Download Effect of growth condition on enzymes of the citric

Antonie van Leeuwenhoek 45 (1979) 521-529
521
Effect of growth condition on enzymes of the citric acid
cycle and the glyoxylate cycle in the photosynthetic
bacterium Rhodopseudomonaspalustris
J. H . ELEY 1, K . KNOBLOCH 2' 3 AND T . - W . H A N 2
Biology Department, University of Colorado,
Colorado Springs, Colorado 80907 and
2 T. H. Morgan School of Biological Sciences, University of Kentucky,
Lexington, Kentucky 40506, U.S.A.
ELEY, J. H., KNO~LOCH,K. and HAN, T.-W. 1979. Effect of growth condition on
enzymes of the citric acid cycle and the glyoxylate cycle in the photosynthetic
bacterium Rhodopseudomonaspalustris. Antonie van Leeuwenhoek 45" 521 529.
The enzymes of the citric acid and glyoxylate cycles as well as RuBP 4 carboxylase were measured in cell-free extracts from Rhodopseudomonas palustris after
growth under chemoheterotrophic, photoheterotrophic and photolithotrophic
conditions. Although the citric acid cycle was found to be complete under all
growth conditions, significant differences in certain enzyme activities occurred
as a function of the different energy sources applied. The glyoxylate cycle also
was complete under all growth conditions with highest isocitrate lyase activity
seen after photoheterotrophic growth on acetate. Photo- and chemoheterotrophic growth on malate reduced the isocitrate lyase. The activity was not
repressed further by photolithotrophic growth on thiosulfate. RuBP carboxylase activity, present under photolithotrophic conditions, was repressed by
chemoheterotrophic growth but was not decreased by the presence of organic
substrates during photoheterotrophic growth.
INTRODUCTION
Among the purple photosynthetic bacteria, the Chromatiaceae (Thiorhodaceae) are predominantly autotrophic w ~il~ the Rhodospirillaceae (Athiorhodaceae) are heterotrophic and typically grow aerobically in the dark (chemohet3Present address: Institut ffir Botanik u. PharmazeutischeBiologie,UniversitfitErlangenNfirnberg, Schlossgarten4, D-8520 Erlangen, Germany.
4Abbreviations: EDTA, ethylenediaminetetraacetic acid; GSH, reducedglutathione; RuBP, ribulose bisphosphate.
522
J. H. ELEY, K. KNOBLOCH AND T.-W. HAN
erotrophically) and anaerobically in the light (photoheterotrophically) on simple organic substrates.
In 1944, van Niel (1944) reported that Rhodopseudomonaspalustris is unique
among the Athiorhodaceae in its ability to oxidize the inorganic compound
thiosulfate during phototrophic growth. Rolls and Lindstrom (1967 a, b) added
thiosulfate to photoheterotrophically growing cultures of Rps. palustris and
observed increased cell yields suggesting that photolithotrophic metabolism of
thiosulfate occurred in the presence of the organic electron donors. In addition,
these experiments revealed that thiosulfate reduced the evolution of CO 2 from
pyruvate and therefore a refixation of the evolved CO 2 may be responsible for
the increased cell yields.
In cell-free extracts from photolithotrophically grown Rps. palustris, Knobloch, Eley and Aleem (1971) have characterized a thiosulfate-linked ATPdependent NAD + reduction which could provide the reducing power needed for
a refixation of CO z released from pyruvate.
Since previous studies provide little quantitative data on the comparative
biochemistry of Rps. palustris as a function of growth condition, the present
study was undertaken to provide quantitative measurements on enzyme activities of the citric acid and glyoxylate cycles and of RuBP carboxylase in extracts
of Rps. palustris after growth under chemoheterotrophic, photoheterotrophic
and photolithotrophic conditions.
MATERIALS AND METHODS
Growth conditions and preparation of extracts
Rhodopseudomonas patustris (ATCC 17001, C.B. van Niet 2.1.6) was grown
under three different conditions: (I) On malate (20 raM) or succinate (20 mM)
under aerobic conditions in the dark; (II) On oL-malate (20 m~), succinate (20
m~) or acetate (20 mM) under anaerobic conditions in the light; (III) On thiosulfate (20 m~) plus bicarbonate under anaerobic conditions in the light.
