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A study of the incorporation of inorganic phosphate by Pseudomonas aeruginosa Campbell, James N.

Abstract

The oxidation of glucose by Pseudomonas aeruginosa is known to follow the sequence: glucose → gluconic acid → 2 ketogluconic acid ↛ pyruvic acid and thence into the Krebs' cycle. Two aspects of this picture which arouse interest are, the missing steps in the pathway and the fact that the initial steps of this pathway do not involve phosphorylated intermediates. Phosphate undoubtedly plays an important part in the metabolism of this organism hence the question is immediately asked as to where phosphorous does become involved. These two questions yield two possible routes of investigation for their own solution; one to follow the fate of glucose as it is metabolized past the 2 ketogluconate level, or two, to follow the fate of inorganic phosphate as it is incorporated. The hope in each case is that the information gleaned will help elucidate how the two phenomena of glucose breakdown and utilization of phosphorous are correlated. Both these routes of investigation were attempted. The investigations along either line are, however, fraught with difficulties. To study the breakdown of glucose, attempts were made to find a cell preparation, or an inhibitor, or a combination of both, which would allow the breakdown of glucose past the 2 ketogluconate level, but not allow its complete oxidation to CO₂ and H₂O, thus accumulating a heretofore unknown intermediate which could be identified. It appears, however, that the system acting on 2 ketogluconate as a substrate is the most labile in this chain, for if inhibition was obtained, it occurred at the 2 ketogluconate level, regardless of the technique employed. The investigation then moved to an attempt to follow the incorporation of inorganic phosphate. Cell free preparations were found to incorporate inorganic phosphate when incubated with glucose and to a lesser extent when incubated with succinate, such incorporation being partially sensitive to dinitrophenol. The curve obtained on measurement of loss of inorganic phosphate indicated that this was not a simple reaction with resultant accumulation of the organic acceptor or a single product of this acceptor, but rather was a complex procedure involving several steps. The fact that cell free preparations took up inorganic phosphate in the absence of added substrate, coupled with the growing opinion that the endogenous respiration in this organism is not a simple reaction, independent of added substrate, showed the necessity of ascertaining first what this organism does with inorganic phosphate in the absence of added substrate, so that when substrate is added, it will be possible to know what phenomena are and what are not, a result of its presence. The addition of inorganic phosphate labelled with P³² to a cell free system in the absence of added substrate resulted in the accumulation of two alkaline labile, acid stable fractions with ultraviolet absorption peaks at 258 - 262 mμ and 263 mμ respectively. Their acid-alkaline behaviour leads to the suggestion that they may be nucleoside-polyphosphate-sugar complexes.

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