8. REACTIONS OF INORGANIC SUBSTANCES
363
C
14 0 2 in the presence of pyruvate or, if pyridine nucleotides are available, of malate. The same extracts can also catalyze the carboxylation
of α-ketoglutarate to yield isocitrate. Bergmann et al. (31) confirmed
these results and showed that H. facilis can carry out ß-carboxylation of
pyruvate or phosphoenolpyruvate in addition to fixing C0 2 on ribulose
diphosphate. Finally, Suzuki and Werkman (34) found that cell-free
extracts of T. thiooxidans contained at the same time the enzymes of
the Calvin cycle and phosphoenolpyruvate carboxylase.
The mechanisms by which chemo-lithotrophic bacteria couple their
litho-oxidative and biosynthetic reactions are little understood. Vogler
and Umbreit (35) studied this problem in T. thiooxidans, which oxidizes elementary sulfur or thiosulfate to sulfuric acid aerobically. They
found that in the presence of sulfur and oxygen and in the absence of
C0 2 from the medium, nonproliferating suspensions of these bacteria
actively incorporate inorganic phosphate. If the cells are then deprived
of sulfur and oxygen and placed in the presence of carbon dioxide, they
fix C0 2 and liberate inorganic phosphate. These findings were interpreted
to mean that the bacterial cells can place into reserve as high-energy
phosphate bonds the energy produced by the oxidation of sulfur and
can then use this energy to fix C0 2 . Baalsrud and Baalsrud (36) took up
this study with the same organism but changed the substrate from elementary sulfur to thiosulfate. They confirmed that under these conditions the oxidation of the substrate is accompanied by a slight decrease
in the content of inorganic phosphate in the medium and thus, most
likely, by phosphorylation. But these authors, and later Newburg (37),
did not find any appreciable C0 2 fixation in the absence of substrate,
thus failing to confirm the observations and interpretation of Vogler
and Umbreit (35).
These results, obtained before the mechanism of C0 2 incorporation
has been clarified, lend considerable weight to the hypothesis that the
coupling of the litho-oxidative and biosynthetic pathways takes place
by means of energy-rich phosphate bonds. The demonstration of a
Calvin cycle in these organisms makes it possible to determine the
nature and number of the reactions into which phosphate esters enter.
According to the Embden-Meyerhof scheme, the reduction to 3-phosphoglyceraldehyde of two molecules of 3-phosphoglyceric acid formed
by the fixation of one molecule of C0 2 to ribulose diphosphate requires
two molecules of adenosine triphosphate (ATP) and two molecules of
reduced diphosphopyridine nucleotide (DPNH + H+):
3-Phosphoglyceric acid + ATP —»1,3-Diphosphoglyceric acid + ADP
1,3-Diphosphoglyceric acid + DPNH + H
+
—> 3-Phosphoglyceraldehyde + DPN
+ + P t
363
C
14 0 2 in the presence of pyruvate or, if pyridine nucleotides are available, of malate. The same extracts can also catalyze the carboxylation
of α-ketoglutarate to yield isocitrate. Bergmann et al. (31) confirmed
these results and showed that H. facilis can carry out ß-carboxylation of
pyruvate or phosphoenolpyruvate in addition to fixing C0 2 on ribulose
diphosphate. Finally, Suzuki and Werkman (34) found that cell-free
extracts of T. thiooxidans contained at the same time the enzymes of
the Calvin cycle and phosphoenolpyruvate carboxylase.
The mechanisms by which chemo-lithotrophic bacteria couple their
litho-oxidative and biosynthetic reactions are little understood. Vogler
and Umbreit (35) studied this problem in T. thiooxidans, which oxidizes elementary sulfur or thiosulfate to sulfuric acid aerobically. They
found that in the presence of sulfur and oxygen and in the absence of
C0 2 from the medium, nonproliferating suspensions of these bacteria
actively incorporate inorganic phosphate. If the cells are then deprived
of sulfur and oxygen and placed in the presence of carbon dioxide, they
fix C0 2 and liberate inorganic phosphate. These findings were interpreted
to mean that the bacterial cells can place into reserve as high-energy
phosphate bonds the energy produced by the oxidation of sulfur and
can then use this energy to fix C0 2 . Baalsrud and Baalsrud (36) took up
this study with the same organism but changed the substrate from elementary sulfur to thiosulfate. They confirmed that under these conditions the oxidation of the substrate is accompanied by a slight decrease
in the content of inorganic phosphate in the medium and thus, most
likely, by phosphorylation. But these authors, and later Newburg (37),
did not find any appreciable C0 2 fixation in the absence of substrate,
thus failing to confirm the observations and interpretation of Vogler
and Umbreit (35).
These results, obtained before the mechanism of C0 2 incorporation
has been clarified, lend considerable weight to the hypothesis that the
coupling of the litho-oxidative and biosynthetic pathways takes place
by means of energy-rich phosphate bonds. The demonstration of a
Calvin cycle in these organisms makes it possible to determine the
nature and number of the reactions into which phosphate esters enter.
According to the Embden-Meyerhof scheme, the reduction to 3-phosphoglyceraldehyde of two molecules of 3-phosphoglyceric acid formed
by the fixation of one molecule of C0 2 to ribulose diphosphate requires
two molecules of adenosine triphosphate (ATP) and two molecules of
reduced diphosphopyridine nucleotide (DPNH + H+):
3-Phosphoglyceric acid + ATP —»1,3-Diphosphoglyceric acid + ADP
1,3-Diphosphoglyceric acid + DPNH + H
+
—> 3-Phosphoglyceraldehyde + DPN
+ + P t