97
Biochimica et Biophysica Acta, 381 (1975) 97--108
Q Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27559 CELLULOSE SYNTHESIS BY A C E T O B A C T E R X Y L I N U M II. INVESTIGATION INTO THE RELATION BETWEEN CELLULOSE SYNTHESIS AND CELL ENVELOPE COMPONENTS
DAVID COOPER and R.St. JOHN MANLEY Department of Chemistry and Pulp and Paper Research Institute, McGill University, Montreal H3C 3G1 (Canada)
(Received May 8th, 1974) (Revised manuscript received September 9th, 1974)
Summary Cell envelope fractions, capable of cellulose synthesis from uridine diphosphate glucose, a-glucose-l-phosphate, glucose-6-phosphate and glucose, have been isolated from A c e t o b a c t e r x y l i n u m suspensions and various enzymatic properties examined. Essential enzymes were f o u n d to be distributed t h r o u g h o u t the cell envelope region, with both inner (cytoplasmic) and outer (cell wall) membranes contributing to cellulose synthesis. The central role of UDPG in cellulose synthesis was confirmed and the results indicated that the nucleoside diphosphate sugar functions solely in the cell envelope region of whole cells. A comparison of properties of the cell envelope system with those of different preparations used by other workers, suggested that the m e t h o d of cell disruption may influence substrate specificity.
Introduction The convenience of utilising suspensions of A c e t o b a c t e r x y l i n u m cells as a system for the study of cellulose biosynthesis has been well documented [1,2]. A number of workers have isolated cell-free e n z y m e systems from this bacterium capable of cellulose synthesis from various substrates [3--10]. From such investigations it became apparent that the m e t h o d of cell disruption may influence both substrate specificity and the extent to which the cellulose synthesizing capacity is maintained. Most results indicated that the ability to form cellulose resides in the particulate fraction of cell-free systems. Furthermore, the results of Colvin et al. [10] using lysed cell preparations, indicated the involvement of the cytoplasmic membrane in the synthesis of the cellulose
98 precursor. However, the evidence was circumstantial and these interesting studies were not pursued further. Depending on the enzyme preparation, cellulose formation has been demonstrated from glucose [3--6,10], fructose I101, UDPG or TDPG [6--91 as substrate, but not from other nucleoside diphosphate sugars or any hexose phosphate. The lack of synthesis from hexose phosphates has led to the suggestion that a bound series of reactions is involved that cannot be entered in the middle [11]. Although many workers agree that a sugar nucleotide may be involved at some stage in cellulose formation, questions have arisen concerning the precise position of this intermediate in the synthetic scheme. For instance, does the final transfer of glycosyl units involve a nucleoside diphosphate sugar or is there an additional c o m p o u n d that assumes this cardinal role, such as a glycolipid intermediate [12--14]? Where does this final transfer occur and is a matrix necessary (i.e. heterogeneous or homogeneous nucleation)? Solutions to these and other problems may come from a study of the contribution of various components of the cell envelope of A. x y l i n u m to cellulose synthesis as a t t e m p t e d in the present work. In Part I [15] it was shown that cellulose synthesising suspensions of A. x y l i n u m contain quantities of a-glucose-l-P and UDPG, and that these compounds behave as though they are intermediates in cellulose synthesis. Furthermore, evidence suggested that pools of these compounds are localised outside the cytoplasm of A. x y l i n u m cells, possibly in the periplasmic space, and that permeability barriers to such phosphorylated compounds exist in the outer cell envelope region. Consequently, the work presented in the present paper, as part of a systematic investigation of cellulose synthesis by A. x y l i n u m was carried out with the following objectives in mind: (i) to isolate a cell-free enzyme system capable of cellulose synthesis from any (or all) of the suggested intermediates found in Part I [15], (ii) to examine the properties of the enzyme system with regard to substrate specificity, variation in pH, and the effect of various co-factors and (iii) to compare the utilisation of substrates, at equivalent molar concentrations, by the cell-free system to that of whole cells. Gentle disruption of washed A. x y l i n u m cells and partial fractionation of cell envelope components, followed by studies of cellulose formation from various substrates, provided evidence that the enzyme(s) responsible for cellulose synthesis are distributed t h r o u g h o u t the cell envelope region. Materials and Methods
Ch ro m a tograp hy Thin-layer chromatography was performed on cellulose or silica gel plates. The same solvents were employed for the washed layers as described earlier [15]. Spray reagents used for phosphate, reducing substances and lipids, and autoradiography of separated radioactive materials have been described earlier [151. Collection of bacteria and preparation o f envelope fractions Surface pellicles were harvested from 48 h cultures and washed bacterial
99 suspensions prepared by the m e t h o d of Hestrin and Schramm [16]. Final suspension was in 0.05 M t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e (Tris) buffer (pH 7.5) containing ethylenediaminetetraacetic acid (EDTA) (1 mM) and MgC12 (10 mM) at 0°C. An aliquot of the cell suspension was passed twice through an Aminco French pressure cell operated in a Carver Laboratory Press, Model B, at a pressure of 20 000 lb/inch 2. The resulting broken cell suspension was then centrifuged at various speeds in a Spinco Model L preparative ultracentrifuge (Beckman Instruments Co.) at 0°C. Fractions isolated were as follows, (i) 0--2000 × g, 10 min (not used), (ii) 2000--10 000 × g, 30 min (fraction B), (iii) 10 000--40 000 × g, 45 min (fraction C) and (iv) > 4 0 000 X g supernatant (fraction D). Electron microscopic observations and viable cell counts indicated that the procedure used was extremely efficient for cell disruption. Each fraction (except D) was washed before use by gentle resuspension in buffer and centrifugation as before. Protein concentrations for each fraction were determined by the biuret method [17], and adjusted to equal levels (usually 20 mg/ml) by dilution with buffer before analysis of cellulose synthesising ability.
Chemicals and radiochemicals Uniformly labeled: D-[~4C]glucose (Schwarz Bioresearch Inc., 240 Ci/mole), UDP [ ~4 C] glucose (250 Ci/mole), a-[ ~4 C] glucose-l-P (210 Ci/mole) and [ 14 C] glucose-6-P (185 Ci/mole) (International Chemical and Nuclear Corporation) were purified before use by thin-layer chromatography on cellulose thin layers. Substrates were diluted to the required specific activity (usually 0.5 Ci/mole) by the addition of non-radioactive chemicals. The counting efficiency for each substrate was determined under the same conditions used for estimation of cellulose as indicated below. All other chemicals were reagent grade or better. Estimation of cellulose Carrier cellulose was prepared from ultrasonically dispersed, purified bacterial cellulose and suspended in distilled water at a concentration of 15 mg/ml. Immediately before termination of cellulose synthesis an aliquot (0.1 ml) of carrier cellulose was added to the incubation mixture containing substrate (1 ml) and enzyme preparation (1 ml). Reactions were terminated by the addition of ethanol (25 ml) and the insoluble residue analysed for cellulose content after treatment with sodium laurylsulphate and sodium hypochlorite as indicated previously [15]. Occasionally the ethanolic supernatant was also analysed. Control mixtures (blanks) were heated for 10 min on a boiling water bath before addition of substrate. Preparation of extracellular enzyme In certain experiments the supernatant fluid from whole cells was used as a source of enzyme for cellulose synthesis from heat inactivated envelope fragments. This 'extracellular' enzyme was prepared by centrifugation of a washedcell suspension of A. xylinum, previously incubated for 10 min with a limiting concentration of glucose (0.01%), at 20 000 × g for 30 min at 0°C, followed by filtration through a 0.22 pm Millipore filter in the cold. The supernatant fluid was stored at 0°C until required (usually less than 1 h).
100
Estimation of glucose-6-phosphate dehydrogenase activity Glucose-6-phosphate dehydrogenase activity was determined spectrophotometrically at 340 nm wavelength using a Beckman Model D.U. spectrophotometer under conditions similar to those described by DeLey and Dochy [18]. A 1 cm cell was used containing NADP (0.3 pmole), glucose-6-P (10 gmole), MgSO4 (5 #mole), Tris--HC1 buffer, pH 8.5 (100 pmole) and 0.5 ml of the enzyme preparation in a total volume of 2.5 ml.
