It was proposed previously that P. islandicum
grows autotrophically using the reductive citric acid cycle based on measurement of the ATP citrate synthase, pyruvate synthase, and 2-oxoglutarate synthase activities in cells reportedly grown autotrophically (21
). This was consistent with the operation of this pathway in T. neutrophilus
, a member of the same family (6
). Our results support this idea with the caveat that the carbon flow appears to be different than the carbon flow at key regulatory steps suggested previously.
The growth rates and maximum concentrations of cells increased with increasing amounts of organic compounds present. P. islandicum
grew heterotrophically on remarkably low concentrations of organic compounds (0.001% yeast extract), albeit to low concentrations of cells. Cultures did not grow on acetate alone, but the maximum concentrations of cells increased up to fourfold in medium containing 0.001% yeast extract and H2
when acetate was added. Growth on acetate without H2
was poor. 14
C-labeled acetate uptake studies showed that acetate was used by the cells, but apparently cells were unable to meet all of their biosynthetic needs without another source of organic compounds, and H2
appeared to ameliorate the need to use acetate as the sole source of electrons. Similarly, acetate assimilation in R. rubrum
was enhanced by addition of pyruvate and was further enhanced by addition of H2
The activity of citrate lyase was more than eight times higher in autotrophically grown cultures than in heterotrophically grown cultures, suggesting that this activity was regulated. ATP citrate synthase activities were not significantly different in autotrophic and heterotrophic samples and were apparently not regulated by the change in conditions. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic samples. Citrate lyase in mesophilic bacteria requires covalent modification via acetylation for activation (11
). In P. islandicum
, citrate lyase activity was observed only when an acetylating compound was present in the assay mixture, suggesting that acetylation may further regulate the activity of this enzyme.
ADP-forming acetate:CoA ligase activity was observed in heterotrophically grown cultures, but AMP-forming acetate:CoA ligase activity was not detected. Conversely, AMP-forming acetate:CoA ligase activity was observed in acetate-grown and autotrophically grown cultures but not in heterotrophically grown cultures. This suggests that ADP-forming acetate:CoA ligase could be used in reverse to make ATP and acetate during heterotrophic growth, but energy in the form of ATP was required to form acetyl-CoA from acetate during growth with acetate and autotrophic growth. During autotrophic growth, the acetate for the reaction came from citrate lyase, and the AMP-forming acetate:CoA ligase reaction completed the conversion of citrate into oxaloacetate and acetyl-CoA (Fig. ). The ATP citrate synthase activity was highest in acetate-grown cells. Therefore, during growth with acetate, the acetyl-CoA formed by AMP-forming acetate:CoA ligase appeared to enter the citric acid cycle for further oxidation in part by ATP citrate synthase, perhaps to generate ATP from ADP (Fig. ).
The activities of malate dehydrogenase, decarboxylating malate dehydrogenase, pyruvate synthase, citrate synthase, and ADP-forming acetate:CoA ligase, which catalyze adjacent metabolic steps, were all significantly lower in autotrophically grown cultures than in cultures grown heterotrophically (Fig. ). The activities of citrate lyase, AMP-forming acetate:CoA ligase, and phosphoenolpyruvate carboxylase were all significantly higher in autotrophically grown cultures (Fig. ). These nine enzymes share many of the same reactants and products. Therefore, their activities appear to be generally coordinated to control carbon flow in the citric acid cycle. In particular, it appeared that acetylated citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, function in a coordinated manner with citrate synthase to regulate the direction of carbon flow. The activities of the other five enzymes of the citric acid cycle, including two enzymes that use CO2, were not affected by a change in the carbon source. The hydrogenase and formate dehydrogenase activities were high in all whole-cell extracts, and phosphoenolpyruvate carboxylase activity was observed only in acetate-grown and autotrophically grown cultures. These results suggest that there may be CO2 uptake at these steps, as well as CO2 reduction to formate via the putatively membrane-bound hydrogenase and formate dehydrogenase. No H2 was detected in the headspace of heterotrophically grown cultures grown in sealed serum bottles, suggesting that P. islandicum did not use pyruvate-formate lyase.
The lack of pyruvate synthase activity when growth was not heterotrophic is interesting since this enzyme is a key enzyme for biosynthesis in the reductive citric acid cycle (10
). It is not known how pyruvate was formed and how biosynthesis from acetyl-CoA occurred in P. islandicum
during autotrophic growth or during growth with acetate without pyruvate synthase. However, one possibility is that the citramalate cycle was used; in this cycle acetyl-CoA is converted into glyoxylate, which enters the citric acid cycle via malate synthase and can be used to make various biosynthetic precursors. It was proposed previously that this pathway is used for biosynthesis in R. rubrum
, which, like P. islandicum
, lacks pyruvate synthase and isocitrate lyase activities when cultures are grown with acetate (23
In conclusion, P. islandicum
fixation via the reductive citric acid cycle may be more like the pathway proposed for some purple photosynthetic bacteria than like the pathways found in green photosynthetic bacteria and Hydrogenobacter
species. The primary enzymes for the formation of oxaloacetate and acetyl-CoA in P. islandicum
appear to be citrate lyase and AMP-forming acetate:CoA ligase and not ATP citrate synthase. It was suggested previously that the reductive citric acid cycle was the origin of intermediary metabolism based on the chemistry of the intermediates (28
). Therefore, further study of this pathway in hyperthermophilic archaea, which themselves may have many ancient traits, may provide insight into the natural history of this pathway.