and (c) ester groups by an alkaline hydroxylamine reaction ~2. These results were normalized to give the relative distribution of material in the chromatographic effluent. Kinetic analysis of the rate of protein release during warm-water treatment of E. coli A and phosphatase assays on the chromatographic effluent were performed as described previously 1. The affinity of the lipopolysaccharide-protein complex for galactose was tested by equilibrium dialysis ~3. A portion of the clarified extract was dialyzed for 24 h at 4 ° against 20 mM D-Ei-~*Clgalactose in o.I M Tris (pH 7.3). Samples of the dialyzed material and its dialyzate were measured for radioactivity with a Packard model 3003 Tri-Carb scintillation spectrometer. Analytical centrifugation was performed with a Spinco model E ultracentrifuge. A portion of the clarified extract was dialyzed for 4 h at 4 ° against o.15 % sodium Biochim. Biophys. Acta, 230 (I97 I) 7 2 - 8 I
74
1). R O G E t { S
deoxycholate in o.I M Tris (pH 7.3). The dialyzed sample and its dialvzate were placed in opposite compartments of a double-sectored cell and centrifuged for 48 rain on speed at 52ooo rev./nfin. The sedimentation constant, s, was calculated ~4 and corrected for viscosity' and density, but not for concentration dependence. The following analyses were performed on portions of the same clarified extract that was analyzed bv sedimentation velocity (Fig. 5)- The dry wt. of the lipopolysaccharide-protein complex in solution was deternfined after exhaustive dialysis (3 days at 4 °) against 2oo volumes of o.I M Tris (pH 7.3). 5-ml portions of the dialyzed extract and the dialyzate were separately dried to constant weight at lO5 °. The difference in weight of the dried samples, 27. 5 rag, was considered to be the dry wt. of the Iipopolysaccharid~protein complex in 5 nil. The corresponding amount of protein was determined with the biuret reagent. A 5-nil sanlple contained 13.0 mg of protein. The protein content of the complex was therefore 49.5 %. A solution containing 7.36 mg of the complex ( 3.64 mg of protein) and ()% trichloroacetic acid was mixed thoroughly and centrifuged. The residue was suspended in constant boiling HC1, evacuated and flushed with purified nitrogen IO times, sealed in an ampoule under nitrogen and hydrolyzed for 2o h at IIO °, The hydrolyzate was analyzed for anlino acids with the method of SPACKMAX et al. ~ using a Beckman model I2o-B amino acid analyzer. A solution containing 7.36 mg of the complex ( 3.72 mg of lipopolysaccharide, by difference) and 14 mM HCI was treated in a boiling-water bath for 3o nlin and centrifuged. The residue was suspended in 5 ml of 5 M HC1 and refluxed overnight. The hydrolyzed material was extracted with three 5-ml portions of ether. After the ether was evaporated, the fatty acids were esterified by boiling for 2 rain with 3 ml of methanol containing BF a. The boiled nfixture was transferred to a separatory funnel containing 2o ml of water. It was extracted 3 times with Io-ml portions of petroleum ether. The combined extracts were washed with water and dried ower anhydrous Na,~S()4. After evaporation, the residue was dissolved in 2 ml of chloroform. Comparable w)luines of buffer, with and without I.O mg of I)L-/!]-hydroxymyristic acid, were also treated in the same manner. Io-/~l samples of the esters were analyzed with an 1" and M model 4o2 chromatograph fitted with a 6 ft • o.I0 in open tubular capillary containing Diatoport S (8o IOO nlesh) coated with diethvleneglycolsuccinate (6°o, w/w). The operating temperature range was I3O I 8 O ; the temperature was programmed to increase at a rate of 3°/min. The combined gas-ttow rates were He, 4 o lb/ineh"-, 7 ° inl/min; air, 20 lb/inch 'a, 28o ml/min; H,,, io lb/inch e, 35 ml/min. All solvents were redistilled and none revealed material in the analytical range. RESULTS
The effectiveness of warm-water treatment depends on the uniform application of pressure by the expanding cell membrane against the weakened and stressed cell wall. Since this pressure is related inversely to the activity of the impernleable solute in the suspending medium, it was reasoned that a differential effect could be achieved by controlling cell turgor with appropriate choices of solute concentration. By this means, the wall layers might be broken sequentially and material released selectiveh as the necessary disrupting pressures were attained. Preliminary experiments 1 proBiochtm. Biophy,~. Acta, 23 ° (~97 l) 72 81
RELEASE OF LIPOPOLYSACCHARIDE--PROTEIN COMPLEX
75
duced the expected results. By decreasing the osmotic pressure of the medium, a differential release of protein was obtained without loss in viability or apparent release of cytoplasmic components. To correlate this effect, the rate of protein release was compared with the average protoplast volume obtained by MARQUISis for Bacillus megaterium. This comparison showed that, within limits, the rate of protein release and the average protoptast volume were both logarithmic functions of the reciprocal of osmolarity (Fig. i). However, about one order of magnitude separated these functions. When two species are compared, some difference in their cell membranes is to be expected, but the noted difference could also be due to the inherent extensibility of the cell wall, which would reduce the effectiveness of warm-water treatment.