Anaerobic growth was in illuminated 10-liter carboys which were filled to
overflowing, stoppered and magnetically stirred. Illumination was provided by a
bank of 150-watt incandescent lamps providing 10000 lux at the culture surface.
Aerobic growth was in similar carboys with sterile air bubbled through the
suspension. All cultures were maintained at 30-35 ~
The basic medium used for all growth conditions was modified after CohenBazire, Sistrom and Stanier (1957) as described previously (Knobloch et al.,
t971).
Cells were collected after 2-3 days growth in a DeLaval continuous flow
centrifuge. After washing twice with 0.05 M Tris-HC1 (pH 8.0), the cells (5-10 g
wet weight) were suspended in 0.05 MTris-HC1 (pH 8.0) containing 5 m~ MgCl2,
0.5 m~ EDTA.Naz, and 0.5 mM GSH.The cells were disrupted by sonication for
CITRIC ACID AND GLYOXYLATECYCLEIN RPS. PALUSTRIS
523
a total time of 2 rain (Biosonik III at maximum output). After centrifugation at
10 000 • g for 30 min to remove cell debris, the supernatant was centrifuged at
40 000 x g for 60 min. The resultant supernatant was dialyzed for 12-15 h at 4'C
against 0.05 MTris-HC 1 (pH 8.0) containing 1.0 mM MgC 12, 0.1 mM EDTA'Na 2
and 0.1 mM GSH and used as the enzyme source.
Enzyme assay' procedures
Enzyme activities were determined spectrophotometrically on a Beckman
Kintrac VII spectrophotometer. Enzymes measured, methods of measurement
and millimolar extinction coefficients (EmM) used in calculations of specific
activities as nanomoles substrate converted per rain and per mg protein are given
below. Citrate synthase (EC 4.1.3.7) was assayed according to the method
described by Ochoa (1955), EmMof 4.5 for the thioester bond in acetyl CoA (230
nm); aconitate hydratase (EC 4.2.1.3) was assayed by the method of Anfinsen
(1955) in which the absorption ofcis-aconitic acid was followed, EmM of 4.88 for
aconitic acid (240 nm); isocitrate dehydrogenase (EC 1.1.1.42) was assayed by
the procedure of Sottocasa, Kuylenstierna, Ernster and Bergstrand (1967), EmM
of 6.22 ; oxoglutarate dehydrogenase (EC 1.2.4.2) was determined by the method
of Kaufman (1955), EmMof 6.22 at 340 nm; succinyl CoA synthetase (EC 6.2.1.5)
was measured by the method of Bridger, Ramaley and Boyer (1969), EmMof 4.5
at 230 nm; succinate dehydrogenase (EC 1.3.99.1) was assayed by the method of
King (1967) using phenazine methosulfate (PMS) coupled with 2,6-dichlorophenolindophenol (DCPIP) reduction at 600 nm, EmM of 20.6; fumarate hydratase
(EC 4.2.1.2) was determined by the procedure of Hill and Bradshaw (1969) in
which the increase in fumarate concentration was followed at 300 nm, EmM of
0.0335 ; and malate dehydrogenase (EC 1.1.1.37) was determined by the method
of Yoshida (1969), EmM of 6.22.
Other enzymes assayed were: isocitrate lyase (EC 4.1.3.1) by the method of
Olson (1959), EmMofglyoxylic semicarbazone at 252 nm of 12.4; malate synthase
(EC 4.1.3.2) by the method of Dixon and Kornberg (1962) based on the decrease
in absorbance at 232 nm consequent on breakage of the thioester bond of acetyl
CoA in the presence of glyoxylate; ribulose bisphosphate (RuBP) carboxylase
(EC 4. i. 1.39) was assayed by the radioactive procedure of Aleem and Huang
(1965).
Protein was determined by the biuret method of Gornall, Bardawill and David
(1949).
All biochemicals were obtained from Sigma Chemical Co., St. Louis, Missouri, U.S.A.
RESULTS
The data show that Rps. palustris has a complete citric acid and glyoxylic acid
4.1
9.8
192
2.5
199
41.6
220
1840
30
146
0
N.T. 2
7,9
189
2.2
200
62.3
t 56
1320
32
125
N.T.
