Journal of General Microbiology (1981), 127, 185-191.
Printed in Great Britain
185
Effects of Carbon Source and Inorganic Phosphate Concentration on the
Production of Alginic Acid by a Mutant of Azotobacter vinelandii and on
the Enzymes Involved in its Biosynthesis
By N I G E L J . H O R A N , ' T R E V O R R . J A R M A N * A N D
E D W I N A. D A W E S 1 *
Department of Biochemistry, University of Hull, Hull HU6 7RX, U.K.
Tate & Lyle Ltd, Group Research and Development, Philip Lyle Memorial Research
Laboratory, P.O. Box 68, Reading RG6 2BX, U.K.
(Received 2 March 1981)
~~
~~
The specific activities of the key enzymes involved in the biosynthesis of the exopolysaccharide alginic acid by Azotobacter vinelandii were determined in extracts of batchcultured organisms grown with different carbon sources in the presence of limited and excess
inorganic phosphate. Alginic acid production was also measured. Glucose, fructose, sorbitol,
mannitol, glycerol and gluconate resembled sucrose in supporting much greater alginate
production in media containing growth-limiting amounts of inorganic phosphate. Mannose
supported only poor growth with no alginate formation, and growth did not occur on acetate.
Increases in the specific activities of phosphomannose isomerase, GDPmannose
pyrophosphorylase and GDPmannose dehydrogenase were accompanied by increased alginic
acid production. Our results accord with the suggestion that alginate formation is controlled
by derepression of key biosynthetic enzymes.
INTRODUCTION
Alginic acid is a commercially important polysaccharide used predominantly as a gelling or
a viscosifying agent and is currently obtained from certain species of marine algae (Jarman,
1979). It is also produced as an exopolysaccharide by Azotobacter vinelandii (Gorin &
Spencer, 1966) and Pseudomonas aeruginosa (Doggett & Harrison, 1969). Little is known of
the mechanism of control of the biosynthesis of exopolysaccharides in general and alginic acid
in particular, although more interest has been shown recently owing to their possible
commercial exploitation (Deavin et al., 1977).
A pathway for the biosynthesis of alginic acid in bacteria has been proposed on the basis of
enzymes detected in A . vinelandii (Pindar & Bucke, 1975). Sucrose is hydrolysed by an
intracellular invertase to fructose and glucose which, after phosphorylation, are interconverted
to mannose 6-phosphate by the appropriate hexose-6-phosphate isomerases. Mannose
1-phosphate is then formed by the action of phosphomannomutase and esterified with GTP
by GDPmannose pyrophosphorylase. Polymannuronic acid is produced by the subsequent
action of GDPmannose dehydrogenase and alginate polymerase. Alginic acid is a partially
acetylated (1 + 4)-linked linear copolymer of D-mannuronic acid units with a small proportion
of L-guluronic acid units (Gorin & Spencer, 1966); an extracellular mannuronate C-5
epimerase selectively epimerizes mannuronic acid units of polymannuronic acid to guluronic
acid.
The amount of alginic acid produced by A . vinelandii in batch culture was dependent on
the initial phosphate concentration (Deavin, 1976), increasing dramatically as the phosphate
concentration was decreased. We have now investigated the effect of phosphate concentration
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186
N. J . HORAN, T. R. JARMAN A N D E. A . D A W E S
and carbon source on the production of alginic acid in batch cultures of A . vinelandii and on
the specific activities of enzymes involved in its biosynthesis.
METHODS
.I.lainrcnance and growth of the organism. The organism used was a mutant of Azotobacter vinelandii
NClR 9068 with enhanced ability to produce polysaccharide: it was supplied by Tate & Lyle Ltd and designated
SMS2B. I t was maintained on the 'limited-phosphate' growth medium (see below) solidified with 2 % (w/v) Bacto
no. I agar. Stock cultures were subcultured monthly. grown at 30 OC for 3 d and stored at 4 "C. Working cultures
were subcultured from the stock cultures every 3 months. The mutant was stable both on agar plates and in liquid
culture. It was routinely cultured in the chemically defined. limited-phosphate medium of Jarman et al. (1978),
and K,HPO, (32 mg I-').
buffered with 50 m~-3-(N-morphowhich contains KH,PO, (8 mg I-')
1ino)propanesulphonic acid (Mops) (final concentration). and with the pH adjusted to 7.2 with NaOH.