Electron microscopy The methods for fixation and embedding of the envelope fractions were similar to those used for whole cells described earlier [15]. However, because of the more delicate nature of these preparations, certain modifications were made [19]. The pellets, after centrifugation, were fixed initially in glutaraldehyde (2.5%) for 2 h before post-fixation in osmium tetroxide and subsequent washing. Results The apparent permeability barrier of the outer cell envelope region of A.
xylinum to phosphorylated compounds, detected in cellulose synthesising suspensions of this bacterium, has been described in Part I [15]. To facilitate a study of the interaction (or interconversion) of detected compounds it was necessary to develop a procedure for isolation of an enzyme preparation capable of cellulose synthesis in which the above mentioned permeability barriers are removed. Considering the results of previous workers it was suspected that the enzyme system responsible for cellulose synthesis required the structural integrity of the cell envelope region and/or its components [10]. For this reason excessively damaging procedures such as ultrasonic disruption or powder grinding were not employed, the more gentle technique of disruption using a French pressure cell being used. Light microscopic observations and viable cell counts indicated that cell rupture was efficiently achieved by double passage of cell suspensions containing excess Mg 2+ through an Aminco French pressure cell operated in a manual press at the m a x i m u m working pressure. An a t t e m p t was made to fractionate the constituents of the ruptured cell suspension by centrifugation at various forces. Electron microscopic observations of thin sections of stained sediments confirmed that at least a partial fractionation was achieved. Large cell debris including heavy cytoplasmic particulate material were observed in the fraction centrifuging at 2000 X g with some cells retaining the usual cell envelope structure of whole cells. Higher centrifugal forces, up to 10 000 × g, resulted in a sediment containing predominantly open membranous structures of single tripartite layers. Particles sedimenting at higher velocities (10 000--40 000 × g) contained many closed, vesicular-like structures, each composed of single tripartite layers; however, some of the more open structures previously described were also present. A crude preparation of envelope fragments, sedimenting between 2000--40 000 × g, consisted of a mixture of both membrane types described above. In earlier experiments performed in the absence of excess Mg 2÷, membrane and wall fragments were observed to be extremely small and poor maintenance of both double track layer structures was evident.
101
TABLE I COMPARISON OF CELLULOSE SYNTHESIS FROM VARIOUS CELL FRACTIONS AND SUBSTRATES A c o n t a i n e d 2 " 1 0 1 0 w h o l e cells, f r a c t i o n s B a n d C, 20 m g p r o t e i n , D, 10 m g p r o t e i n , E, 20 m g t o t a l p r o t e i n a n d F, 30 m g t o t a l p r o t e i n p e r i n c u b a t i o n m i x t u r e . S u b s t r a t e c o n c e n t r a t i o n w a s 3.5 g m o l e / m l in a t o t a l v o l u m e o f 2.0 m l ( p H 7 . 5 ) as d e s c r i b e d in t h e M e t h o d s s e c t i o n . T h e a c t i v i t y o f t h e d e h y d r o g e n a s e in t h e s u p e r n a t a n t o f d i s r u p t e d cells, D, w a s t a k e n as 1 0 0 % f o r c o m p a r i s o n p u r p o s e s . F o r f u r t h e r d e t a i l s see M e t h o d s s e c t i o n . Fraction
p m o l e g l u c o s e i n c o r p o r a t e d / 3 h (× 103) glucose
A B C D E = B + C F=B+C+D
164 9 4 < 1 14 8
UDPG 3 24 17 1 36 29
Glucose-l-P 9 15 12 < 1 25 12
Glucose-6-P 3 4 2 < 1 7 2
Glucose-6-P dehydrogenase activity 2 4 5 100 ---
It has been recently demonstrated by other workers that envelope fractions of Gram-negative bacteria, prepared in a similar way to those in this work, possess both types of structure described above [19]. Furthermore, it has been shown t h a t the lighter fraction consists of material derived from the cytoplasmic membrane and the heavier fraction from the outer cell wall region. It should be emphasised that efficient separations were n o t obtained, so that some contamination of one fraction with another was always apparent. However, the fractions indicated contained a high percentage of a particular structure. Each fraction was analysed for glucose-6-P dehydrogenase activity: the enzyme was detected only in fraction D which is to be expected of this soluble enzyme (Table I). As a final note it should be remembered that alkali-insoluble polymeric material (containing ~4C-labeled glucose) as described in the following results, is referred to as cellulose (see Methods section).
Comparison of fractions Initial experiments were designed to compare the cellulose synthesising ability of various envelope fractions with whole cells from 1 4 C-labeled glucose, UDPG and a-glucose-l-P as substrates at pH 6.0 and pH 8.0. As shown in Table II, cellulose was formed by whole cells from glucose, with increased yield at the lower pH value as expected from the results of previous workers [16]. This formation was markedly inhibited at higher pH values. Small, but finite, transfer of glucosyl units from UDPG into cellulose was detected using whole cells, w i t h o u t a t t e n d a n t formation of gluconate. The lack of gluconate formation indicated that UDPG was n o t hydrolysed to glucose. This effect was investigated further in additional experiments described later. With a-glucose-l-P as substrate, larger amounts of cellulose were formed compared to UDPG although some hydrolysis to glucose was observed with concomitant production of gluconate. Data presented in Table II show the marked decrease in cellulose formation from glucose when cell fragments are used as e n z y m e source. Synthesis
102 TABLE
II
CELLULOSE AND GLUCONATE FORMATION FROM [14C]GLUCOSE, UDP [14C]GLUCOSE a-[14C] GLUCOSE-I-P USING VARIOUS CELL FRACTIONS AS ENZYME SOURCE
AND
E a c h i n c u b a t i o n m i x t u r e c o n t a i n e d s u b s t r a t e ( 3 . 5 ~ t m o l e / m l ) a n d an a l i q u o t o f e n z y m e p r e p a r a t i o n in a t o t a l v o l u m e o f 2 . 0 m l as d e s c r i b e d in t h e M e t h o d s s e c t i o n . F r a c t i o n A c o n t a i n e d 2 " 1 0 1 0 w h o l e cells, f r a c t i o n s B a n d C, 2 0 m g p r o t e i n a n d D, 1 0 m g p r o t e i n p e r i n c u b a t i o n m i x t u r e . B o i l e d e n z y m e p r e p a r a t i o n s w e r e u s e d as c o n t r o l s a m p l e s . T h e i n c u b a t i o n t e m p e r a t u r e for t h i s a n d all o t h e r e x p e r i m e n t s d e s c r i b e d in this paper was 30°C. Free glucose and gluconate were estimated visually after thin-layer chromatography as d e s c r i b e d i n t h e M e t h o d s s e c t i o n . + a n d designate presence or absence of component, respectively. t = trace quantity.
Incubation mixture
Free glucose 15 min pH 8.0
glucose UDPG
+
pmole
15 min pH 8.0 pH 8.0
t t t
t t t t
180 min pH 6.0
+
-15 min pH 8.0
t
+ +
+
+
+
+
t
(B)
glucose UDPG ( ~ - g l u cose- 1 - P
+ t
t t t
t
t
t
(C)
+
+
+
+
+
+
t -
UDPG c~-glucose-l-P
t
t
t
(D)
+
+ t +
+ t t
glucose UDPG
~-glucose-l-P
-
t -
(X 1 0 3 )
1 8 0 rain
pH 8.0
+
~-glucose-l-P
glucose
[ 14C] glucose
incorporated
180 inin pH 6.0
(A)
Gluconate - --
~
t ~ ~ ~
pH 6.0
pH 8.0
19 1 2
422 3 19
98 5 11
1 3 5
10 6 12
7 20 16
1
5
4
2 3
6 8
15 10
1 1 1
~ ~ ~
1 1 1
( ~ ~
1 1 1
from glucose was again more efficient at pH 6.0. In contrast to the results of experiments in which glucose was used as a substrate, UDPG was found to be more efficient in cellulose formation in or by cell envelope fragments (E and C), with greater synthesis occurring at pH 8.0. Similar trends were observed with a-glucose-l-P as substrate, although the decrease in activity at lower pH values was less pronounced. With UDPG and a-glucose-l-P, slightly higher incorporation of [ 1 4 C ] g l u c o s e was observed from fraction B compared to C whereas no cellulose was formed from any substrate from fraction D. Gluconate was produced from all fractions except D with glucose as substrate and this gluconate accumulated in the incubation medium, indicating the absence of gluconokinase activity. In a similar way a-glucose-l-P formed small concentrations of gluconate from all fractions except D, indicating some hydrolysis to glucose; because of this, it is difficult to assign cellulose formation entirely to the original substrate or its hydrolysis product. However, formation o f small concentrations of UDPG was observed at early incubation times in or by envelope fractions B and C and a-glucose-l-P, indicating the presence of UDPG pyrophosphorylase. This formation was stimulated by the presence of UTP or ATP as observed in later experiments described below. The efficiency of cellulose formation of each cell envelope fraction, and combinations of them, using various substrates, were compared as indicated in Table I. Similar trends were found to those observed in Table II. In addition, [14C] glucose-6-P was observed to incorporate finite, but much lower, concert-
103
trations of [14C]glucose into cellulose from both whole cells and envelope fragments compared to a-glucose-l-P and UDPG. An interesting feature of this experiment was the stimulation of incorporation of [14C]glucose from all substrates in or by the combined cell envelope fraction (E = B + C) compared to the individual fractions. Also, the addition of the broken cell supernatant (D) to this latter preparation was found to inhibit this stimulation. Since greater amounts of cellulose were obtained from the combined envelope fraction (2000--40 000 × g), this enzyme system was studied in more detail.