6.6
29.5
327
3.9
49
5.4
206
434
2 t .6
25
5.3
6.1
12.3
180
3.0
51.6
2.4
169
366
26.9
24
N.T.
succinate
malate
malate
succinate
Photoheterotrophic
Chemoheterotrophic
Growth condition and substrate
7.3
2.6
220
5.5
N.T.
1.1
163
734
79.4
20.2
N.T.
acetate
Photolithotrophic
5.1
2.0
186
3.1
64
2.7
117
370
39.2
30
4.3
thiosulfate
Activities are expressed as nmoles per min and per m g protein. Values reported for specific activities are average values, determined for two to four
independently prepared extracts.
2 N.T. indicates not tested.
Citrate synthase
Aeonitate hydratase
Isocitrate dehydrogenase
Oxoglutarate dehydrogenase
Succinyl-CoA synthetase
Succinate dehydrogenase
Fumarate hydratase
Maiate dehydrogenase
Isocitrate lyase
Malate synthase
RuBP carboxylase
Enzyme
Table 1. Specific activities l of citric acid and glyoxylic acid cycle enzymes in cell-free extracts from Rps. palustris after growth under various conditions
7~
9
.~
t,J
CITRIC ACID AND GLYOXYLATE CYCLE IN RPS. PALUSTRIS
525
cycle under chemoheterotrophic, photoheterotrophic or photolithotrophic
growth conditions although quantitative differences do exist which depend upon
the conditions of growth as well as upon the substrates applied. (Table 1).
In general, it can be stated that highest specific activities of the citric acid cycle
enzymes were found during chemoheterotrophic growth and almost identical
activities were seen whether the substrate was malate or succinate. The activities
were generally lowest under photolithotrophic growth with only slightly higher
activities seen for several enzymes under photoheterotrophic conditions.
Citrate synthase had low activity under all growth conditions while aconitate
hydratase activity was also quite low except for an increase shown under photoheterotrophic growth. Isocitrate dehydrogenase activity was comparatively high
under all growth circumstances and was found to be NADP-specific.
The specific activity ofoxoglutarate dehydrogenase was the lowest of the citric
acid cycle enzymes under chemoheterotrophic growth and was consistently low
under all growth conditions applied.
After chemoheterotrophic growth, succinyl CoA synthetase and malate dehydrogenase (NAD-specific) showed a 4-fold increase, while succinate dehydrogenase revealed an even larger stimulation compared to the rates obtained from
light-grown cells.
Among the enzymes of the glyoxylate bypass, the isocitrate lyase activity was
found to be most active under photoheterotrophic growth on acetate. This
activity was reduced remarkably when acetate was replaced by malate or by
succinate in the light or in the dark. - Photolithotrophic growth on thiosulfate
resulted in an isocitrate lyase activity which was increased somewhat compared
to the rates obtained after growth on malate or succinate, but it did not reach the
isocitrate lyase activity obtained after photoheterotrophic growth on acetate.
The malate synthase activity in light-grown cells was found to be in the range
of the isocitrate lyase activity. Under chemoheterotrophic conditions in the
dark, however, its activity was increased about sixfold.
RuBP carboxylase activity in Rps. palustris could be demonstrated under
photolithotrophic growth on thiosulfate as well as under photoheterotrophic
growth conditions.
DISCUSSION
The operation of at least a portion of the citric acid cycle was demonstrated in
a number ofchemoautotrophic bacteria (Cooper, 1964; Johnson and Abraham,
1969; Charles, 1971) and in several members of the Chromatiaceae (Fuller et al.,
1961) and the Rhodospirillaceae (Morita, 1961; Ormerod and Gest, 1962).
Kornberg and Lascelles (1960) examined several Rhodospirillaceae after chemoand photoheterotrophic growth on malate or succinate and found the citric acid
cycle to be sufficient in providing both energy and carbon skeletons for growth.
526
J. H. ELEY, K. KNOBLOCH AND T.-W. HAN
The low activities of the citric acid cycle enzymes reported here for photolithotrophic growth conditions are consistent with data obtained on extracts
from several obligate autotrophs and facultative heterotrophs after growth
under autotrophic conditions (Smith, London and Stanier, 1967; Peeters, Liu
andAleem, 1970; Charles, 1971).