Sucrose was present at 20 g I-'. Control experiments showed that Mops did not affect alginate production. The
'excess-phosphate' medium lacked KH,PO, and Mops but contained 1 g K,HPO, I-' and the pH was adjusted to
7 . 2 with HCI. Cultures (l00ml) were grown in 250ml Erlenmeyer flasks at 30 "C with gyratory shaking
(200 rev. min-'1. Inoculation was always from liquid cultures after at least three passages from plates.
Bacterial d v weight determination. Cultures ( 5 ml) were pipetted on to preweighed glass-fibre filters (Whatman
GF/F) supported on the apparatus described by Midgley & Dawes (1973). Vacuum was applied and the filters
were washed with distilled water ( 5 ml). When cultures were too viscous to filter, appropriate dilutions were made
in 50 m~-piperazine-N,N'-bis(2-ethanesulphonic
acid) (Pipes)/NaOH. pH 8-0. The filters were dried to a constant
weight at 60 "C (about 3 h). All assays were performed in duplicate. Cultures containing more than 3 g alginate I-'
became very viscous and samples of approximately the required volume were initially taken by pouring the culture,
with aseptic precautions. into a measuring cylinder. Accurate sampling then followed.
Po!iisaccharide determination. This was done after precipitation from culture supernatants with propan-2-01
according to the method of Jarman et al. (1978). Comparison with commercial standards was always made by
infrared spectrophotometry (Couperwhite & McCallum. 1975).
Po!~.--?-h?Idro.~~bul?lrare
determination. This was done by a modification of the method of Law & Slepecky
( 196 1 as described by Carter & Dawes ( 1979).
Inorganic phosphate determination. This was done as described by Clarke & Morris (1976).
Sucrose determination. Sucrose was determined as its trimethylsilyl ether derivative. prepared according to
Sweeley et af. (1963). and assayed by gas-liquid chromatography using a column of OV-17 on Chrom Q at a
temperature of 250 " C and a nitrogen flow rate of 20 ml min-I. Trehalose was the internal standard. Under these
conditions. sucrose appeared after 5 min.
Preparation of bacterial e.rfracts. Cultures were harvested by centrifuging at 28000 g and 4 "C for 20 min. If
the culture viscosity was high, sedimentation was facilitated by adding NaCl and EDTA to final concentrations of
100 m M and IOmM. respectively. The organisms were washed twice with, and resuspended in, either (1)
50m~-Pipes/NaOH. pH 8.0. or (2) 0.1 M-K'/Na' phosphate. pH 8.0. or (3) 5 0 m ~ - T r i s / H C 1+ 5 0 m ~ glutathione + 10 mM-MgCI,. pH 7.5 (subsequently referred to as buffers 1, 2 and 3, respectively), at a density of
0 . 3 g wet wt bacteria ml '. The organisms were then disrupted by passage twice through a chilled French press at
69 MPa. Cell debris was removed by centrifuging at 35000 g and 4 "C for 30 min. The extract was then
centrifuged at 105 OOO g and 4 OC for 2 h to remove the highly active membrane-bound NADPH oxidase and the
supernatant was stored on ice.
Enzyme assays. Optimum conditions for the following assays were ascertained in relation to both the buffer used
for extraction and for the assay. For glucokinase (EC 2.7.1 .2). fructokinase (EC 2 . 7 . 1 .4) and GDPmannose
pyrophosphorylase (EC 2.7.7.13) assays. organisms were suspended in buffer 3 for disruption and assayed in the
same buffer. The kinases were assayed by the method of Pindar & Bucke (1975) and GDPmannose
pyrophosphorylase by the spectrophotometric method of Munch-Petersen ( 1962). Phosphomannose isomerase
(EC 5 . 3 . 1 .8) was assayed according to Gracy & Noltmann (1968) using buffer 1 with extracts prepared in the
same buffer. GDPmannose dehydrogenase (EC 1 . I . 1.132) was both extracted and assayed in buffer 2 by the
method of Couperwhite & McCallum (1975). Mannokinase (EC 2 . 7 . 1 -7) was assayed precisely as described by
Anderson & Sapico ( 1 975) and mannose isomerase (EC 5 . 3 . 1 .7) by the method of Doudoroff (1962). Protein
was determined by the Lowry method.
