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FEMS MicrobiologyLetters, 4 (1978) 261-264
©Copyright Federation of European MicrobiologicalSocieties
Pubfishedby Elsevier/North-HollandBiomedical Press
261
CYANOBACTERIA GROWN U N D E R P H O T O A U T O T R O P H I C , P H O T O H E T E R O T R O P H I C ,
AND H E T E R O T R O P H I C REGIMES: S U G A R METABOLISM AND CARBON DIOXIDE
FIXATION
F. JOSET-ESPARDELLIER, C. ASTIER, E.H. EVANS * and N.G. CARR *
Laboratoire de Photosynth~se, Centre National de la Recherche Scientiflque, 91 Gif sur Yvette, France, and
• Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, U.K.
Received 19 July 1978
1. Introduction
Whilst all cyanobacteria are capable of photoautotrophic growth, species vary with respect to their
photoheterotrophic and heterotrophic capacities. The
entry of reduced carbon molecules may be shown [1]
although it is noteworthy that only sugars support
photoheterotrophy, as demonstrated by growth in
the presence of DCMU [2] and that sugars are the
substrates for the relatively few cases of heterotrophic growth. Several reasons for this restriction in
cyanobacteria of heterotrophic metabolism have been
suggested, usually with the implicit assumption that
a unitary cause does exist. The transport of organic
materials into autotrophic prokaryotes has recently
been reviewed by Smith and Hoare [3] and the lack
of repression and derepression of synthesis of
enzymes involved in carbon metabolism of obligately
phototrophic cyanobacteria is extensively documented [4]. This paper examines some aspects of
sugar metabolism in two facultative cyanobacteria,
after growth under photoautotrophic, photoheterotrophic and heterotrophic conditions.
2. Methods
2.1. Organisms and growth
Aphanocapsa sp. was strain 6714 from Prof. R.Y.
Stanier's collection and is now deposited in the Ame-
Abbreviation: DCMU,dichlorophenyl dimethylurea.
rican Type Culture Collection as No. 27178; this was
grown on Aliens medium [5] with twice the stated
amount of nitrate and in the presence or absence of
glucose (0.2% w/v) at 34°C. Chlorogloeafritschii,
Culture Centre of Algae and Protozoa, Cambridge,
No. 1411/la, was grown on medium C [6] with and
without sucrose (0.1 M) at 34°C.
All photoautotrophic and photoheterotrophic
cultures were grown in an orbital shaker with illumination approx. 5000 lux and the gas phase enriched
with CO2. Heterotrophic cultures of C. fritschii were
wrapped with foil to exclude light and maintained
and grown at 34°C without agitation. Aphanocapsa
6714 was transferred from photoautotrophic to
heterotrophic conditions ten generations prior to
enzymic analysis.
2.2. Enzyme assays
A harvested, washed suspension of organisms was
resuspended in 0.5 M phosphate buffer pH 7.0 and
disrupted by extrusion through a chilled French
pressure cell at 10 tons psi. Unbroken cells and debris
were removed by centrifugation at 14 000 rev./min
for 30 rain.
Protein concentration in the supernatant was measured as described [7]. Acetothiokinase, isocitrate
dehydrogenase, malate dehydrogenase [7], ~,-oxoglutarate dehydrogenase [8], glucose-6-phosphate dehydrogenase, gluconate-6-phosphate dehydrogenase,
fructose-6-phosphate isomerase, glyceraldehyde-3phosphate dehydrogenase, fructose diphosphate aldolase [9], glucose dehydrogenase [10], NADH oxidase
262
[ 11 ], and ribulose biphosphate carboxylase [ 12]
were measured as previously described.
3. Results and Discussion
Although the heterotrophic growth rate of both
organisms was considerably slower than that obtained
under phototrophic conditions, there were only
minor alterations in the activities of enzymes involved
in sugar and carboxylic acid metabolism (Table I).
Desalting of the extracts of Aphanocapsa 6714 on
Sephadex G25 caused little changes in enzyme activity. There was slight variation observed between
photoautotrophically and photoheterotrophicaUy
grown cultures.
The most significant alterations were the higher
activities of malate dehydrogenase, glucose-6-phos-
phate dehydrogenase and ribulose biphosphate carboxylase after heterotrophic growth of C fritschii. In
these conditions, the organisms contain less than 10%
the normal amount of phycocyanin, a photopigment
which accounts for up to 20% of the protein of
phototrophic cells. This reduction in a major protein
species could, in part, account for the increased specific activities observed.
When [14C]HCO3 was added to a dark heterotrophic culture of C fritschii, a steady rate of incorporation was observed, which was approx. 1% that
obtained with a photoheterotrophic culture. Presumably the supply of NADPH and/or ATP was limiting
the activity of the ribulose biphosphate carboxylase,
The rate of incorporation of [14C]HCO3 appears to
be, at least in part, via ribulose biphosphate carboxylase, in that phosphoglyceric acid is an early product
of incorporation (Fig. 2).
