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Journal of General Microbiology (1986), 132, 2601-2603.
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Metabolic Reactions Responsible for Glucose Stimulation of Alkaline
Phosphatase in Vibvio choIevae
By S . MITRA, A. GHOSH A N D R. K . GHOSH*
Indian Institute of Chemical Biology, Jadavpur, Calcutta 700 032, India
(Received 29 January 1986; revised 7 May 1986)
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Alkaline phosphatase activity in Vibrio cholerae strain 569B grown in low-phosphate medium
was stimulated if glucose or glycerol was used as the carbon source. No such stimulation was
observed, however, if tricarboxylic acid cycle intermediates like succinate or citrate were used.
Experiments using specific enzyme inhibitors strongly indicated that the metabolic reactions of
the glycolytic pathway from glyceraldehyde 3-phosphate to 2-phosphoglycerateplay a key role
in the stimulation process.
INTRODUCTION
Alkaline phosphatase (APase) of Vibrio cholerae is different from that found in other bacteria
in that it is monomeric (Roy et al., 1 9 8 2 ~ )However,
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like the enzymes found in other organisms,
its synthesisis repressible by phosphate. Derepression can be achieved by loweringthe phosphate
content of the growth medium. The presence of glucose in low-phosphate medium (LP)
stimulatesenzyme synthesis further (Roy et al., 19823). Similar observations have been reported
for the Gram-positive organisms Bacillus subtilis (Ghosh & Ghosh, 1972) and Bacillus
licheniformis (Hydrean et al., 1977). The mechanism of glucose stimulation, however, has
remained obscure. We show here that the metabolic reactions of glycolysisfrom glyceraldehyde
3-phosphate to 2-phosphoglycerate play a major role.
METHODS
Organism andgrowth media. Vibrio cholerae Inaba 569B strain used in this study was obtained from the Cholera
Research Centre, Calcutta, India. Cultures were stored and maintained under the conditions described by Roy et
al. (19826).
Cultures were grown at 37 "C in LP (phosphate-depletednutrient broth) as described by Roy et al. (19826).In
some experimentsglucose or other intermediatesof carbohydratemetabolism were added. Growth was assayed by
measuring the OD540 using a Gilford model 250 spectrophotometer. (An
of 1-0 corresponded to 1.2 x lo9
cells ml-* .)
Growth conditwns. Cells were grown in 40 ml LP for 18 h with shaking (180 r.p.m.) at 37 "C and then used to
inoculate 100 ml LP medium in a 500 ml Erlenmeyer flask to give an initial ODS40of 0.12. The suspension was
incubated at 37 "C with shaking (180 r.p.m.) and samples were removed at appropriate intervals for the assay of
enzyme activity and growth.
APase assay. APase activity in whole or toluene-treated cells was assayed as described by Roy et al. (1982b).
RESULTS A N D DISCUSSION
Derepression of APase synthesis in the presence of diflerent carbon sources
In order to see if carbon sources other than glucose would stimulate APase in V . cholerae, we
measured APase activity in LP in the presence of different carbon sources. Different degrees of
stimulation were obtained with different carbon sources (Fig. 1). APase activity was four times
Abbreviatwns: LP, low-phosphate medium; APase, alkaline phosphatase.
0001-3235 @ 1986 SGM
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2602
S. M I T R A , A . GHOSH AND R. K. GHOSH
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Fig. 1. APase activity in V .choleruestrain 569B. Cells were grown in LP (O), LP 2% (w/v) succinate
2% (w/v) citrate (o),
LP
1% (v/v) glycerol (i) and LP
0.1% (w/v) glucose (a).
(a),
LP
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h
5
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800
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n"
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x
0.2
%
v
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600
U
(I)
-a
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400
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cd
%
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200
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Time (h)
Fig. 2. Effect of various inhibitors of glycolysis on growth (inset) and APase synthesis in V . cholerue
strain 569B. Cells were grown in LP
glucose (a) or LP (b) for 3 h and then inhibitors were added
(arrows). Samples were withdrawn at different times to monitor growth and APase activity. 0 ,LP
0.1 % (w/v) glucose; 0, LP alone; A, LP
glucose 2.5 mM-potassium fluoride or LP alone 2.5
mM-potassium fluoride; 0 , LP + glucose
0.075 mM-iodoacetate or LP alone
0.075 mM
iodoacetate.