When enzyme assays gave negative results (Table 1). a portion ( 5 ml) of the bacterial extract was dialysed for
4 h against the extraction buffer and another portion (2 ml) was passed through a column ( 1 5 x 0.5 cm) of
Sephadex G-25. The diffusate and the void volume eluate were then re-assayed. In all cases results were still
negative. Finally, the validity of the assays was checked with samples of crude extracts from Arthrobacter ciscosus
prepared as described by Preiss (1966) and stored deep frozen. These extracts contained the enzymes
phosphomannose isomerase. GDPmannose pyrophosphorylase and GDPmannose dehydrogenase.
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Alginate biosynthesis by A . vinelandii
187
Chemicals and biochemicals. Analytical reagent grade chemicals were used wherever possible. Non-physiological
buffers were obtained from Sigma. Mannose 6-phosphate was purchased from Sigma as the insoluble barium salt
and converted to the sodium salt by addition of a slight molar excess of sodium sulphate; barium sulphate was
removed by centrifuging.
RESULTS
Production of alginic acid during batch growth
During growth of the organism in limited-phosphate medium with sucrose (20 g 1-l) as the
sole carbon source, production of alginic acid commenced shortly after the onset of
exponential growth (Fig. 1). It was produced at a low rate until growth ceased due to
phosphate exhaustion; then alginic acid production increased markedly and continued for a
further 40 h. Cessation of alginic acid production could not be attributed to exhaustion of the
carbon source since the culture supernatant still contained 6 g sucrose 1-'. Values of 5 to 7 g
alginate (g dry wt bacteria)-' were usually observed, compared with 1.5 g alginate (g dry wt
bacteria)-' achieved by the wild-type organism (Deavin et at., 1977). This resulted in a culture
of high viscosity causing severe oxygen limitation, with an attendant rise in poly3-hydroxybutyrate content which is associated with the Azotobacteraceae under this
limitation (Dawes & Senior, 1973).
Alginate production was very low (below 500 pg ml-', the limit of assay) during
exponential growth in the excess-phosphate medium and growth ceased with phosphate
(0.8 g 1-l) and sucrose (8 g 1-*) still available in the medium. Then alginate production
increased for 30 h and values of 0.8 to 1.2 g alginate (g dry wt bacteria)-' were routinely
observed. At this concentration of exopolysaccharide the culture viscosity was low and the
bacterial poly-3-hydroxybutyrate content attained a maximum value of 7 %.
Enzymes of alginate biosynthesis
Because of the marked increase in alginate production after the cessation of growth (Fig. l),
the enzymes involved in its formation were assayed at different phases of the growth cycle to
see if changes in polysaccharide concentration could be correlated with changes in enzyme
activity. The enzymes glucokinase, fructokinase, phosphomannose isomerase, GDPmannose
pyrophosphorylase and GDPmannose dehydrogenase were first assayed by Pindar & Bucke
(1975) who observed very low activities. A systematic study of the extraction and assay of
each enzyme was therefore undertaken and at least tenfold increases in specific activity, as
compared with those reported by Pindar & Bucke (1975), were obtained by changing the
extraction buffers (see Methods). Adopting the best extraction and assay buffer, each enzyme
0.24
E:
E
5
W
0.18
a
s
c
a
0.12 .y
z
0.06 2
cd
0
Q
1
.rn
2
Time (h)
Fig. 1. Growth, alginic acid production and poly-3-hydroxybutyrate formation by A . vinelandii in
limited-phosphate batch culture on sucrose. Bacterial dry weight (0).
alginic acid (A), poly3-hydroxybutyrate (0)and inorganic phosphate concentration (0).