Properties of the e n z y m e system (E) Addition of ATP or UTP stimulated the incorporation from [14 C] glucose and []4C]glucose-l-P substrates to levels approaching those found using UDp[14C]glucose (Table III). UTP was observed to be more efficient than ATP with glucose as substrate whereas neither nucleotide triphosphate stimulated cellulose formation from UDPG to any great extent. It is also interesting to note that the addition of UDP or UMP had little effect on cellulose formation from UDPG. Initial rates of cellulose formation from UDp[14C] glucose were directly proportional to protein levels and increased with increasing substrate concentration (Figs l a and l b , respectively). The o p t i m u m concentration of UDPG substrate was approximately 4--5 mM. An interesting feature of the incubations with UDPG at pH 8.0 was the formation of large concentrations of a c o m p o u n d that co-chromatographed with reference cyclic glucose phosphate (prepared from alkaline hydrolysis of UDPG). An estimate of the extent of degradation of UDPG during a 3 h incubation was obtained from results using boiled enzyme preparations (zero time samples). They indicated that at pH 8.0 up to 60% of the supplied UDPG was degraded to cyclic glucose phosphate. Maximum initial rates were obtained at high pH values (pH > 8.0) as indicated in Table IV, although most of the experiments described in the TABLE III EFFECT OF ATP, UTP, UDP AND UMP ON CELLULOSE USING VARIOUS SUBSTRATES
FORMATION
FROM ENZYME
SYSTEM E
Each incubation mixture (pH 7.5) contained substrate (3.5 ttmole/ml), enzyme preparation (20 mg protein) a n d a d d i t i v e (5 p m o l e / m l ) i n a t o t a l v o l u m e o f 2 . 0 m l . F o r f u r t h e r d e t a i l s see M e t h o d s s e c t i o n . - - , n o t assayed.
Additive
Control I II ATP UTP ATP + UTP UDP UMP
pmole glucose incorporated/2
h (X 1 0 3 )
Glucose
UDPG
Glucose-l-P
10 11 16 20 20 ---
20 19 22 21 22 21 20
15 15 19 22 21 ---
104
12C
I,¢1 k-
80~
z
-a bJ
40
o a. nO
z ~u o
0
L
l
i
5
15
25
PROTEIN CONCENTRATION ( m g / 2 m l ) I
i
]
I00
...1 I _~
50
::k 0
I
L
L
5
I0
15
UDPG CONCENTRATION
(/J.mole/2ml)
Fig. 1. D e p e n d e n c e of t h e initial r a t e of cellulose f o r m a t i o n f r o m U D P [ 14C] glucose a n d cell e n v e l o p e f r a g m e n t s (E) on (a) p r o t e i n c o n c e n t r a t i o n a n d (b) U D P G c o n c e n t r a t i o n . E a c h s a m p l e c o n t a i n e d cellopent a o s e (5 # m o l e / m l ) in a t o t a l v o l u m e of 2.0 m l ( p H 7.5). I n c u b a t i o n t i m e was 15 m i n a t 3 0 ° C . F o r f u r t h e r details see M e t h o d s s e c t i o n .
present work were performed at lower pH values because of the pronounced degradation of UDPG in alkaline buffer systems.
Utilisation of UDPG by EDTA-treated whole cells Results presented in Part I [15] indicated that the permeability of whole cells of A. xylinurn to phosphorylated c o m p o u n d s could be facilitated by brief TABLE IV T H E V A R I A T I O N O F I N I T I A L R A T E OF C E L L U L O S E S Y N T H E S I S F R O M UDP [14C] G L U C O S E A N D ENZYME SYSTEM E WITH pH E a c h i n c u b a t i o n m i x t u r e c o n t a i n e d U D P [ 14C] glucose (2.5 p m o l e / m l ) , c e l l o p e n t a o s e (5 p m o l e / m l ) and e n z y m e p r e p a r a t i o n (20 m g p r o t e i n ) in a t o t a l v o l u m e o f 2.0 ml. Cellulose w a s e s t i m a t e d as d e s c r i b e d in the M e t h o d s section. pH
#zmole glucose inc o r p o r a t e d / 1 5 m i n (× 104)
5.5 7.0 7.5 8.0 8.4
4.6 32.9 47.9 58.5 59.6
105 TABLE V C E L L U L O S E S Y N T H E S I S F R O M U D p [ 1 4 C ] G L U C O S E BY W H O L E C E L L S T R E A T E D W I T H 1 m M EDTA W a s h e d cells ( 4 . 1 0 1 0 c e l l s / i n c u b a t i o n ) w e r e t r e a t e d for 2 rain w i t h 1 m M E D T A , f o l l o w e d b y a d d i t i o n of excess Mg 2+ ( 1 0 raM). I n c u b a t i o n m i x t u r e s c o n t a i n e d U D P [ 14C] glucose (3.5 p m o l e / m l ) in a t o t a l v o l u m e o f 2.0 ml. I n c u b a t i o n s w e r e for 120 rain at p H 7.5. I, II = d u p l i c a t e e x p e r i m e n t s . p m o l e glucose i n c o r p o r a t e d (X 10 3)
Treatment
I Boiled cells Untreated Treated
~
1 4.5 24.1
II ~
1 3.1 25.4
washing with low concentrations of EDTA. Also, experiments described above suggest that UDPG may be utilised only slowly by whole cells to form cellulose, without a t t e n d a n t hydrolysis to glucose. Therefore, to test the possibility that EDTA may loosen the outer region of the cell wall and allow uptake of UDPG for participation in cellulose synthesis, the following experiment was performed. Washed whole cells were treated for 2 min with 1 mM EDTA followed by the addition of excess Mg 2÷ (10 mM) and compared to untreated cells for cellulose synthesis from UDP[ 14C/glucose. The results are presented in Table V. A 6- to 8-fold increase in cellulose formation was observed for EDTAtreated cells compared to untreated, under conditions in which no gluconate was formed. Discussion
General The experiments described above were designed primarily to establish the extent to which the cellulose synthesising system of A. xylinum depends upon individual components of the cell envelope. The following discussion emphasises the findings t h a t both inner and outer membranes of the cell envelope are important in cellulose synthesis. Also, from consideration of the results presented in Part I [15] ~ possible locations of various e n z y m e activities are suggested.
Properties of the cell envelope preparations The procedure used for the disruption and fractionation of washed cell envelopes of A. xylinum, similar to that described by Schnaitman for E. coli [19], was found to be partially successful in the separation of components into two morphologically distinct fractions. Greater resolution of these fractions may have resulted from an additional step of layering on sucrose gradients, however this lengthy technique was n o t applied in the present work because of the possible increased loss of protein (and possibly essential enzymes) from the envelopes. Nevertheless, sufficient separation of the crude envelope preparations was observed to allow classification into two fractions, one of which was enriched in outer cell wall fragments, and the other enriched with fragments
106 derived from the cytoplasmic membrane [19 ] (fractions B and C, respectively). A comparative study of cellulose and gluconate synthesis from glucose, a-glucose-l-P and UDPG by the cell envelope fractions and whole cells led to many interesting observations. Cell envelope fractions were capable of transferring glycosyl units from UDPG and a-glucose-l-P into cellulose. The formation of cellulose by hexose phosphates in vitro has not been reported previously, and it is possible that the observed synthesis from c~-glucose-l-P is a special characteristic of envelope fragments prepared as in the present study [20]. Since no gluconate was produced during incubations with UDPG it was concluded that no hydrolysis to glucose occurred. However, extensive hydrolysis to cyclic glucose phosphate was observed, especially at high values of pH (pH > 8.0). With a-glucose-l-P as substrate, some gluconate formation was apparent indicating the presence of a hexose phosphatase. Cellulose was formed in low yields from glucose and cell envelope fragments compared to whole cells and cellulose synthesis was stimulated by the addition of ATP indicating the importance of the phosphorylation process. In this respect the observation of UDPG formation during incubation of the envelope fractions with a-glucoseI-P (especially in the presence of ATP) is evidence for the particulate nature of UDPG pyrophosphorylase. This enzyme has been regarded as cytoplasmic since it could be demonstrated in the supernatant fluid of ultrasonically disrupted cells [6]; however, this solubility may be the result of the more efficient disruption afforded by the ultrasonic technique. It is interesting to note that the enzymes responsible for the synthesis of nucleotide precursors, and those responsible for their subsequent polymerisation in techoic acid synthesis by Staphylococcus lactis, were found to occur in a particulate enzyme preparation composed of membrane fragments [21]. In the present work glucose-6-P dehydrogenase was found exclusively in the supernatant fluid of disrupted cells confirming the solubility of this enzyme, similar to the findings of other workers [22]. No cellulose was produced from any substrate using the disrupted cell supematant (Fraction D) confirming the results of other workers that the cellulose synthesising ability of A. xylinum resides exclusively in the cell envelope region [3--10]. Enzymes responsible for gluconate formation from glucose were also found to be particulate with maximum activity evident in the cytoplasmic membrane-rich fraction, in agreement with the suggestion of Deley and Dochy [18]. The repeated observation of gluconate accumulation from whole and broken cells and glucose as described in the present work (see also Part I [15] ), may be interpreted as a lack of gluconokinase activity not previously reported for A. xylinum cells. Thus, for the A. xylinum strain used here, gluconokinase may be induced in the presence of gluconate only when glucose levels are extremely low, similar to the behaviour of certain strains of E. coli [23], without effect on the glycolytic pathway. The constitutive ATP dependent glucokinase of the glucose grown cells used in the present investigation is most probably therefore the major mechanism of hexose phosphate formation [24]. A recent study of hexose phosphorylation in A. xylinum by Benziman et al. [25] has indicated the possible regulatory role of this process in both cellulose synthesis and oxidative dissimilation of carbohydrate via the pentose