~0
1.0
~/
B.megaterium
/
protoplosts
/ •8
o
g
~" o
7 _ /
D /
E. coli A
/ 0"1
I
I0
100
-2 ~"
oi
I/0smolarity
Fig. 1. Corre]ation b e t w e e n rate of release of p r o t e i n d u r i n g w a r m - w a t e r t r e a t m e n t of E. cull A a n d a v e r a g e p r o t o p l a s t v o l u m e of B. rnegaterium as f u n c t i o n s of osmolarity. T h e p r o t o p l a s t v o l u m e s were d e t e r m i n e d b y MARQUIS (see ref, 16).
As expected, the impermeable sucrose was more suitable in effecting osmotic shock than the partially permeable Tris buffer. However, at high concentrations, Tris contributed to the dissociation of the surface structure (cf. ref. 17). The release of lipopolysaccharide was not complete, although a discrete proportion was obtained by this warm-water treatment. In all of the following experiments, warm-water extracts were prepared with o.I M Tris (pH 7.3) because only a single sequence of protein release was obtained under these conditions 1. A warm-water extract was analyzed with the cysteine-H zSO 4 reaction and the periodate-thiobarbiturate reaction. The spectrum of the cysteineH2SO 4 reaction products, which was recorded after 24 h at 23 °, revealed peaks that corresponded to heptose (~max505-510 rim9), 3-deoxyoctulosonate (~max 385-39 ° nml°) and hexose, possibly glucose (~max 41°-415 nm9) - The secondary peak at about 590-600 nm was probably due to 3-deoxyoctulosonate and glucose9,10. The spectrum of the periodate-thiobarbiturate reaction products revealed peaks that corresponded to 3-deoxyoctulosonate (~m~ 548 rim1°' 1~) and a substance like that present in irradiated mouse skin which is not extracted with organic solvents (~max 450 nmlS). When the periodate treatment was performed at 55 °, instead of 23 °, the peak at 548 nm was intensified 3-fold without shifting its ~mx. This increase may have been due to hydrolysis of certain bound forms of 3-deoxyoctulosonate. It seems unlikely that colitose (3,6-dideoxy-L-galactose), whose reaction product absorbs at 532 nm ~s, after periodate treatment, was present. Colitose was not expected because, in the absence of galactose, the synthesis of the polysaccharide chain should have been Biochim. Biophys. Acta, 23o (1971) 72-81
7()
D. ROGERS
interrupted before colitose could be incorporated. Nevertlleless, a minor amount of colitose has been recovered from the lipopolysaccharide of a galactose-negative culture ~:'. Heptose-containing material was precipitated completely with trichloroacetic acid and with I0 mM Mg 2~, Ca 2= and Zn 2~. Its reactivity as heptose was enhanced three-fold by hydrolysis in 14 mM HC1 for I h at I00 °. Dilute acid hydrolysis als,~ liberated lipid, which was presumably lipid A 2°. During warm-water treatment, the rate of release of both protein and heptosecontaining material corresponded exactly (Fig. 2). In addition, Mg~- stabilized both protein and heptose-containing material against warm-water treatment. o I'C cL e :z:
dO8
OI A
0"8
o o
m
a.O.6
o 0"6
o
~t
+MgCl 2
o M i n u t e s at 4 8 *
80 Effluent
160 Frocliorts
240
Fig. 2. Release of protein and heptose-containing material (luring w a r m - w a t e r t r e a t m e n t of heptose; . - - . w i t h o u t Mg('l,z: . . . . . . . , w i t h MgC12. E . coli A. The concentration of MgC12 was o.ot M. ~,, protein; • ,
Fig. 3. C h r o m a t o g r a p h y of a w a r m - w a t e r e x t r a c t of E. coli A o n DEAl'; cellulose. The effluent was analvzed for protein ( 2 ) , heptose (O) and 3-deoxyoctulosonate (@).
