DISCUSSION ON BACTERIAL RESPIRATION 261 



man reaction. We have found some evidence for the formation of 

 citric acid in the presence of acetic acid by Micrococcus lysodeik- 

 ticus. 



On the other hand, many bacteria do not appear to metaboHze 

 citric acid; this is difficult to reconcile with the Krebs cycle as pro- 

 posed, although it is probable that it is not citric acid as such which 

 is the intermediate. 



At present we are at work on C" acetic acid as a tracer. Certain 

 experimental evidence with the four-carbon acids has at times sug- 

 gested that we are dealing with phosphorylated compounds, prob- 

 ably of a very labile nature. Thus an acetone preparation is made 

 magnesium- and cocarboxylase-deficient by alkaline phosphate 

 washing of the cells as determined by testing on pyruvic acid. The 

 deficient preparation does not decarboxylate oxalacetic acid; how- 

 ever, the addition of magnesium ions completely restores the ac- 

 tivity. The reaction yields carbon dioxide and pyruvic acid. When 

 malic acid replaces oxalacetic acid, the deficient preparation under 

 the same conditions does not oxidize malic acid to carbon dioxide 

 and pyruvic acid, but only to oxalacetate, which accumulates and 

 does not inhibit the bacterial malic dehydrogenase as it does tissue 

 dehydrogenase. The complete preparation (deficient plus magnesium 

 ions or the unwashed acetone preparation) oxidizes malate to carbon 

 dioxide and pyruvic acid with traces of oxalacetic acid. Thus with 

 laboratory oxalacetic acid the reaction goes to carbon dioxide and 

 pyruvate, whereas with "physiological" oxalacetic acid (from malate) 

 the reaction appears to maintain an equilibrium. 



Is the "physiological" oxalacetic acid different from that prepared 

 in the laboratory, possibly a phosphorylated compound? We investi- 

 gated the problem and at one time thought that it was. Definite 

 stimulation by phosphate has been demonstrated for fumaric or 

 malic acid. As yet we have not shown a phosphate uptake, or isolated 

 an organic phosphate; however, we may be dealing with a labile 

 carbonyl phosphate in the sense of Lipmann. It is suggested that the 

 carbonyl group is bound in the physiological oxalacetate formed 

 from malic acid, since traces of oxalacetate are known to inhibit 

 malic oxidation in tissue. If malate is oxidized in an atmosphere of 

 C^^Oa and O2, the oxalacetic acid contains C^^ in the carboxyl group 

 adjacent to the methylene group. No chemical exchange takes place. 

 The enzymatic decarboxylation of oxalacetic acid in the presence of 

 heavy carbon dioxide also yields heavy carbon oxalacetic acid. This 

 is a form of carbon dioxide utilization. 