107 and citrate cycles. The stimulation of cellulose synthesis in vitro from glucose and a-glucose-l-P by ATP or UTP addition and the small but finite synthesis from glucose alone, as described in the present work, is in agreement with the suggestion of the dependence of glucokinase action on coupled ATP formation during the oxidation of glucose to gluconate [24]. Evidence for the involvement of the cytoplasmic membrane in the synthesis of a cellulose precursor has been presented by Colvin et al. using lysed cell preparations [10]. However, their conclusion was based upon the fact that precipitated envelopes treated with trypsin (which resulted in the loss of the cytoplasmic membrane) failed to form cellulose from glucose. Since it is well known that trypsin destroys the regular lattice structure of cell walls by the digestion of all proteins [26] (a fact conceded by the authors) the above conclusion is open to doubt. The findings of the present investigation do not contradict, but rather extend the ideas formulated by C o l o n and co-workers. They indicate that not only the cytoplasmic membrane is required for cellulose synthesis but also the outer cell wall region is important, since fractions in which this latter component is predominant are as, and even more, efficient than fractions containing predominantly cytoplasmic membrane derived material (Tables I and II). The stimulation of incorporation after re-constitution of the total envelope fraction (E = B + C) provides additional evidence for the presence of an enzyme system that requires either the structural integrity of the cell envelope or components {enzymes and/or substrates) from both inner and outer regions of the cell envelope. No cellulose was formed from the supernatant fraction of the disrupted cells, and addition of this fraction to the combined envelope fraction resulted in the loss of stimulation provided by the combination. The source of this inhibiting action is u n k n o w n but is evidently the property of a cytoplasmic component, since later experiments demonstrated the stimulatory effect of the extracellular supernatant fluid, from whole cells, on cellulose synthesis by envelope fractions (see Part III, [27] ). The results also indicate that cellulose synthesis from the combined cell envelope fraction and UDPG is unaffected by the presence of UTP, ATP, UDP and UMP. The initial rate of synthesis was directly proportional to protein levels and increased with increasing substrate concentration with an o p t i m u m concentration of UDPG at approximately 4--5 mM and a pH o p t i m u m greater than pH 8.0. These findings are similar to those obtained by Glaser who used a particulate fraction frdm ultrasonically disrupted cells as enzyme source [6]. An interesting feature of the experiments was the high substrate (UDPG) concentration required for efficient cellulose synthesis. Taking into consideration errors associated with cell counting and estimations of cell size, the optimum UDPG pool size observed in Part I [15] corresponds to a concentration in the periplasm of approximately 2--5 raM. The calculation assumes that UDPG may occupy the whole of the periplasm and is obviously an oversimplification since the boundaries of the periplasm are not distinct. Although the observation may be fortuitous, it is interesting to note that this concentration range is the same order of magnitude as that required by the cell-free e n z y m e system (i.e. 4--5 mM). The observation that UDPG does n o t serve as glycosyl donor using the supernatant fluid from whole cells, whereas EDTA-treated whole cells
108
synthesise cellulose from this substrate in the presence of excess Mg 2+, provides additional evidence that UDPG functions solely in the cell envelope region. This conclusion stems from work presented in Part I [15] in which brief treatments with EDTA were found to increase the permeability of the outer cell envelope region of A. xylinum to phosphorylated compounds. References 1 S h a f i z a d e h , F. a n d McGinnis, G , D . ( 1 9 7 1 ) A d v . C a r b o h y d r . C h e m . B i o c h e m . 2 6 , 2 9 7 - - 3 4 9 2 Colvin, J . R . ( 1 9 7 1 ) Cellulose a n d Cellulose D e r i v a t i v e s , Vol. V, Part I V , pp. 6 9 5 - - 7 1 8 , Wiley-Interscience, N e w Y o r k 3 Colvin, J . R . ( 1 9 5 7 ) A r c h . B i o c h e m . Biophys. 70, 2 9 4 - - 2 9 5 4 G r o m e t , Z., S e h r a m m , M. a n d H e s t r i n , S. ( 1 9 5 7 ) B i o c h e m . J. 67, 6 7 9 - - 6 8 9 5 G r e a t h o u s e , G . A . ( 1 9 5 7 ) J. A m . C h e m . Soe. 79, 4 5 0 3 - - 4 5 0 4 6 Glaser, L. ( 1 9 5 8 ) J. Biol. C h e m . 2 3 2 , 6 2 7 - - 6 3 6 7 y o n Leisinger, Th. ( 1 9 6 6 ) P a t h o l . Microbiol. 29, 7 5 6 - - 7 6 6 8 B a r b e r , G . A . , Elbein, A . D . a n d Hassid, W.Z. ( 1 9 6 4 ) J. Biol. C h e m . 239, 4056---4061 9 B e n - H a y y i m , G. a n d O h a d , I. ( 1 9 6 5 ) J. Cell Biol, 25, 1 9 1 - - 2 0 7 10 Dennis, D.T. a n d Colvin, J . R . ( 1 9 6 5 ) Cellular U l t r a s t r u c t u r e of W o o d y Plants, pp. 1 9 9 - - 2 1 2 , S y r a c u s e U n i v e r s i t y Press, N e w Y o r k 11 Dennis, D . T . a n d Colvin, J . R , ( 1 9 6 4 ) Pulp P a p e r Mag. Can. 64, T - 3 9 5 - - 3 9 9 1 2 Colvin, J . R . a n d L e p p a r d , G . G . ( 1 9 7 1 ) J. P o l y m e r Sci, 36C, 4 1 7 - - 4 2 4 13 M a n l e y , R . S t . J . , J o n k e r , J,W., C o o p e r , D. a n d P o u n d , T.C. ( 1 9 7 1 ) Nat. N e w Biol. 229, 8 8 - - 8 9 14 D a n k e r t , M., G a r c i a , R. a n d R e e o n d o , E. ( 1 9 7 2 ) B i o c h e m i s t r y of t h e G l y c o s i d i c L i n k a g e , pp. 1 9 9 - 2 0 6 , A c a d e m i c Press, N e w Y o r k 15 C o o p e r , D. a n d M a n l e y , R . S t . J . ( 1 9 7 5 ) B i o e h i m . Biophys. A c t a 3 8 1 , 7 8 - - 9 6 16 H e s t r i n , S. a n d S c h r a m m , M. ( 1 9 5 4 ) B i o c h e m . J. 58, 3 4 5 - - 3 5 2 17 L a y n e , E. ( 1 9 5 7 ) M e t h o d s E n z y m o l . 3, 4 4 7 - - 4 5 0 18 D e l e y , J. a n d D o c h y , R. ( 1 9 6 0 ) B i o e h i m . B i o p h y s . A e t a 42, 538---541 19 S c l m a i t m a n , C.A. ( 1 9 7 0 ) J. Baeteriol. 1 0 4 , 8 9 0 - - 8 9 8 20 Ellar, D.J. ( 1 9 7 0 ) O r g a n l s a t i o n a n d C o n t r o l in P r o k a x y o t i e a n d E u k a r y o t i c Cells, pp. 1 6 7 - - 2 0 1 , C a m b r i d g e U n i v e r s i t y Press, L o n d o n 21 B a d d i l e y , J., B l u m s o m , N.L. a n d Douglas, J. ( 1 9 6 8 ) B i o c h e m . J. 1 1 0 , 5 6 5 - - 5 7 1 22 W e b b , T.E. a n d Colvin, J . R . ( 1 9 6 2 ) Can. J, Microbiol. 8, 8 4 1 - - 8 4 6 23 C o h e n , S,S. ( 1 9 5 1 ) J. Biol. C h e m . 1 8 9 , 6 1 7 - - 6 2 8 24 W e i n h o u s e , H. a n d B e n z i m a n , M. ( 1 9 7 1 ) B i o c h e m . B i o p h y s . Res, C o m m u n . 43, 2 3 3 - - 2 3 8 25 B e n z i m a n , M. a n d R i v e t z , B. ( 1 9 7 2 ) J. Bacteriol. 1 1 1 , 3 2 5 - - 3 3 3 26 H o r n e , R.W., Davies, D . R , , N o r t o n , K. a n d G t t r n e y - S m i t h , M. ( 1 9 7 1 ) N a t u r e 232, 4 9 3 - - 4 9 5 27 C o o p e r , D. a n d M a r d e y , R . S t . J . ( 1 9 7 5 ) B i o e h i m . B i o p h y s . A e t a 3 8 1 . 1 0 9 - - 1 1 9
Biochimica et Biophysica Acta, 381 (1975) 97--108
Q Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27559 CELLULOSE SYNTHESIS BY A C E T O B A C T E R X Y L I N U M II. INVESTIGATION INTO THE RELATION BETWEEN CELLULOSE SYNTHESIS AND CELL ENVELOPE COMPONENTS
DAVID COOPER and R.St. JOHN MANLEY Department of Chemistry and Pulp and Paper Research Institute, McGill University, Montreal H3C 3G1 (Canada)
(Received May 8th, 1974) (Revised manuscript received September 9th, 1974)
Summary Cell envelope fractions, capable of cellulose synthesis from uridine diphosphate glucose, a-glucose-l-phosphate, glucose-6-phosphate and glucose, have been isolated from A c e t o b a c t e r x y l i n u m suspensions and various enzymatic properties examined. Essential enzymes were f o u n d to be distributed t h r o u g h o u t the cell envelope region, with both inner (cytoplasmic) and outer (cell wall) membranes contributing to cellulose synthesis. The central role of UDPG in cellulose synthesis was confirmed and the results indicated that the nucleoside diphosphate sugar functions solely in the cell envelope region of whole cells. A comparison of properties of the cell envelope system with those of different preparations used by other workers, suggested that the m e t h o d of cell disruption may influence substrate specificity.