A warm-water extract was chromatographed on DEAE cellulose with tile result shown in Fig. 3. The distribution of protein, heptose and 3 deoxyoctulosonate corresponded closely and indicated one major peak and two minor peaks. The first minor peak came off the column at about the break-through point. This peak may have consisted of material aggregated by the presence of o.I mM Mg('l., that was present in the buffer used in this experiment only. In the absence of added MgC12, only one minor peak is observed. Assays for ester groups, which are not shown in this figure, also indicated a close correspondence. A similar distribution of these components was observed with material that was precipitated with Mg2., Ca 2~ and Zn 2~, except for the absence of material usually appearing as a shoulder to the major peak (Fractions
Io5 125). Also expected in the warm-water extract were tllose components a that can be washed from the cells at o °, even in the presence of Mg2-. Among these components is a phosphatase, whose distribution in the DEAE-cellulose effluent is shown in Fig. 4This activity was located in the shoulder to the major peak (Fractions lO5-125). Analysis of the warm-water extract for homogeneity was performed by analytical centrifugation. This analysis revealed both a nlajor component and a minor component, whose Sobs were 2.3 S and 4.9 S, respectively. A similar analysis in the presence of IO mM MgC12 revealed four peaks, whose Sobs were 3.45 S, 5.7 S, 15.9 S and 24.5 S. When the warm-water extract was treated briefly with o.15 Oo sodium B i o c k i m . B i o p h y s . A c l a . 23o (197 t) 72-81
77
RELEASE OF LIPOPOLYSACCHARIDE--PROTEIN COMPLEX
deoxycholate before analytical centrifugation, only a single component was revealed (Fig. 5). The Sobs and s20, w were 3.25 S and 5.35 S, respectively. After treatment for 2 days with either o.15 % sodium deoxycholate (Fig. 6) or 5 mM EDTA (Fig. 7), the lipopolysaccharide-protein complex was dissociated by chromatography on DEAE-cellulose into three subfractions, which contained varying proportions of protein and lipopolysaccharide. However, complete resolution of these subfractions was not obtained. I.C
i
ii
_
il
i
.~° 0"4
i
i i
-6 ¢c0. 2
::~
./" ,,, 0 . . . . .
J'
0
'\
" .........
80 Effluent
160 Fractions
240
Fig. 4- Distribution of p h o s p h a t a s e activity in t h e effluent from DEAE-cellulose. The p h o s p h a t a s e substrate was glucose 6-phosphate (see ref. i). The d o t t e d line represents the distribution of protein in the effluent. Fig. 5. Analytical centrifugation of a w a r m - w a t e r e x t r a c t of E. coli A t h a t was precipitated with (NH4)~SO 4 and dialyzed briefly against o.15 % sodium deoxycholate (4 h at 4 °). The buffer was o.i M Tris (pH 7.3). The dialyzed sample and its dialyzate were placed in opposite c o m p a r t m e n t s of a double-sectored cell and centrifuged for 48 min on speed at 52000 rev./min at 5 °. Sedimentation is toward the left. i.o I
1.0
2"
0`8
=0-8
=°0`6 <
~0`6
~0`4
.~0"4
0.2
¢0-2
it ¢ =
=
o
S I
a/
eo
o/
120 tr ffluont
o
160 Fractions
200
eO
120
Eftluen!