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I88
N . J. H O R A N , T. R. J A R M A N A N D E . A. DAWES
Table 1. Erect of period of growth on enzyme activities and alginate production in batch
culture on sucrose with limited phosphate
Due to the low bacterial densities obtained with the limited-phosphate medium the results were
secured with separate cultures and not with a single culture sampled at intervals. These results
represent an average of two assays. which differed by less than 5 %. performed on the same cell extract.
Specific activity lnmol min-I (mg protein)-I
Growth period
(h)
Alginate
(g I-')
Fxponential phase
20
25
42
40
0.44
0.90
1-00
Station ary phase
52
65
70
86
84
85
I00
3.0
4.0
4.5
4.9
5.2
6.0
0.17
1.8
G~UCOkinase
Fructokinase
1
GDPmannose
Phosphomannose
pyroGDPmannose
isomerase
phosphorylase dehydrogenase
14
19
24
28
16
12
19
24
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
29
31
27
28
32
33
35
25
28
23
26
2s
30
22
3.0
2.0
2.5
3.4
3.8
4-0
4.0
4.0
ND.
5.0
6.0
7.0
7.7
8.0
8.0
-
-
-
2-5
2.5
-
3.0
-
Not detected; -, not performed.
was characterized with respect to optimum pH and substrate concentration and the following
results were obtained: glucokinase, pH 7.2. K , 0 . 1 7 mM (glucose) and 0.11 mM (ATP);
fructokinase, pH 7.0. K , 0 . 2 7 mM (fructose) and 0-21 mM (ATP); phosphomannose
isomerase, pH 8.0, K,, 0-20 mM: GDPmannose dehydrogenase, pH 8.0, K,, 0.15 mM. It was
not possible to characterize GDPmannose pyrophosphorylase which had a half-life of 2 h on
ice but the optimum pH appeared to be about 8.0.When the enzymes were assayed in
different phases of growth (Table 1) good correlation was achieved between specific activity
and the amount of alginate formed.
EfSect of phosphate concentration and carbon source on alginate production
Deavin (1976) showed that the amount of alginate produced was dependent on the initial
inorganic phosphate concentration when sucrose was the sole carbon source. Table 2
indicates that this is true for a range of substrates. Alginate was not produced when mannose
was the sole carbon source. or with sorbitol in the presence of a high phosphate concentration.
Bacterial extracts were again examined for a possible correlation between enzyme activity and
alginate production (Table 3). The results revealed a wide variation in the specific activities of
the key enzymes which mirrored the observed pattern of alginate production. A surprising
result was the low activity of phosphomannose isomerase when mannose was the sole carbon
source. Since mannose isomerase could not be detected, growth of the organism on mannose
as the sole carbon source was examined. The low activity of phosphomannose isomerase was
reflected in the doubling time obtained (7.5 h with mannose. cf. 5 h with sucrose).
As Jarman et GI. (1978) had suggested that some control of alginate biosynthesis might be
exerted via phosphomannose isomerase. this enzyme was studied further in crude extracts.
Activity was not affected by the divalent metal ions Mg2+, Mn2+, Zn2+ or Co2+, while
2 . 5 rnM-Ca'' inhibited activity by 50%. Of the metabolites involved in alginate biosynthesis,
mannose 6-phosphate. GDPmannose and polymannuronic acid were not inhibitory. Mannose
1-phosphate and GTP both inhibited competitively with K i values of 1.0 mM and 200 p ~ ,
respectively.
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Alginate biosynthesis by A. vinelandii
189
Table 2. Eflect of carbon source on alginate biosynfhesis under conditions of limited and
excess phosphate
To ensure that there was no growth associated with a carryover of sucrose, the organism was
subcultured twice on agar plates containing the relevant carbon source. Liquid cultures were inoculated
from these plates. All carbon sources were present at 20 g I-' and bacteria were harvested at the
cessation of growth.