Introduction The convenience of utilising suspensions of A c e t o b a c t e r x y l i n u m cells as a system for the study of cellulose biosynthesis has been well documented [1,2]. A number of workers have isolated cell-free e n z y m e systems from this bacterium capable of cellulose synthesis from various substrates [3--10]. From such investigations it became apparent that the m e t h o d of cell disruption may influence both substrate specificity and the extent to which the cellulose synthesizing capacity is maintained. Most results indicated that the ability to form cellulose resides in the particulate fraction of cell-free systems. Furthermore, the results of Colvin et al. [10] using lysed cell preparations, indicated the involvement of the cytoplasmic membrane in the synthesis of the cellulose
98 precursor. However, the evidence was circumstantial and these interesting studies were not pursued further. Depending on the enzyme preparation, cellulose formation has been demonstrated from glucose [3--6,10], fructose I101, UDPG or TDPG [6--91 as substrate, but not from other nucleoside diphosphate sugars or any hexose phosphate. The lack of synthesis from hexose phosphates has led to the suggestion that a bound series of reactions is involved that cannot be entered in the middle [11]. Although many workers agree that a sugar nucleotide may be involved at some stage in cellulose formation, questions have arisen concerning the precise position of this intermediate in the synthetic scheme. For instance, does the final transfer of glycosyl units involve a nucleoside diphosphate sugar or is there an additional c o m p o u n d that assumes this cardinal role, such as a glycolipid intermediate [12--14]? Where does this final transfer occur and is a matrix necessary (i.e. heterogeneous or homogeneous nucleation)? Solutions to these and other problems may come from a study of the contribution of various components of the cell envelope of A. x y l i n u m to cellulose synthesis as a t t e m p t e d in the present work. In Part I [15] it was shown that cellulose synthesising suspensions of A. x y l i n u m contain quantities of a-glucose-l-P and UDPG, and that these compounds behave as though they are intermediates in cellulose synthesis. Furthermore, evidence suggested that pools of these compounds are localised outside the cytoplasm of A. x y l i n u m cells, possibly in the periplasmic space, and that permeability barriers to such phosphorylated compounds exist in the outer cell envelope region. Consequently, the work presented in the present paper, as part of a systematic investigation of cellulose synthesis by A. x y l i n u m was carried out with the following objectives in mind: (i) to isolate a cell-free enzyme system capable of cellulose synthesis from any (or all) of the suggested intermediates found in Part I [15], (ii) to examine the properties of the enzyme system with regard to substrate specificity, variation in pH, and the effect of various co-factors and (iii) to compare the utilisation of substrates, at equivalent molar concentrations, by the cell-free system to that of whole cells. Gentle disruption of washed A. x y l i n u m cells and partial fractionation of cell envelope components, followed by studies of cellulose formation from various substrates, provided evidence that the enzyme(s) responsible for cellulose synthesis are distributed t h r o u g h o u t the cell envelope region. Materials and Methods
Ch ro m a tograp hy Thin-layer chromatography was performed on cellulose or silica gel plates. The same solvents were employed for the washed layers as described earlier [15]. Spray reagents used for phosphate, reducing substances and lipids, and autoradiography of separated radioactive materials have been described earlier [151. Collection of bacteria and preparation o f envelope fractions Surface pellicles were harvested from 48 h cultures and washed bacterial
99 suspensions prepared by the m e t h o d of Hestrin and Schramm [16]. Final suspension was in 0.05 M t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e (Tris) buffer (pH 7.5) containing ethylenediaminetetraacetic acid (EDTA) (1 mM) and MgC12 (10 mM) at 0°C. An aliquot of the cell suspension was passed twice through an Aminco French pressure cell operated in a Carver Laboratory Press, Model B, at a pressure of 20 000 lb/inch 2. The resulting broken cell suspension was then centrifuged at various speeds in a Spinco Model L preparative ultracentrifuge (Beckman Instruments Co.) at 0°C. Fractions isolated were as follows, (i) 0--2000 × g, 10 min (not used), (ii) 2000--10 000 × g, 30 min (fraction B), (iii) 10 000--40 000 × g, 45 min (fraction C) and (iv) > 4 0 000 X g supernatant (fraction D). Electron microscopic observations and viable cell counts indicated that the procedure used was extremely efficient for cell disruption. Each fraction (except D) was washed before use by gentle resuspension in buffer and centrifugation as before. Protein concentrations for each fraction were determined by the biuret method [17], and adjusted to equal levels (usually 20 mg/ml) by dilution with buffer before analysis of cellulose synthesising ability.
Chemicals and radiochemicals Uniformly labeled: D-[~4C]glucose (Schwarz Bioresearch Inc., 240 Ci/mole), UDP [ ~4 C] glucose (250 Ci/mole), a-[ ~4 C] glucose-l-P (210 Ci/mole) and [ 14 C] glucose-6-P (185 Ci/mole) (International Chemical and Nuclear Corporation) were purified before use by thin-layer chromatography on cellulose thin layers. Substrates were diluted to the required specific activity (usually 0.5 Ci/mole) by the addition of non-radioactive chemicals. The counting efficiency for each substrate was determined under the same conditions used for estimation of cellulose as indicated below. All other chemicals were reagent grade or better. Estimation of cellulose Carrier cellulose was prepared from ultrasonically dispersed, purified bacterial cellulose and suspended in distilled water at a concentration of 15 mg/ml. Immediately before termination of cellulose synthesis an aliquot (0.1 ml) of carrier cellulose was added to the incubation mixture containing substrate (1 ml) and enzyme preparation (1 ml). Reactions were terminated by the addition of ethanol (25 ml) and the insoluble residue analysed for cellulose content after treatment with sodium laurylsulphate and sodium hypochlorite as indicated previously [15]. Occasionally the ethanolic supernatant was also analysed. Control mixtures (blanks) were heated for 10 min on a boiling water bath before addition of substrate. Preparation of extracellular enzyme In certain experiments the supernatant fluid from whole cells was used as a source of enzyme for cellulose synthesis from heat inactivated envelope fragments. This 'extracellular' enzyme was prepared by centrifugation of a washedcell suspension of A. xylinum, previously incubated for 10 min with a limiting concentration of glucose (0.01%), at 20 000 × g for 30 min at 0°C, followed by filtration through a 0.22 pm Millipore filter in the cold. The supernatant fluid was stored at 0°C until required (usually less than 1 h).
100
Estimation of glucose-6-phosphate dehydrogenase activity Glucose-6-phosphate dehydrogenase activity was determined spectrophotometrically at 340 nm wavelength using a Beckman Model D.U. spectrophotometer under conditions similar to those described by DeLey and Dochy [18]. A 1 cm cell was used containing NADP (0.3 pmole), glucose-6-P (10 gmole), MgSO4 (5 #mole), Tris--HC1 buffer, pH 8.5 (100 pmole) and 0.5 ml of the enzyme preparation in a total volume of 2.5 ml.