,
,
160 Fractions
,
200
Fig. 6, Effect of sodium deoxycholate on t h e c h r o m a t o g r a p h y of a w a r m - w a t e r e x t r a c t of E. coli A on DEAE-cellulose. The w a r m - w a t e r e x t r a c t was dialyzed against o.1 M Tris (pH 7.3) containing o.i5 % sodium deoxycholate (2 days at 4 °). The effluent was analyzed for protein (A), heptose ( • ) and 3-deoxyoctulosonate (O). Fig. 7. Effect of E D T A on the c h r o m a t o g r a p h y of a w a r m - w a t e r e x t r a c t of E. call A on D E A E cellulose. The w a r m - w a t e r e x t r a c t was dialyzed against o.I M Tris (pH 7-3) containing 5 mM E D T A (2 days at 4 o). The effluent was analyzed for protein (A), heptose ( • ) , 3-deoxyoctulosonate (©) and ester groups (V). Biochim. Biophys. Acta, 23o (1971) 72-8i
78
D . R()GI~R~
TABLE
I
ANALYSIS
FOR
The material
Peak
THE
FATTY
Retention time (rain)
I 2
ACID
was hydrolyzed,
2.8 5.0
COMPONENTS
OF
THE
esterified and analyzed
In *
LIPOPOLYSACCHARIDE
Relative peak height .Sample
o.I 3 O.211
Control
I]. 5 I9. 5
-PROTEIN
COMPLEX
l)y g a s c h r o m a t o g r a p h y .
O 4.O
t,¢ "
Pcrtcnta~,e ,,f total /arty acids
<~:O
O.I 5
,~" t J
lO :O
0 3 I
I
3
0.8
0. 31
I "5
4 .5 {) 7
8. 5 9.9 I o.8 1-.o
o.39 o-45 o.49 (>.M
)o.o 42.{>
o 7.o
12 : o
().44
7 -'5
I 1.1}
5.0
14 : o
o.4~
4
2.o
11
t
N
1 3.2
{).{)O
().O
i).O
()
0
14.()
o.{){)
) o'5
1{} II 12 13
15. 7 17.2 10. I 28. 3
o.71 o.79 (1.87 1.29
I().(}
7 .o IO.O
15"5
I{},O
43.O ,q.o
{) 9,o
* Stearic
TABLE
a c i d { 18 :o}
O
Tentative identification
3
{).7 I
l{): 1
0.79
fl-{)H 14:o 18 : i
o.S 7 1.2()
4 nl
31 ~)
J .oo.
I1
ANALYSIS
FOR
The complex
A miuo acid
AMINO
ACID
was hydrolyzed
COMPONENTS
OF
and analyzed
THE
LIPOPOLYSACCHARIDE--PROTE1N
with an automatic
mg residue (3.64 mg protein) *
Lys
i . 7 (}
o . 2 2{)
o . 51 I . 1~
o.o7o
3.23
o-372
Thr
1.o 3
o. 10 5
i .5 °
,). 13 l o. 4 1 3 o.272 o. 14 °
Ser * * Glu*" Pro Gly *"
Val * * Met 1 le
Leu Tyr Phe Cy~ NH a Trp
amino
l~moles residue (3.64 mg protein) "
His A r g *" Asp''
.\la
I0:o
COMPLEX
acid analyzer.
O. l 8 4
3.z°
2.4o 2.45 3-o9
o. 22o
2 .o{) 11.5q ) .63 2.43 o.95 ~.oo . . *** ""*
o.2o 7 o.o77 o. 1 8 5 I).275 o. i `55 o. 147 .
Total
.
.