Limited-phosphate medium
Excess-phosphate medium
I
Carbon source
Bacterial dry weight
(g 1-7
Alginate
(g I-')
3.0
2.2
2.0
1.8
0
0
Sucrose
Glucose
Fructose
Mannose
Sorbitol
Mannitol
Glycerol
Gluconate
Acetate
2.5
2-3
0-6
2- 1
1.7
f
3
A
Alginate
(g I-')
1.0
5-6
3-4
1.7
0.8
0.4
1.2
1-2
1-2
1-4
0.8
1.8
0.5
3.5
2-4
No growth
No growth
-
\
Bacterial dry weight
(g 1-9
5-0
0
4.1
3.9
3.4
5-2
-
Table 3. Eflect of carbon source and phosphate concentration in the growth medium on the
activities of the enzymes of alginate biosynthesis during the stationary phase of growth
The numbers of extracts prepared are shown in parentheses after the medium compositions: duplicate
assays were performed to within 5 % for each extract. The results show the range of activities
encountered.
Specific activity [nmol min-' (mg protein)-']
f
Enzyme
Glucokinase
Fructokinase
Mannokinase
Mannose isomerase
Phosphomannose isomerase
GDPmannose pyrophosphorylase
GDPmannose dehydrogenase
Alginate [g (g dry wt bacteria)-'
I
A
\
Sucrose:
Limitedphosphate (8)
Sucrose:
Excessphosphate (2)
Mannose:
Limitedphosphate (2)
Sorbitol:
Excessphosphate (3)
18-24
13-20
-
18-22
11-14
40
18
6-8
16-20
26-34
26-28
ND
ND
ND
-
8
6-8
4-6
2-5
3
2
1.5
ND
ND
ND
ND
ND
5-7
0-5
0
0
ND, Not detected: -, not performed.
DISCUSSION
Sutherland (1 977, 1979) has discussed potential control mechanisms for exopolysaccharide
biosynthesis in a variety of organisms. Some of these are pertinent in interpreting our results
for alginate biosynthesis, namely possible competition for essential intermediates involved
both in cell wall biosynthesis and exopolysaccharide biosynthesis, control at the enzyme level
by feedback inhibition, and induction or derepression of enzyme synthesis.
Common intermediates suggested by Sutherland include GDPmannose and the isoprenoid
lipids. D-Mannose is widely distributed in bacterial lipopolysaccharides (Wilkinson, 1977) and
is incorporated from the nucleotide sugar GDPmannose, thus requiring constitutive
precursor-forming enzymes (phosphomannose isomerase and GDPmannose pyrophosphorylase). Our findings that the organism grows adequately without both of these enzymes suggest
that A. vinelandii lipopolysaccharide lacks mannose, an observation which correlates with
Olins & Warner's (1967) identification of glucose, a 2-keto-3-deoxy sugar, ribose, rhamnose
and hexosamine, but not mannose, in the cell wall lipopolysaccharide of A . vinelandii
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190
N. J. HORAN, T. R. JARMAN A N D E. A. D A W E S
ATCC 9 104. Certainly with many other Gram-negative organisms, precursor-forming
enzymes are constitutive, whether exopolysaccharides are formed or not. Norval &
Sutherland (1973) examined enzymes involved in the synthesis of precursors for exopolysaccharide formation in mucoid wild-type and non-mucoid mutants of Klebsiella aerogenes.
Loss of ability to form exopolysaccharide had little effect on the activity of the enzymes
tested. Recently Williams & Wimpenny (1980) reported that in Pseudomonas NCIB 11264,
specific activities of enzymes involved in exopolysaccharide precursor formation did not
reflect either the amount of exopolysaccharide produced, or the rate at which it incorporated
glucose.
The marked increase in alginate biosynthesis observed at the cessation of growth of the
mutant with enhanced ability to synthesize alginate is in contrast to the finding of Deavin et
a/. ( 1977) that in the wild-type organism alginate production paralleled growth and did not
continue when growth stopped. Sutherland (1977) proposed that such increases in
exopolysaccharide synthesis on cessation of growth could be due to the release of isoprenoid
cofactors, used preferentially for peptidoglycan and lipopolysaccharide synthesis during
growth, for exopolysaccharide synthesis. The involvement of polyisoprenoid cofactors in
alginate synthesis is not proven, however. Scott (1979), using bacitracin (an inhibitor of
isoprenoid lipid dephosphorylation), isotopic intermediates and column chromatography,
demonstrated the involvement of such lipids in peptidoglycan and lipopoly saccharide
synthesis in A . vinelandii but could not obtain evidence for their involvement in alginate
synthesis.