Electron microscopy The methods for fixation and embedding of the envelope fractions were similar to those used for whole cells described earlier [15]. However, because of the more delicate nature of these preparations, certain modifications were made [19]. The pellets, after centrifugation, were fixed initially in glutaraldehyde (2.5%) for 2 h before post-fixation in osmium tetroxide and subsequent washing. Results The apparent permeability barrier of the outer cell envelope region of A.
xylinum to phosphorylated compounds, detected in cellulose synthesising suspensions of this bacterium, has been described in Part I [15]. To facilitate a study of the interaction (or interconversion) of detected compounds it was necessary to develop a procedure for isolation of an enzyme preparation capable of cellulose synthesis in which the above mentioned permeability barriers are removed. Considering the results of previous workers it was suspected that the enzyme system responsible for cellulose synthesis required the structural integrity of the cell envelope region and/or its components [10]. For this reason excessively damaging procedures such as ultrasonic disruption or powder grinding were not employed, the more gentle technique of disruption using a French pressure cell being used. Light microscopic observations and viable cell counts indicated that cell rupture was efficiently achieved by double passage of cell suspensions containing excess Mg 2+ through an Aminco French pressure cell operated in a manual press at the m a x i m u m working pressure. An a t t e m p t was made to fractionate the constituents of the ruptured cell suspension by centrifugation at various forces. Electron microscopic observations of thin sections of stained sediments confirmed that at least a partial fractionation was achieved. Large cell debris including heavy cytoplasmic particulate material were observed in the fraction centrifuging at 2000 X g with some cells retaining the usual cell envelope structure of whole cells. Higher centrifugal forces, up to 10 000 × g, resulted in a sediment containing predominantly open membranous structures of single tripartite layers. Particles sedimenting at higher velocities (10 000--40 000 × g) contained many closed, vesicular-like structures, each composed of single tripartite layers; however, some of the more open structures previously described were also present. A crude preparation of envelope fragments, sedimenting between 2000--40 000 × g, consisted of a mixture of both membrane types described above. In earlier experiments performed in the absence of excess Mg 2÷, membrane and wall fragments were observed to be extremely small and poor maintenance of both double track layer structures was evident.
101
TABLE I COMPARISON OF CELLULOSE SYNTHESIS FROM VARIOUS CELL FRACTIONS AND SUBSTRATES A c o n t a i n e d 2 " 1 0 1 0 w h o l e cells, f r a c t i o n s B a n d C, 20 m g p r o t e i n , D, 10 m g p r o t e i n , E, 20 m g t o t a l p r o t e i n a n d F, 30 m g t o t a l p r o t e i n p e r i n c u b a t i o n m i x t u r e . S u b s t r a t e c o n c e n t r a t i o n w a s 3.5 g m o l e / m l in a t o t a l v o l u m e o f 2.0 m l ( p H 7 . 5 ) as d e s c r i b e d in t h e M e t h o d s s e c t i o n . T h e a c t i v i t y o f t h e d e h y d r o g e n a s e in t h e s u p e r n a t a n t o f d i s r u p t e d cells, D, w a s t a k e n as 1 0 0 % f o r c o m p a r i s o n p u r p o s e s . F o r f u r t h e r d e t a i l s see M e t h o d s s e c t i o n . Fraction
p m o l e g l u c o s e i n c o r p o r a t e d / 3 h (× 103) glucose
A B C D E = B + C F=B+C+D
164 9 4 < 1 14 8
UDPG 3 24 17 1 36 29
Glucose-l-P 9 15 12 < 1 25 12
Glucose-6-P 3 4 2 < 1 7 2
Glucose-6-P dehydrogenase activity 2 4 5 100 ---
It has been recently demonstrated by other workers that envelope fractions of Gram-negative bacteria, prepared in a similar way to those in this work, possess both types of structure described above [19]. Furthermore, it has been shown t h a t the lighter fraction consists of material derived from the cytoplasmic membrane and the heavier fraction from the outer cell wall region. It should be emphasised that efficient separations were n o t obtained, so that some contamination of one fraction with another was always apparent. However, the fractions indicated contained a high percentage of a particular structure. Each fraction was analysed for glucose-6-P dehydrogenase activity: the enzyme was detected only in fraction D which is to be expected of this soluble enzyme (Table I). As a final note it should be remembered that alkali-insoluble polymeric material (containing ~4C-labeled glucose) as described in the following results, is referred to as cellulose (see Methods section).
Comparison of fractions Initial experiments were designed to compare the cellulose synthesising ability of various envelope fractions with whole cells from 1 4 C-labeled glucose, UDPG and a-glucose-l-P as substrates at pH 6.0 and pH 8.0. As shown in Table II, cellulose was formed by whole cells from glucose, with increased yield at the lower pH value as expected from the results of previous workers [16]. This formation was markedly inhibited at higher pH values. Small, but finite, transfer of glucosyl units from UDPG into cellulose was detected using whole cells, w i t h o u t a t t e n d a n t formation of gluconate. The lack of gluconate formation indicated that UDPG was n o t hydrolysed to glucose. This effect was investigated further in additional experiments described later. With a-glucose-l-P as substrate, larger amounts of cellulose were formed compared to UDPG although some hydrolysis to glucose was observed with concomitant production of gluconate. Data presented in Table II show the marked decrease in cellulose formation from glucose when cell fragments are used as e n z y m e source. Synthesis
102 TABLE
II
CELLULOSE AND GLUCONATE FORMATION FROM [14C]GLUCOSE, UDP [14C]GLUCOSE a-[14C] GLUCOSE-I-P USING VARIOUS CELL FRACTIONS AS ENZYME SOURCE
AND
E a c h i n c u b a t i o n m i x t u r e c o n t a i n e d s u b s t r a t e ( 3 . 5 ~ t m o l e / m l ) a n d an a l i q u o t o f e n z y m e p r e p a r a t i o n in a t o t a l v o l u m e o f 2 . 0 m l as d e s c r i b e d in t h e M e t h o d s s e c t i o n . F r a c t i o n A c o n t a i n e d 2 " 1 0 1 0 w h o l e cells, f r a c t i o n s B a n d C, 2 0 m g p r o t e i n a n d D, 1 0 m g p r o t e i n p e r i n c u b a t i o n m i x t u r e . B o i l e d e n z y m e p r e p a r a t i o n s w e r e u s e d as c o n t r o l s a m p l e s . T h e i n c u b a t i o n t e m p e r a t u r e for t h i s a n d all o t h e r e x p e r i m e n t s d e s c r i b e d in this paper was 30°C. Free glucose and gluconate were estimated visually after thin-layer chromatography as d e s c r i b e d i n t h e M e t h o d s s e c t i o n . + a n d designate presence or absence of component, respectively. t = trace quantity.
Incubation mixture
Free glucose 15 min pH 8.0
glucose UDPG
+
pmole
15 min pH 8.0 pH 8.0
t t t
t t t t
180 min pH 6.0
+
-15 min pH 8.0
t
+ +
+
+
+
+
t
(B)
glucose UDPG ( ~ - g l u cose- 1 - P
+ t
t t t
t
t
t
(C)
+
+
+
+
+
+
t -
UDPG c~-glucose-l-P
t
t
t
(D)
+
+ t +
+ t t
glucose UDPG
~-glucose-l-P
-
t -
(X 1 0 3 )
1 8 0 rain
pH 8.0
+
~-glucose-l-P
glucose
[ 14C] glucose
incorporated
180 inin pH 6.0
(A)
Gluconate - --
~
t ~ ~ ~
pH 6.0
pH 8.0
19 1 2
422 3 19
98 5 11
1 3 5
10 6 12
7 20 16
1
5
4
2 3
6 8
15 10
1 1 1
~ ~ ~
1 1 1
( ~ ~
1 1 1
from glucose was again more efficient at pH 6.0. In contrast to the results of experiments in which glucose was used as a substrate, UDPG was found to be more efficient in cellulose formation in or by cell envelope fragments (E and C), with greater synthesis occurring at pH 8.0. Similar trends were observed with a-glucose-l-P as substrate, although the decrease in activity at lower pH values was less pronounced. With UDPG and a-glucose-l-P, slightly higher incorporation of [ 1 4 C ] g l u c o s e was observed from fraction B compared to C whereas no cellulose was formed from any substrate from fraction D. Gluconate was produced from all fractions except D with glucose as substrate and this gluconate accumulated in the incubation medium, indicating the absence of gluconokinase activity. In a similar way a-glucose-l-P formed small concentrations of gluconate from all fractions except D, indicating some hydrolysis to glucose; because of this, it is difficult to assign cellulose formation entirely to the original substrate or its hydrolysis product. However, formation o f small concentrations of UDPG was observed at early incubation times in or by envelope fractions B and C and a-glucose-l-P, indicating the presence of UDPG pyrophosphorylase. This formation was stimulated by the presence of UTP or ATP as observed in later experiments described below. The efficiency of cellulose formation of each cell envelope fraction, and combinations of them, using various substrates, were compared as indicated in Table I. Similar trends were found to those observed in Table II. In addition, [14C] glucose-6-P was observed to incorporate finite, but much lower, concert-
103
trations of [14C]glucose into cellulose from both whole cells and envelope fragments compared to a-glucose-l-P and UDPG. An interesting feature of this experiment was the stimulation of incorporation of [14C]glucose from all substrates in or by the combined cell envelope fraction (E = B + C) compared to the individual fractions. Also, the addition of the broken cell supernatant (D) to this latter preparation was found to inhibit this stimulation. Since greater amounts of cellulose were obtained from the combined envelope fraction (2000--40 000 × g), this enzyme system was studied in more detail.