. .., """ 3.239
• 7.3 {) n a g c o m p l e x = 3.{)4 m g p r o t e i n ( b i u r e t ) . indicated amino acids, in the form of polypeptide (biuret), are also found in the lipopolysaccharide moiety of the complex (see ref. 2 U. "** } ' r e s e n t , b u t n o t d e t e r m i n e d ; tryptophan w a s n o t e d i n t h e s p e c t r u m a t 2t}z n m . " * The
Biochim. Biophys. Acta, 2 3 0 ( I 9 7 I) 7 2 - 8 I
RELEASE OF LIPOPOLYSACCHARIDE-PROTEIN COMPLEX
79
Another characteristic component of lipopolysaccharide is/~-hydroxymyristic acid, which was detected by means of gas chromatography after hydrolysis and esterification (Table I). Peak 12 corresponded to the observed appearance of /5hydroxymyristic acid. The relative distribution of fatty acids reported in this paper and by ELBEIN AND HEATH19 and by BURTON AND CARTER20 all differ quite strikingly from each other. The biuret assay of the lipopolysaccharide-protein complex had indicated that protein was 49.5 % of the dry weight. An amino acid analysis of the hydrolyzed complex confirmed this estimate (Table II). A full complement of amino acids was obtained and the recovered residue weights accounted for 45 % of the dry wt. of the complex and 9° % of the biuret assay value. This account did not include the contributions of tryptophan, half cysteine and NHs. A numerical analysis 22 of the ratio of hydrophilic-to-apolar amino acid residues and the ratio of charged-to-small amino acid residues revealed that the protein component of the lipopolysaccharide-protein complex resembled more closely membranous lipoprotein than soluble lipoprotein and oligomeric protein than monomeric protein. All attempts failed to show any affinity between the lipopolysaccharide-protein complex and galactose. DISCUSSION
Current conceptions of the cell wall of E. coli and other gram-negative bacteria consider a multilayered, asymmetric structure zz-26. The outer, L-layer of the cell wall contains lipopolysaccharide and lipoprotein, and by electron microscopy it appears as two visible layers separated by a transparent layer. The intermediate, G-layer contains globular protein and mucopeptide, and it appears as a transparent layer and a visible layer. Since the L-layer can be removed with phenol or detergents, it may be only loosely connected to the G-layer. These treatments, however, fragment the L-layer. The isolated lipopolysaccharide contains less than I0 % protein, which may be attached covalently to the lipopolysaccharide~1. In cell envelopes, in addition to hydrophobic bonding, lipids and proteins may be bound by polar bonds involving Mg 2÷ (see ref. 27). The L-layer is also stabilized by divalent cations because osmotic shock with EDTA releases about one-half of the lipopolysaccharide 28. Much of this release is prevented with Mg2+. The lipopolysaccharide-protein complex that is isolated by warm-water treatment may be derived from the outer, L-layer of the cell wall because of the similarity in composition and the mode of isolation. The complexity of this material, which is evident from chromatography in the presence of sodimn deoxycholate or EDTA, may reflect the composition and structule of this layer in its original state. Warm-water treatment in 0.I M Tris, in the absence of added Mg2+, may prove to be a specific probe of the outer, L-layer of the cell wall and discriminate it from the intermediate, G-layer. Among the proteins that are released by osmotic shock are several nutrient binding proteins 29-31. Since the isolated lipopolysaccharide-protein complex lacks any affinity for galactose, nutlient binding proteins as a class may be located beneath the lipopolysaccharide-protein complex. This possibility is substantiated by the inability of antiserum for the leucine-binding protein to inhibit uptake by the intact Biochim. Biophys. Acta, 23 ° (1971) 72-8I
~O