Phenotype rather than genotypic changes account for the observed differences in alginate
production by A . uinelandii, since organisms grown on mannose or sorbitol and high
phosphate resume normal alginate production, after a short lag on being transferred to the
sucrose limited-phosphate medium, by derepression of enzymes involved in its biosynthesis. A
similar situation was found with colanic acid synthesis in Escherichia coli K12 (Grant et al.,
1970; reviewed by Markovitz, 1977). Certain non-mucoid strains of this organism become
mucoid when grown in the presence of p-fluorophenylalanine. The non-mucoid strains have
repressed (but detectable) levels of enzymes responsible for colanic acid formation. These
enzymes are controlled by regulator genes, mutation in which leads to derepression and
increased colanic acid synthesis.
We conclude that the biosynthesis of alginate is controlled through the specific activities of
the enzymes involved in its biosynthesis. These are regulated by repression and derepression
by an unknown mechanism. Regulation through feedback inhibition appears to be
insignificant. although GDPmannose pyrophosphorylase is subject to feedback regulation by
GDPmannose in some organisms (Kornfield & Ginsburg, 1966). Attempts are being made to
stabilize and purify this enzyme from A . vinelandii to determine if control of alginate synthesis
might also be exerted by this means.
We thank the Science Research Council for the award of a CASE studentship to N. J . H.
REFERENCES
ANDERSON.R. L. & SAPICO,V. L. (1975). D-Fructose
(D-mannose) kinase. Methods in Enzynology 42.
39-43.
CARTER,
I. S. & DAWES.
E. A. ( 1979). Effect of oxygen
concentration and growth rate on glucose metabolism. poly-phydroxybutyrate biosynthesis and respiration of Azotobacrer beijerinckii. Journal of
General Microbiology I 10. 393-400.
CLARKE,D. J . & MORRIS.J. G. (1976). Partial
purification of a dicyclohexylcarbodiimide-sensitive
membrane adenosine triphosphatase complex from
the obligately anaerobic bacterium Clostridium
pasteurianum. Biochemical Journal 154,725-729.
COUPERWHITE,
I. & MCCALLUM,M. F. (1975).
Polysaccharide production and the possible occurrence of GDP-D-mannose dehydrogenase in
Azorobacrer vinelandii. A ntonie van Leeuwenhoek
48.25-32.
DAWES.E. A. & SENIOR,
P. J. (1973). The role and
regulation of energy reserve polymers in microorganisms. Advances in Microbial Ph.vsioIogy 10,
135-266.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 18:30:31
Alginate biosynthesis by A . vinelandii
19 1
of transport of glucose and methyl a-glucoside in
DEAVIN,L. (1976). The production of polysaccharide
Pseudomonas aeruginosa. Biochemical Journal 132,
by Azotobacter vinelandii. Ph.D. thesis, Brunel
141-154.
University, U.K.
A. (1962). GDPM pyrophosDEAVIN, L., JARMAN,T. R., LAWSON,C. J., MUNCH-PETERSEN,
RIGHELATO,
R. C. & SLOCOMBE,
S. (1977). The
phorylase. Methods in Enzymology 5, 17 1-1 74.
production of alginic acid by Azotobacter vinelandii NORVAL,M. & SUTHERLAND,
I. W. (1973). The
in batch and continuous culture. In Extracellular
production of enzymes involved in exopolyMicrobial Polysaccharides, pp. 14-26. Edited by P.
saccharide synthesis in Klebsiella aerogenes, Types
A. Sandford & A. Laskin. Washington: American
1 and 8. European Journal of Biochemistry 35,
Chemical Society.
209-2 15.
DOGGETT, R. G. & HARRISON,G. M. (1969). OLINS, A. L. & WARNER, R. C. (1967).