Properties of the e n z y m e system (E) Addition of ATP or UTP stimulated the incorporation from [14 C] glucose and []4C]glucose-l-P substrates to levels approaching those found using UDp[14C]glucose (Table III). UTP was observed to be more efficient than ATP with glucose as substrate whereas neither nucleotide triphosphate stimulated cellulose formation from UDPG to any great extent. It is also interesting to note that the addition of UDP or UMP had little effect on cellulose formation from UDPG. Initial rates of cellulose formation from UDp[14C] glucose were directly proportional to protein levels and increased with increasing substrate concentration (Figs l a and l b , respectively). The o p t i m u m concentration of UDPG substrate was approximately 4--5 mM. An interesting feature of the incubations with UDPG at pH 8.0 was the formation of large concentrations of a c o m p o u n d that co-chromatographed with reference cyclic glucose phosphate (prepared from alkaline hydrolysis of UDPG). An estimate of the extent of degradation of UDPG during a 3 h incubation was obtained from results using boiled enzyme preparations (zero time samples). They indicated that at pH 8.0 up to 60% of the supplied UDPG was degraded to cyclic glucose phosphate. Maximum initial rates were obtained at high pH values (pH > 8.0) as indicated in Table IV, although most of the experiments described in the TABLE III EFFECT OF ATP, UTP, UDP AND UMP ON CELLULOSE USING VARIOUS SUBSTRATES
FORMATION
FROM ENZYME
SYSTEM E
Each incubation mixture (pH 7.5) contained substrate (3.5 ttmole/ml), enzyme preparation (20 mg protein) a n d a d d i t i v e (5 p m o l e / m l ) i n a t o t a l v o l u m e o f 2 . 0 m l . F o r f u r t h e r d e t a i l s see M e t h o d s s e c t i o n . - - , n o t assayed.
Additive
Control I II ATP UTP ATP + UTP UDP UMP
pmole glucose incorporated/2
h (X 1 0 3 )
Glucose
UDPG
Glucose-l-P
10 11 16 20 20 ---
20 19 22 21 22 21 20
15 15 19 22 21 ---
104
12C
I,¢1 k-
80~
z
-a bJ
40
o a. nO
z ~u o
0
L
l
i
5
15
25
PROTEIN CONCENTRATION ( m g / 2 m l ) I
i
]
I00
...1 I _~
50
::k 0
I
L
L
5
I0
15
UDPG CONCENTRATION
(/J.mole/2ml)
Fig. 1. D e p e n d e n c e of t h e initial r a t e of cellulose f o r m a t i o n f r o m U D P [ 14C] glucose a n d cell e n v e l o p e f r a g m e n t s (E) on (a) p r o t e i n c o n c e n t r a t i o n a n d (b) U D P G c o n c e n t r a t i o n . E a c h s a m p l e c o n t a i n e d cellopent a o s e (5 # m o l e / m l ) in a t o t a l v o l u m e of 2.0 m l ( p H 7.5). I n c u b a t i o n t i m e was 15 m i n a t 3 0 ° C . F o r f u r t h e r details see M e t h o d s s e c t i o n .
present work were performed at lower pH values because of the pronounced degradation of UDPG in alkaline buffer systems.
Utilisation of UDPG by EDTA-treated whole cells Results presented in Part I [15] indicated that the permeability of whole cells of A. xylinurn to phosphorylated c o m p o u n d s could be facilitated by brief TABLE IV T H E V A R I A T I O N O F I N I T I A L R A T E OF C E L L U L O S E S Y N T H E S I S F R O M UDP [14C] G L U C O S E A N D ENZYME SYSTEM E WITH pH E a c h i n c u b a t i o n m i x t u r e c o n t a i n e d U D P [ 14C] glucose (2.5 p m o l e / m l ) , c e l l o p e n t a o s e (5 p m o l e / m l ) and e n z y m e p r e p a r a t i o n (20 m g p r o t e i n ) in a t o t a l v o l u m e o f 2.0 ml. Cellulose w a s e s t i m a t e d as d e s c r i b e d in the M e t h o d s section. pH
#zmole glucose inc o r p o r a t e d / 1 5 m i n (× 104)
5.5 7.0 7.5 8.0 8.4
4.6 32.9 47.9 58.5 59.6
105 TABLE V C E L L U L O S E S Y N T H E S I S F R O M U D p [ 1 4 C ] G L U C O S E BY W H O L E C E L L S T R E A T E D W I T H 1 m M EDTA W a s h e d cells ( 4 . 1 0 1 0 c e l l s / i n c u b a t i o n ) w e r e t r e a t e d for 2 rain w i t h 1 m M E D T A , f o l l o w e d b y a d d i t i o n of excess Mg 2+ ( 1 0 raM). I n c u b a t i o n m i x t u r e s c o n t a i n e d U D P [ 14C] glucose (3.5 p m o l e / m l ) in a t o t a l v o l u m e o f 2.0 ml. I n c u b a t i o n s w e r e for 120 rain at p H 7.5. I, II = d u p l i c a t e e x p e r i m e n t s . p m o l e glucose i n c o r p o r a t e d (X 10 3)
Treatment
I Boiled cells Untreated Treated
~
1 4.5 24.1
II ~
1 3.1 25.4
washing with low concentrations of EDTA. Also, experiments described above suggest that UDPG may be utilised only slowly by whole cells to form cellulose, without a t t e n d a n t hydrolysis to glucose. Therefore, to test the possibility that EDTA may loosen the outer region of the cell wall and allow uptake of UDPG for participation in cellulose synthesis, the following experiment was performed. Washed whole cells were treated for 2 min with 1 mM EDTA followed by the addition of excess Mg 2÷ (10 mM) and compared to untreated cells for cellulose synthesis from UDP[ 14C/glucose. The results are presented in Table V. A 6- to 8-fold increase in cellulose formation was observed for EDTAtreated cells compared to untreated, under conditions in which no gluconate was formed. Discussion
General The experiments described above were designed primarily to establish the extent to which the cellulose synthesising system of A. xylinum depends upon individual components of the cell envelope. The following discussion emphasises the findings t h a t both inner and outer membranes of the cell envelope are important in cellulose synthesis. Also, from consideration of the results presented in Part I [15] ~ possible locations of various e n z y m e activities are suggested.
Properties of the cell envelope preparations The procedure used for the disruption and fractionation of washed cell envelopes of A. xylinum, similar to that described by Schnaitman for E. coli [19], was found to be partially successful in the separation of components into two morphologically distinct fractions. Greater resolution of these fractions may have resulted from an additional step of layering on sucrose gradients, however this lengthy technique was n o t applied in the present work because of the possible increased loss of protein (and possibly essential enzymes) from the envelopes. Nevertheless, sufficient separation of the crude envelope preparations was observed to allow classification into two fractions, one of which was enriched in outer cell wall fragments, and the other enriched with fragments
106 derived from the cytoplasmic membrane [19 ] (fractions B and C, respectively). A comparative study of cellulose and gluconate synthesis from glucose, a-glucose-l-P and UDPG by the cell envelope fractions and whole cells led to many interesting observations. Cell envelope fractions were capable of transferring glycosyl units from UDPG and a-glucose-l-P into cellulose. The formation of cellulose by hexose phosphates in vitro has not been reported previously, and it is possible that the observed synthesis from c~-glucose-l-P is a special characteristic of envelope fragments prepared as in the present study [20]. Since no gluconate was produced during incubations with UDPG it was concluded that no hydrolysis to glucose occurred. However, extensive hydrolysis to cyclic glucose phosphate was observed, especially at high values of pH (pH > 8.0). With a-glucose-l-P as substrate, some gluconate formation was apparent indicating the presence of a hexose phosphatase. Cellulose was formed in low yields from glucose and cell envelope fragments compared to whole cells and cellulose synthesis was stimulated by the addition of ATP indicating the importance of the phosphorylation process. In this respect the observation of UDPG formation during incubation of the envelope fractions with a-glucoseI-P (especially in the presence of ATP) is evidence for the particulate nature of UDPG pyrophosphorylase. This enzyme has been regarded as cytoplasmic since it could be demonstrated in the supernatant fluid of ultrasonically disrupted cells [6]; however, this solubility may be the result of the more efficient disruption afforded by the ultrasonic technique. It is interesting to note that the enzymes responsible for the synthesis of nucleotide precursors, and those responsible for their subsequent polymerisation in techoic acid synthesis by Staphylococcus lactis, were found to occur in a particulate enzyme preparation composed of membrane fragments [21]. In the present work glucose-6-P dehydrogenase was found exclusively in the supernatant fluid of disrupted cells confirming the solubility of this enzyme, similar to the findings of other workers [22]. No cellulose was produced from any substrate using the disrupted cell supematant (Fraction D) confirming the results of other workers that the cellulose synthesising ability of A. xylinum resides exclusively in the cell envelope region [3--10]. Enzymes responsible for gluconate formation from glucose were also found to be particulate with maximum activity evident in the cytoplasmic membrane-rich fraction, in agreement with the suggestion of Deley and Dochy [18]. The repeated observation of gluconate accumulation from whole and broken cells and glucose as described in the present work (see also Part I [15] ), may be interpreted as a lack of gluconokinase activity not previously reported for A. xylinum cells. Thus, for the A. xylinum strain used here, gluconokinase may be induced in the presence of gluconate only when glucose levels are extremely low, similar to the behaviour of certain strains of E. coli [23], without effect on the glycolytic pathway. The constitutive ATP dependent glucokinase of the glucose grown cells used in the present investigation is most probably therefore the major mechanism of hexose phosphate formation [24]. A recent study of hexose phosphorylation in A. xylinum by Benziman et al. [25] has indicated the possible regulatory role of this process in both cellulose synthesis and oxidative dissimilation of carbohydrate via the pentose
107 and citrate cycles. The stimulation of cellulose synthesis in vitro from glucose and a-glucose-l-P by ATP or UTP addition and the small but finite synthesis from glucose alone, as described in the present work, is in agreement with the suggestion of the dependence of glucokinase action on coupled ATP formation during the oxidation of glucose to gluconate [24]. Evidence for the involvement of the cytoplasmic membrane in the synthesis of a cellulose precursor has been presented by Colvin et al. using lysed cell preparations [10]. However, their conclusion was based upon the fact that precipitated envelopes treated with trypsin (which resulted in the loss of the cytoplasmic membrane) failed to form cellulose from glucose. Since it is well known that trypsin destroys the regular lattice structure of cell walls by the digestion of all proteins [26] (a fact conceded by the authors) the above conclusion is open to doubt. The findings of the present investigation do not contradict, but rather extend the ideas formulated by C o l o n and co-workers. They indicate that not only the cytoplasmic membrane is required for cellulose synthesis but also the outer cell wall region is important, since fractions in which this latter component is predominant are as, and even more, efficient than fractions containing predominantly cytoplasmic membrane derived material (Tables I and II). The stimulation of incorporation after re-constitution of the total envelope fraction (E = B + C) provides additional evidence for the presence of an enzyme system that requires either the structural integrity of the cell envelope or components {enzymes and/or substrates) from both inner and outer regions of the cell envelope. No cellulose was formed from the supernatant fraction of the disrupted cells, and addition of this fraction to the combined envelope fraction resulted in the loss of stimulation provided by the combination. The source of this inhibiting action is u n k n o w n but is evidently the property of a cytoplasmic component, since later experiments demonstrated the stimulatory effect of the extracellular supernatant fluid, from whole cells, on cellulose synthesis by envelope fractions (see Part III, [27] ). The results also indicate that cellulose synthesis from the combined cell envelope fraction and UDPG is unaffected by the presence of UTP, ATP, UDP and UMP. The initial rate of synthesis was directly proportional to protein levels and increased with increasing substrate concentration with an o p t i m u m concentration of UDPG at approximately 4--5 mM and a pH o p t i m u m greater than pH 8.0. These findings are similar to those obtained by Glaser who used a particulate fraction frdm ultrasonically disrupted cells as enzyme source [6]. An interesting feature of the experiments was the high substrate (UDPG) concentration required for efficient cellulose synthesis. Taking into consideration errors associated with cell counting and estimations of cell size, the optimum UDPG pool size observed in Part I [15] corresponds to a concentration in the periplasm of approximately 2--5 raM. The calculation assumes that UDPG may occupy the whole of the periplasm and is obviously an oversimplification since the boundaries of the periplasm are not distinct. Although the observation may be fortuitous, it is interesting to note that this concentration range is the same order of magnitude as that required by the cell-free e n z y m e system (i.e. 4--5 mM). The observation that UDPG does n o t serve as glycosyl donor using the supernatant fluid from whole cells, whereas EDTA-treated whole cells
108
synthesise cellulose from this substrate in the presence of excess Mg 2+, provides additional evidence that UDPG functions solely in the cell envelope region. This conclusion stems from work presented in Part I [15] in which brief treatments with EDTA were found to increase the permeability of the outer cell envelope region of A. xylinum to phosphorylated compounds. References 1 S h a f i z a d e h , F. a n d McGinnis, G , D . ( 1 9 7 1 ) A d v . C a r b o h y d r . C h e m . B i o c h e m . 2 6 , 2 9 7 - - 3 4 9 2 Colvin, J . R . ( 1 9 7 1 ) Cellulose a n d Cellulose D e r i v a t i v e s , Vol. V, Part I V , pp. 6 9 5 - - 7 1 8 , Wiley-Interscience, N e w Y o r k 3 Colvin, J . R . ( 1 9 5 7 ) A r c h . B i o c h e m . Biophys. 70, 2 9 4 - - 2 9 5 4 G r o m e t , Z., S e h r a m m , M. a n d H e s t r i n , S. ( 1 9 5 7 ) B i o c h e m . J. 67, 6 7 9 - - 6 8 9 5 G r e a t h o u s e , G . A . ( 1 9 5 7 ) J. A m . C h e m . Soe. 79, 4 5 0 3 - - 4 5 0 4 6 Glaser, L. ( 1 9 5 8 ) J. Biol. C h e m . 2 3 2 , 6 2 7 - - 6 3 6 7 y o n Leisinger, Th. ( 1 9 6 6 ) P a t h o l . Microbiol. 29, 7 5 6 - - 7 6 6 8 B a r b e r , G . A . , Elbein, A . D . a n d Hassid, W.Z. ( 1 9 6 4 ) J. Biol. C h e m . 239, 4056---4061 9 B e n - H a y y i m , G. a n d O h a d , I. ( 1 9 6 5 ) J. Cell Biol, 25, 1 9 1 - - 2 0 7 10 Dennis, D.T. a n d Colvin, J . R . ( 1 9 6 5 ) Cellular U l t r a s t r u c t u r e of W o o d y Plants, pp. 1 9 9 - - 2 1 2 , S y r a c u s e U n i v e r s i t y Press, N e w Y o r k 11 Dennis, D . T . a n d Colvin, J . R , ( 1 9 6 4 ) Pulp P a p e r Mag. Can. 64, T - 3 9 5 - - 3 9 9 1 2 Colvin, J . R . a n d L e p p a r d , G . G . ( 1 9 7 1 ) J. P o l y m e r Sci, 36C, 4 1 7 - - 4 2 4 13 M a n l e y , R . S t . J . , J o n k e r , J,W., C o o p e r , D. a n d P o u n d , T.C. ( 1 9 7 1 ) Nat. N e w Biol. 229, 8 8 - - 8 9 14 D a n k e r t , M., G a r c i a , R. a n d R e e o n d o , E. ( 1 9 7 2 ) B i o c h e m i s t r y of t h e G l y c o s i d i c L i n k a g e , pp. 1 9 9 - 2 0 6 , A c a d e m i c Press, N e w Y o r k 15 C o o p e r , D. a n d M a n l e y , R . S t . J . ( 1 9 7 5 ) B i o e h i m . Biophys. A c t a 3 8 1 , 7 8 - - 9 6 16 H e s t r i n , S. a n d S c h r a m m , M. ( 1 9 5 4 ) B i o c h e m . J. 58, 3 4 5 - - 3 5 2 17 L a y n e , E. ( 1 9 5 7 ) M e t h o d s E n z y m o l . 3, 4 4 7 - - 4 5 0 18 D e l e y , J. a n d D o c h y , R. ( 1 9 6 0 ) B i o e h i m . B i o p h y s . A e t a 42, 538---541 19 S c l m a i t m a n , C.A. ( 1 9 7 0 ) J. Baeteriol. 1 0 4 , 8 9 0 - - 8 9 8 20 Ellar, D.J. ( 1 9 7 0 ) O r g a n l s a t i o n a n d C o n t r o l in P r o k a x y o t i e a n d E u k a r y o t i c Cells, pp. 1 6 7 - - 2 0 1 , C a m b r i d g e U n i v e r s i t y Press, L o n d o n 21 B a d d i l e y , J., B l u m s o m , N.L. a n d Douglas, J. ( 1 9 6 8 ) B i o c h e m . J. 1 1 0 , 5 6 5 - - 5 7 1 22 W e b b , T.E. a n d Colvin, J . R . ( 1 9 6 2 ) Can. J, Microbiol. 8, 8 4 1 - - 8 4 6 23 C o h e n , S,S. ( 1 9 5 1 ) J. Biol. C h e m . 1 8 9 , 6 1 7 - - 6 2 8 24 W e i n h o u s e , H. a n d B e n z i m a n , M. ( 1 9 7 1 ) B i o c h e m . B i o p h y s . Res, C o m m u n . 43, 2 3 3 - - 2 3 8 25 B e n z i m a n , M. a n d R i v e t z , B. ( 1 9 7 2 ) J. Bacteriol. 1 1 1 , 3 2 5 - - 3 3 3 26 H o r n e , R.W., Davies, D . R , , N o r t o n , K. a n d G t t r n e y - S m i t h , M. ( 1 9 7 1 ) N a t u r e 232, 4 9 3 - - 4 9 5 27 C o o p e r , D. a n d M a r d e y , R . S t . J . ( 1 9 7 5 ) B i o e h i m . B i o p h y s . A e t a 3 8 1 . 1 0 9 - - 1 1 9