Smith et al. (1967) found that the facultative chemoautotroph Thiobacillus
intermedius lacks oxoglutarate dehydrogenase after autotrophic growth. On the
other hand, the organism contains the complete citric acid cycle when grown
heterotrophically. Studies with the facultative chemoautotrophs Thiobacillus
denitrificans and Thiobacillus novellus have shown that oxoglutarate dehydrogenase activity was lost completely or greatly reduced during autotrophic growth
(Charles, 1971 ; Peeters et al., 1970).
Our results show that Rps. palustris differs in its metabolism from the facultative heterotrophic Thiobacilli in not showing repression of oxoglutarate dehydrogenase activity during photolithotrophic growth. Rather, oxoglutarate dehydrogenase activity was present under all growth conditions but was of consistently low activity.
The glyoxylate cycle involves several enzymes in common with the citric acid
cycle and has two additional enzymes, isocitrate lyase and malate synthase, not
involved in the operation of the cycle. Previous studies suggest that members of
the Rhodospirillaceae differ in their use and formation of isocitrate lyase (Kornberg and Lascelles, 1960). Albers and Gottschalk (1976) have demonstrated
isocitrate lyase activities to be present after photoheterotrophic growth on acetate in five strains, including Rps. palustris (ATCC 17002), out of twelve Rhodospirillaceae tested. Likewise, Krasil'nikova et al. (1974) found isocitrate lyase
activity to be developed by Rps. palustris (strain Nakamura) after growth on
acetate either in the light or in the dark.
The same Rps. palustris strain, however, did not reveal isocitrate lyase activity
after growth on a (not specified) medium with thiosulfate and bicarbonate
(Krasil'nikova et al., 1974). In contrast, our data show that in Rps. palustris
photolithotrophic growth did not reduce isocitrate lyase activity; the enzymatic
rate was increased somewhat compared to its level during chemo- or photoheterotrophic growth on malate or succinate.
The second key enzyme of the glyoxylic acid cycle, malate synthase, has been
reported in extracts from several Rhodospirillaceae, including Rps. palustris
(van Niel 2.1.7 and 2.3.11) after growth under chemo- and photo-heterotrophic
conditions and therefore has been suggested as being constitutive in the Rhodospirillaceae (Kornberg and Lascelles, 1960). Our results show malate synthase
activity to be equal to that of isocitrate lyase under photoheterotrophic and
photolithotrophic growth conditions while its activity increases about 6-fold
during chemoheterotrophic growth.
RuBP carboxylase, which catalyzes the formation of 3-phosphoglyceric acid
from ribulose-1, 5-bisphosphate and CO 2, has been demonstrated in extracts
CITRIC ACID AND GLYOXYLATE CYCLE IN RPS. PALUSTRIS
527
from autotrophic bacteria and several studies have shown the enzyme to be
adaptive and thus to be formed under autotrophic growth conditions (Fuller and
Gibbs, 1959; Gale and Beck, 1967; Pearce, Leach and Cart, 1969). The growth of
facultative autotrophs in the presence of organic substrates has been shown to
result in the repression of RuBP carboxylase (Kornberg et al., 1960; Hurlbert
and Lascelles, 1963). Rps. palustris (strain 2.1.7, C. B. van Niel) revealed a
pronounced repression of the RuBP carboxylase activity when transferred from
photoheterotrophic to dark, highly aerated conditions in the presence of malate
plus glutamate; however, the repression was not complete after aerobic growth
(Lascelles, 1960).
In our experiments, RuBP carboxylase could not be detected after chemoheterotrophic growth, whereas the enzymatic activity was observed at about similar rates after photoheterotrophic and photolithotrophic growth conditions.
Likewise, the Rps. palustris strain isolated by Qadri and Hoare (1968) revealed
similar RuBP carboxylase activities after photoheterotrophic and hydrogenasemediated, formate-dependent photo-autotrophic growth (Stokes and' Hoare,
1969). Chernyad'ev and Doman (1971) reported activities in whole cells of Rps.
palustr&, Japanese strain, which during photosynthetic growth on formate were
sufficient to ensure autotrophic fixation of carbon dioxide.
It is a pleasure to thank Dr M. I. H. Aleem for discussions which led to the
present study.
Received 24 October 1978
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