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Cashel, M. (1969) J. Biol. Chem. 244, 3133-3141
Cooper, S. & Helmstetter, C. E. (1968) J. Mol. Biol. 31, 519-540
Donachie, W. D. (1968) Nature (London) 219, 1077-1079
Doolittle, W. F. (1972) J. Bacteriol. 111, 316-324
Doolittle, W. F. (1973) J. Bacteriol. 113, 1256-1263
Grierson, D. & Smith, H. (1973) Eur. J. Biochem. 36, 280-285
Herdman, M., Faulkner, B. M. & Carr, N. G. (1970) Arch. Mikrobiol. 73, 238-249
Leach, C. K. & Cam, N. G. (1974)J. Gen. Microbiol. 81, 47-58
Mann, N. & Carr, N. G. (1974) J. Gen. Microbiol. 83, 399-405
Mann, N., Carr, N. G. & Midgley, J. E. M. (1975) Biochim. Biophys. Acta in the press
Pigott, G. H. & Carr, N. G. (1971) Arch. Mikrobiol. 79, 1-6
Pritchard, R. H., Barth, P. T. & Collins, J. (1969) Symp. SOC.
Gen. Microbiol. 19,263-297
Schaechter, M., Maabe, 0. & Kjeldgaard, N. U. (1958) J. Gen. Microbiol. 19,592-606
Seitz, U. & Seitz, U. (1973) Arch. Mikrobiol. 90, 213-222
Szalay, A., Munsche, D., Wollgiehn, R. & Parthier, B. (1972) Biochem. J. 129, 135-140
Travers, A., Kamen, R. & Cashel, M. (1970) Coldspring Harbor Symp. Quant. Biol. 35,383-390
CO MMUNICAT10NS
Dark-Light Transitions with a Heterotrophic Culture of a Blue-Green Alga
E. HILARY EVANS and NOEL G. CARR
Department of Biochemistry, University of Liverpool, P.O. Box 147,
Liverpool L69 3BX,
U.K.
Although a significant number of blue-green algae are capable of heterotrophic growth
in the dark (Khoja & Whitton, 1971), our knowledge of such metabolism is limited, and
in particular very little is known of the mechanism by which phototrophic growth is
re-established. Chlorogloea fritschii Mitra, probably incorrectly named (Bourrelly,
1970), can grow heterotropically on sugars, with sucrose as the favoured substrate
(Fay, 1965). Dark growth led to substantial loss of the accessory photosynthetic pigment
phycocyanin (Fay, 1965; Lex etal., 1974) and thecapacity for light-induced O2evolution
was lost (Lex et al., 1974; Evans & Carr, 1974). On re-introduction into light the ability
to evolve O2was regained rapidly with a half-recovery time of about 13h (Evans & Carr,
1974). There was a 2-3h lag before any increase was seen in the ratio of chlorophyll to
protein.
Sucrose was assimilated by both dark- and light-grown Chlorogloea fritschii but at a
much greater rate in the light (Fay, 1965). Since sugar uptake has been suggested to be a
measure of photophosphorylation in algal cells (see Simonis & Urbach, 1973), this result
may reflect the increased capacity for ATP production by Cfilorogloenfritschii on transference into light. There was a 2 h lag before the sucrose uptake, after transfer from dark
to light, began to recover. The present paper investigates the effect of the protein-synthesis inhibitor chioramphenicol on the re-acquisition of light-induced O2evolution and
the rate of sucrose uptake.
Methods
Chlorogloea fritschii was grown at 32-34°C in medium C described by Kratz &
Myers (1955), supplemented with l0mM-sucrose, in total darkness, for 5-6 weeks. The
culture employed had been maintained heterotrophically in the dark for over 3 years.
Dark-to-light transitions involved transfer of dark-grown cultures to an illuminated
shaker with fluorescent lights (Grolux type) and maintained at 34°C. [U-14C]Sucrose
uptake was measured as trichloroacetic acid-precipitable material. O2 evolution was
measured with a Rank electrode, and chlorophyll was estimated at Essoafter extraction
of a ultrasonically broken suspension.