I). R O G E R S
cellaL It is possible that warm-water treatment might be modified to probe deeper into the cell wall. When warm-water treatment is performed in I0 mM Tris, instead of I00 mM Tris, a second sequence of protein release occurs 1. Among the pr-teins released under these conditions is a binding protein for galactoseL Further work may be able to establish the location of binding proteins among the globular proteins at the outerface of the G-layer or within the periplasmic space. The process of nutrient transport into a cell has been recognized as a surface phenomenon and the cell membrane, the osmotic barrier, is considered to hold at least some of the transport components a3. Certain components have already been isolated from membrane fragments a4, 35. Still other components mav be located outside of the cell membrane because thev can be released by osmotic shock 29 a~ a pr.cess that apparently does not damage the cell membrane or release cytoplasmic material 3°,a7. In fact, a treated cell can regain its lost function when the binding protein is reinstalled in the cell 29. For a multicomponent transport system, the anisotr~q~ic distribution of its components would contribute to the vectored movement ~)f the transported nutrient 38. Therefore, the transport components should be located experimentally within the cell surface in order to fully understand the process. ACKNOWLEDGEMENTS
This study was made while the author was a Special Fellow of the U.S. Public Health Service, National Institutes of Health and was supported by a Public Health Service Fellowship 5 I:3 AM-II 083. I am grateful for the warm hospitality that was provided by the late Vernon H. Cheldelin. I thank Robert R. Becker for many useful discussions and Robert L. Howard for performing the amino acid analysis. REFERENCES I D. ROGERS, Science, I 5 9 (1968) 531. 2 I). ROGERS, J. Bacteriol., 88 (1964) 279. 3 D. ROGERS .aND S . - H . YO, J. Bacteriol., 8 4 ( I 9 0 2 ) 8 7 7 . 4 0 . L/3DERITZ .aND O. WESTPHAL, Angew. Chem. Intern. Ed., 5 (1966) I 9 8 . 5 R . S. ALM, t{. J . P . WILLIAMS AND A. TISELIUS, Acta Chem. Scan&, 6 (1952) 8 20. 6 0 . H . LOWRY, N. J. ROSEBROUGH, A. [~. FARR AND R . J. RANDALL, ./. Biol. Chem., I03 (It#3l) 265 . 7 A. G. GO~NALL, C. J . BaRDAWILL AND M. 3I. DAVID, .[. Biol. Chem., 177 (1949) 751 • 8 B . J . ADKINS .aND J. F. FOSTER, Biochemistry, 5 ( r 9 6 6 ) 2579. q Z. DlSCHE, J. Biol. Chem., 2 o 4 (1953) 983. i o M. J . OSBORN, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 4991~ A. WEISSBACH AND J . HURWlTZ, J . Biol. Chem., 2 3 4 (1959) 705 . 1 2 R . F . GOODU, N. F. LEBLANC AND C. M. WRIGHT, Anal. Chem., 27 (1955) 125J. 13 R . M. ROSENBERG ANI) I. M. KLOTZ, in P. ALFXANDER AND R . J . BLOCK, A Laboratory 3lamtal of Analytical Methods of Protein Chemistry, Vol. 2, Pergamon Press, New York, 196o, p. 1 3 t . 14 H . K . SCHACHMAN, in S. P . COLOXVICK AND N. O. I~_APL.aN, Methods in E,zymologl,, Vol. 4, Academic Press, New York, I 9 5 7 , p. 32. I 5 D. H . SPACKMAN, \ ¥ . H . STEIN AND S. ~'~OORE, Anal. Che~l., 3o ( t 9 5 8 ) 1 I9(1. I 0 R. E. I~.IARQUIS, Arch. BiochenL Biophys., i i 8 (1967) 323 . 17 H . C. NEU, D. F . ASHMAN AND T. D. PRICE, J. Bacteriol., 93 (1967) 130o18 I-~. M. \¥ILBUR, F. BERNHEIM AND (). ~ r . SHAPIRO, Arch. Biochem. Biophys., 24 (1949) 3o5. ~9 A. D. ELBEIN AND E. C. HEATH, J . Biol. Chem., 2 4 0 (1965) 1919. 20 A. J . BURTON AND H . E. CARTER, Biochemistry, 3 (1964) 4 T M 21 A. ~OV~OTNY, A. ~ . NO\VOTNY AND M. P. BRIGHAM, Bacteriol. Proc., p. ~)ti, ~Of~8. 22 1?. T. HATCH AND A. L. BRUCE, Nature, 218 (1968) IlOU. 23 S. DE PETRIS, J. Ultrastruct. Res., 19 (1967) 45.
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RELEASE OF LIPOPOLYSACCHARIDE-PROTEIN COMPLEX 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38
8I
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