TABLE 1
Enzyme activity after growth under different regimes
Activities expressed are the mean of several determinations.
Enzyme (nmol/mg/min)
Generation time (h)
+10%
(a) Chlorogloeafritschii
Growth
Photoautotrophic
30
Malate dehydrogenase
Photoheterotrophic
25
2.6
Isocitrate dehydrogenase
Glucose-6-phosphate
dehydrogenase
(b) Aphanoeapsa 6 714
Growth
Heterotrophic
Photoautotrophie
Photoheterotrophic
Heterotrophic
80
6
6
27
2.9
16
16
l0
11
16.6
90
200
260
21
70
60
50
52
298
270
250
170
100
220
I000
640
230 b
180
I10
330
Gluconate-6-phosphate
dehydrogenase
Glyceraldehyde-3 -phosphate
dehydrogenase
-
-
108
171
Fructose-6-phosphate
-
-
-
2.4.103
1.8 • 103
8.0.10
68
isomerase
Fmctose-diphospate aldolase
Ribulose biphosphate
carboxylase a
Acetate thiokinase
31
a Expressed as dpm/mg protein/min.
b This assay was performed only once.
20
-
27
20
30
3
73
1.8.10 s
6.104
1.8.10 s
-
-
-
263
TIME AFTER TRANSFER
TO DARK (HOURS)
Fig. 1. Variation in malate dehydrogenase followed a lightdark shift. CMorogioen frischii. A photoheterotrophic
culture was transferred to darkness, maintained at 34’C and
aerated with air: COz (5 : 5, v/v). Aiiquots were taken at
time intervals and organisms disrupted and the enzyme measured as in Methods.
The increase in malate dehydrogenase activity in
dark grown C. ftitschii occurred within 12 h after
transfer from photoautotrophic to heterotrophic
growth, in a culture that had a mean generation time
of 30 h. When C. ftitschii was transferred from photoheterotrophic to dark heterotrophic growth conditions, a marked transient in malate dehydrogenase
activity was observed over the first 6 h; subsequently
an increased activity was established (Fig. 1). This
variation in malate dehydrogenase activity was observed after transfer of C fitschii to darkness in the pres.
ence of chloramphenicol(l0 &ml), indicating that
the alteration in this enzyme was due to activation
rather than alteration in rate of enzyme synthesis.
The slight increase in malate dehydrogenase activity seen in heterotrophic cells of Aphanucapsa 67 14
(Table I) has proven not to be significant, after a
kinetics experiment was performed. No change in
activity was detectable during the first two generations after transfer from photoautotrophy to heterotrophy. Glucose dehydrogenase was absent from
Aphanocupsa under all growth conditions; this
Fig. 2. Distribution of 14C 2 min after incorporation of
[ 14C]HCOs into dark, heterotrophic C?ilorogloeo ftitschiL
Isotope-containing compounds were separated by a twodimensional system of thin-layer electrophoresis and chromatography, as described by Schiirmann [ 161. Areas of the
autoradiogram were identified by co-chromatography with
authentic material in the same electrofluoretic and chromatographic system. 1, glutamine; 2, asparagine; 3, aspartate; 6,
glutamate; 7, phosphoglyceric acid; 11, phosphoenol pyruvate; 12, glucose&phosphate.
enzyme was observed by Pulich and Van Baalen [lo]
in extracts from two filamentous species of cyanobacteria capable of dark heterotrophic growth.
a-Oxoglutarate dehydrogenase was also absent, the
lack of a complete tricarboxylic acid cycle is consistent with other reports of cyanobacterial metabolism.
Since the observation of heterotrophic growth in
Aphanocapsa 67 14 by Rippka [ 131 some attention
has been paid to its assimilation of sugar and considerable evidence indicates that the oxidative pentose pathway is the route [ 141. The data presented
here show that the supply of exogenous glucose has
no appreciable effect on the synthesis of enzymes
concerned with its dissimilation. Likewise ribulose
biphosphate carboxylase is not repressed when
glucose is supplied exogenously and the organism
grown heterotrophically. The actual operation of the
Calvin cycle appears to continue at a very reduced
264
rate in dark heterotrophic conditions. This lack o f
regulation at the level of enzyme synthesis is consistent with observations on other obligately photoautotrophic cyanobacteria. The only difference
described up to now between obligate phototrophs
and Aphanocapsa 6714 is the existence of greater
transport rates o f D-glucose in the latter [ 15].
Acknowledgements
We are indebted to Miss Ann Dickson and Miss
Barbara Fuller for skilled assistance, and to the
Science Research Council for support.
References
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419-448.
[4] Carr, N.G. (1973) In: The Biology of Blue-Green Algae
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