VOl. 3
BIOCHEMICAL SOCIETY TRANSACTIONS
3 74
400
-0"
D
a
o 300
8
h
0
8
8a
-s
200
0
?3
v
U
.-0
Y
3
100
?i
6
0
I
I
I
2
4
6
Time (h)
Fig. 1. Increase of O2 evolufion by Chlorogloeafritschii a/ier dark-light transition
A 4ml culture (cell density approx. 3mg/ml) was used. e, No additions;
chloramphenicol/ml.
0 , +20pg
of
Results and discussion
Fig. 1 shows the recovery of light-induced O2 uptake by dark-grown Chforogloea
frirschii on reintroduction into light, in the presence and in the absence of chloramphenicol (20pglml). The very low initial evolution of O2 increased at the same rate
for the f m t 30min in each case.
The culture containing chloramphenicol showed a lower rate of increase for the following hour, when it reached a steady rate, which was maintained for some hours. The
control experiment showed a greater increase in rate, after the first 30min, reaching a
higher steady value after about 4h. The introduction of chloramphenicol (20pglml)
immediately stops the incorporation of [U-14C]leucine into trichloroacetic acidinsoluble material.
These results suggest that at least some of the photosynthetic apparatus concerned
with Photosystem 2 is present in dark-grown Chlorogloeafritschii despite long-term
maintenance under heterotrophic dark conditions. This is in accord with the findings of
Cheniae & Martin (1973) with Chlorella. They suggested that Photosystem 2 requires
photoactivation, but that all the components are present in dark-grown cells.
However, the rate of O2 evolution in Chlorogloeajkitschii recovered to only half the
control value in the presence of chloramphenicol. This suggests that, if Photosystem 2
was intact, there was some inhibition in photosynthetic reactions requiring protein
synthesis de novo for full efficiency. Support for this view comes from examination of the
recovery of the light-induced rate of sucrose uptake (Fig. 2). In the presence of chloramphenicol there was no recovery and the culture maintained the dark rate of entry for
several hours. This suggests that, if sucrose uptake is a measure of ATP production by
1975
555th ;MEETING, ABERYSTWYTH
0
2
4
375
b
Time (h)
Fig. 2. Photoenhancement of [ U-14C]sucroseuptake by Chlorogloea fritschii a f e r darklight transition
A l m l culture was treated with trichloroacetic acid (5%), and the precipitate was
filtered off and its I4Cradioactivity counted. 8 , no additions in the light; o, no additions
in the dark; A , +20pg of chloramphenicol/ml in the light.
Chlorogloea fritschii, the efficiency of photophosphoryiation was very low despite 02evolving capacity. Glucose uptake in the presence of 3-(3,4-dichlorophenyl)-l,I-dimethylurea has also been used as a measure of cyclic photophosphorylation in green
algae (Senger, 1970). 3-(3,4-Dichlorophenyl)-l,l-dimethylurea (200fiM)does not inhibit
the recovery of the light-induced sucrose uptake in Chlorogloea fritschii. Thus this process may reflect the recovery of Photosystem 1.
The present data suggest that a component for light-induced sucrose uptake, but not
O2evolution, requires protein synthesis after transference of Chlorogloea fritschii from
dark to light. Because light-enhanced sucrose uptake is 3-(3,4-dichlorophenyl)-J ,1dimethylurea-insensitive,it may be that a component of Photosystem 1 is inactive. This
contrasts with the findings that higher-algal chloroplasts such as those of Chlorella,
Chlamydomonas reinhardii and Scenedesmus, when grown in the dark, make all components necessary for recovery of light growth (Myers, 1940; Senger & Bishop, 1969;
Schor et al., 1970). Mutants of Chlamydomonas reinhardii, however, were shown to
acquire the photosynthetic apparatus by a stepwise process (Hoober et al., 1969), and
this behaviour is also shown by higher-plant chloroplasts (reviewed by Kirk, 1971).