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higher in cells grown in LP glucose compared to those grown in LP alone, and growth in LP
glycerol caused a 2.5-fold enhancement. No stimulation of enzyme activity was obtained when
succinate or citrate was the carbon source.
Involvement of glycolytic pathway in the glucose derepression of APase
The above experiments indicated that glucose or glycerol, which are metabolized through the
glycolyticpathway, stimulated APase under derepression conditions, whereas tricarboxylic acid
cycle intermediates did not. Thus it appeared that the metabolic reactions of glycolysis were
somehow involved in the observed stimulation. Further, since glycerol enters the glycolytic
pathway through the formation of glyceraldehyde 3-phosphate7 it can be postulated that
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2603
Alkaline phosphatase of Vibrio cholerae
metabolic reactions subsequent to this step were involved in the stimulation. In order to identify
the metabolic reactions responsible, specific inhibitors were added to cultures grown in LP or
LP glucose, and APase activity was monitored. The addition of 2.5 mM-potassium fluoride,
which inhibits enolase in vivo (Kanapka & Hamilton, 1971) and is also active against V . cholerae
enolase in uitro (our unpublished observation), had no effect on the derepression of APase
activity by glucose. However, derepression was substantially reduced in the presence of 0.75
mM-iodoacetate(Fig. 2a) an inhibitor of glyceraldehyde-3-phosphatedehydrogenase (Harris &
Waters, 1976). Similar observations were made with cells grown in LP
glycerol (data not
shown), but the inhibitors had no effect when cells were grown in LP (Fig. 2b). Thus it seemed
reasonable to conclude that the metabolic reactions of the glycolytic pathway from
glyceraldehyde 3-phosphate to 2-phosphoglyceric acid play, in an as yet undetermined way, a
major role in the stimulation of APase activity by glucose in V . cholerae.
One criticism of the above experiments is that iodoacetate, a general -SH inhibitor, could
inhibit enzymes other than triose phosphate dehydrogenase. However, the observation that
iodoacetate at the concentration used had no effect on cell growth (Fig. 2 inset), or on APase
activity when V . cholerae was grown in the absence of glucose, effectively rules out this
possibility.
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REFERENCES
GHOSH,A. & GHOSH,B. K. (1972). Alkaline phosphatase derepression in vegetative cells of Bacillus
subtilis by glucose and its reversal by lactate.
Biochemical and Biophysical Research Communications 46,296-304.
HARRIS,J. I. &WATERS,M.(1976). Glyceraldehyde-3phosphate dehydrogenase. In The Enzymes, vol. 13,
part C, pp. 1-49. Edited by P. D. Boyer. New York:
Academic Press.
HYDREAN,
C., GHOSH,
A., NALLIN,M. & GHOSH,
B. K.
(1977). Interrelationship of carbohydrate metabolism and alkaline phosphatase synthesis in Bacillus
lichen$ormis 749/C. Journal of Biological Chemistry
252, 68064812.
KANAPKA,
J. A. & HAMILTON,
I. R. (1971). Fluoride
inhibition of enolase activity in vim and its relationship to the inhibition of glucose 6-phosphate
formation in Streptococcus salivarius. Archives of
Biochemistry and Biophysics 146, 167-1 74.
ROY, N. K., GHOSH,R. K. & DAS, J. (1982a).
Monomeric alkaline phosphatase of Vibrio cholerae.
Journal of Bacteriology 150, 1033-1039.
J. (19826).
ROY, N. K., GHOSH,R. K. & DAS,
Repression of the alkaline phosphatase of Vibrio
cholerae. Journal of General Microbiology 120, 349353.
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