152 METABOLISM AND PHYSIOLOGY 



The results obtained support the hypothesis of Sadler and Stanier (5) 

 and provide a possible biochemical basis for the inability of C. 

 thiosulfatophilum to utilize acetate in the absence of CO2. 



EXPERIMENTAL PROCEDURE 



The green sulfur anaerobe Chlorobium thiosulfatophilum, strain L, 

 was cultivated as described by Larsen (7). The growth temperature 

 was 26 °C, Chloropseudomonas ethylicum was grown at 30°C in a 

 medium containing ethanol and CO2 as the carbon sources (8). Chro- 

 matium, strain D, was grown at 35°C in Hendley's medium (9). R. 

 rubrum was cultivated as described by Kohlmiller and Gest (10). 

 Rhodomicrohium vannielii was grown in a medium containing acetate 

 as the carbon source (11), 



Since some of the growth media contained sulfide, an inhibitor of 

 aconitate hydratase (12), the harvested cells were washed several 

 times with 0,05 M Tris buffer, pH 7,8. Cells were broken by passage 

 through a French pressure cell (13). The extracts were centrifuged 

 at 144,000 X g for 60 minutes and the supernatant fluid assayed for 

 enzymic activity. The protein content of the extracts was determined 

 by the biuret reaction (14). 



All enzymic measurements were made with a Gary recording spec- 

 trophotometer using a slide- wire showing full-scale deflection at an 

 absorbancy of 0.1. The assay temperature was 23 °C. Two methods 

 were employed to assay aconitate hydratase. In one, the enzyme was 

 directly assayed by the change in absorbancy at 240 mfi (15). Gitrate, 

 cis-aconitate, or isocitrate were used as substrates. In the other, an 

 excess of purified pig heart isocitrate dehydrogenase and TPN were 

 included in the reaction mixture and the reduction of TPN was meas- 

 ured by the absorbancy change at 340 m/i (16). Gitrate or cis-aconitate 

 were the substrates. Isocitrate dehydrogenase was measured by the 

 reduction of TPN in the presence of isocitrate (16), Succinic dehy- 

 drogenase was assayed by the reduction of cytochrome c in the pres- 

 ence of catalytic amounts of iV-methylphenazonium sulfate (17). The 

 absorbancy change at 240 m/y using malate as substrate was recorded 

 to estimate fumarate hydratase activity (15). Malate dehydrogenase 

 was determined by DPNH oxidation in the presence of oxalacetate (18), 

 Aceto-GoA- kinase was assayed by hydroxamic acid formation (19). 



RESULTS 



The Conversion of Acetate to Isocitrate 



The incorporation of acetate into isocitrate is a prerequisite for 

 any oxidation via the citric acid cycle. Incorporation of acetate into 