Bourrelly, P. (1970) Les Algues d'Eau Douce 111: Les AIgues Bleues et Rouges, les Eugleniens
Peridiniens et Cryptomonadines, N. Boubee et Cie., Paris
Cheniae, G . M. & Martin, I. F. (1973) Photochem. Photobiol. 17,441-459
Evans, E. H. & Carr, N. G. (1914) Proc. Int. Congr. Photosynth., 3rd, 1861-1866
Vol.' 3
376
BIOCHEMICAL SOCIETY TRANSACTIONS
Fay, P. (1965) J. Gen. Microbiol. 39, 11-20
Hoober, J. K., Siekevitz, P. & Palade, G. E. (1969) J. Biol. Chem. 244,2621-2631
Khoja, T. & Whitton, B. A. (1971) Arch. Mikrobiol. 79, 280-282
Kirk, J. T. 0. (1971) Annu. Rev. Biochem. 40, 160-196
Kratz, W.A. & Myers, J. (1955) Amer. J. Bot. 42, 282-287
Lex, M., Dickson, A. E. & Carr, N. G. (1974) Brit. Phycol. J. 9,221
Myers, J. (1940) Plant Physiol. 15, 575-588
Schor, S., Siekevitz, P. & Palade, G. E. (1970) Proc. Nat. Acad. Sci. US.66, 174-180
Senger, H. (1970) PZantu 92,327-332
Senger, H. & Bishop, N. I. (1969) Nature (London) 221,975-979
Simonis, W.& Urbach, W. (1973) Annu. Rev. Plant Physiol. 24, 89-114
The Occurrence of the Pyruvate Dehydrogenase Complex and the
Pyruvate-Ferredoxin Oxidoreductase in Blue-Green Bacteria
HERMANN BOTHE
Ruhr- Universitit Bochum, Lehrstuhl Biochemie der PJlanzen,
0-4630Bochum, Postfach 2148, German Federal Republic
Two different reactions are known for the formation of acetyl-CoA and COz from pyruvate and CoA. One is catalysed by the multienzyme pyruvate dehydrogenase complex.
The role of the cofactors involved in this system, thiamin pyrophosphate, lipoic acid,
FAD and NAD+, is well established. The other oxidative decarboxylation of pyruvate is
catalysed by the pyruvateferredoxin oxidoreductase, which has mainly been described
in anaerobic bacteria. In this case the remaining electrons are transferred not to NAD+
but to ferredoxin.
In blue-green bacteria the nature of the enzyme systems that catalyse the decarboxylation of keto acids with concomitant formation of acyl-coenzymes is uncertain. The
pyruvate dehydrogenase complex has not yet been demonstrated [cf. Smith (1973),
Tables 1 and 21. Recently a ferredoxin-dependent decarboxylation of pyruvate was
demonstrated in Anabaena variabilis (Leach & Carr, 1971) and Anabaena cylindrica
(Bothe & Falkenberg, 1973). Extracts from Anabaenacylindricaalsocatalysethe synthesis
of pyruvate from COz, acetyl-CoA and reduced ferredoxin, and (with a high rate) the
exchange reaction between COz and the carboxyl group of pyruvate. Thus all the reactions typical of the pyruvateferredoxin oxidoreductase have been demonstrated in
Anabaena cylindrica.
The activity of the pyruvateferredoxin oxidoreductase is about fivefold higher in
Anabaena grown on NO3- or atmospheric Nz as compared with cells grown on NH3.
From this it was concluded that one physiological function of this enzyme is probably
to provide reduced ferredoxin for the reduction of NO3- or Nz to NH3 (Bothe et a/.,
1974).
The pyruvate dehydrogenase complex, on the other hand, is present in the blue-green
bacterium Anacystis nidulans. Extracts from this organism catalyse a pyruvate- and
CoA-dependent formation of NADH that is completely blocked by 5mM-arsenite. It
was also found in Ankistrodesmus braunii, Chlamydomonas reinhardtii, Micrococcus
denitriJcans, Azotobacter vinelandii and Escherichia coli. In contrast, the tests were
negative for Anabaena cylindrica, Clostridium pasteurianum, Clostridium kluyveri and
Chlorobium thiosulfatophilum, which cleave pyruvate and CoA to acetyl-CoA and COz
probably exclusively via the pyruvate-ferredoxin oxidoreductase.
The conclusions from the enzymic determinations are supported by the results of a
microbiological test on lipoic acid done with a strain of Streptococcus faecalis deficient
in this coenzyme (Bothe & Nolteernsting, 1975). High concentrations of lipoic acid
were found in Anacystis, Ankistrodesmus, Chlamydomonas, Micrococcus, Azotobacter
1975