Significance of the pulmonary flow associated with
Physicochemical studies on a lipopolysaccharide
chronic pulmonary disease in cystic fibrosis.
from the cell wall of Azotobacter vinelandii. Journal
Proceedings of the 5th International Cystic Fibrosis
of Biological Chemistry 242,4994-500 1.
Conference, pp. 175-188. Edited by D. Lawson. PINDAR,D. F. & BUCKE,C. (1975). The biosynthesis
London: Cambridge University Press.
of alginic acid by Azotobacter vinelandii. Biochemical Journal 152,617-622.
DOUDOROFF,M. (1962). Mannose isomerase of
Pseudomonas saccharophila. Methods in En- PREISS,J. ( 1966). GDP-mannose pyrophosphorylase
zymology 5,335-338.
from Arthrobacter. Methods in Enzymology 8,
GORIN, P. A. J. & SPENCER,J. F. T. (1966).
271-275.
Exocellular alginic acid from Azotobacter vinelandii. SCOTT,G. (1979). Glycosyl transfer mechanisms in the
Canadian Journal of Chemistry 44,993-998.
biosynthesis of alginic acid. Ph.D. thesis, University
GRACY,R. W. & NOLTMANN,
E. A. (1968). Studies on
of Nottingham, U.K.
phosphomannose isomerase. Journal of Biological SUTHERLAND,
I. W. (1977). Microbial exopolysaccharide synthesis. In Extracellular Microbial
Chemistry 243,3 16 1-3 168.
Polysaccharides, pp. 40-57. Edited by P. A.
GRANT,W. D., SUTHERLAND,
I. W. & WILKINSON,
J.
F. (1970). Control of colanic acid synthesis. Journal
Sandford & A. Laskin. Washington: American
Chemical Society.
of Bacteriology 103, 89-96.
JARMAN,
T. R. (1979). Bacterial alginate synthesis. In. SUTHERLAND,1. W. (1979). Microbial exopolyMicrobial Polysaccharides and Polysaccharases, pp.
saccharides: control of synthesis and acylation. In
35-50. Edited by R. C. W. Berkeley, G. W. Gooday
Microbial Polysaccharides and Polysaccharases, pp.
& D. C. Ellwood. London: Academic Press.
1-34. Edited by R. C. W. Berkeley, G. W. Gooday
& D. C. Ellwood. London: Academic Press.
JARMAN,T. R., DEAVIN, L., SLOCOMBE,S. &
RIGHELATO,
R. C. (1978). Investigation of the effect SWEELEY,
C. C., BENTLEY,
R., MAKITA,M. & WELLS,
of environmental conditions on the rate of exopolyW. W. (1963). Gas-liquid chromatography of
trimethylsilyl derivatives of sugars and related
saccharide synthesis in Azotobacter vinelandii.
substances. Journal of the American Chemical
Journal of General Microbiology 107, 59-64.
Society 85, 2497-2507.
KORNFIELD,
R. H. & GINSBURG,
V. (1966). Control of
synthesis of guanosine 5'-diphosphate D-mannose WILKINSON,
S. G. (1977). Composition and structure
and guanosine 5 '-diphosphate Dfucose in bacteria.
of bacterial lipopolysaccharides. In Surface CarBiochimica et biophysica acta 117, 79-87.
bohydrates of the Prokaryotic Cell, pp. 97-175.
LAW, J. H. & SLEPECKY,R. A. (1961). Assay of
Edited by 1. W. Sutherland. London: Academic
poly-&hydroxybutyric acid. Journal of Bacteriology
Press.
8533-36.
WILLIAMS,A. G. & WIMPENNY,
J. W. T. (1980).
MARKOVITZ,
A. (1977). Genetics and regulation of
Extracellular polysaccharide biosynthesis by
bacterial capsular polysaccharide biosynthesis and
Pseudomonas NCIB 11264. Studies on precursorradiation sensitivity. In Surface Carbohydrates of
forming enzymes and factors affecting exopolysaccharide production by washed suspensions.
the Prokaryotic Cell, pp. 415-482. Edited by I. W.
Journal of General Microbiology 116, 133-141.
Sutherland. London: Academic Press.
MIDGLEY,
M. & DAWES,E. A. (1973). The regulation
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