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J Bacteriol. 1974 June; 118(3): 999-1009
Copyright © 1974 American Society for Microbiology. All Rights Reserved.

Pyruvate Carboxylase Deficiency in Pleiotropic Carbohydrate-Negative Mutant Strains of Pseudomonas aeruginosa

^a Department of Microbiology, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23298
^b Department of Microbiology, University of Alabama in Birmingham, Birmingham, Alabama 35233
Department of Biology, University of Alabama in Birmingham, Birmingham, Alabama 35233

ABSTRACT

Cell extracts of Pseudomonas aeruginosa strain PAO were found to contain pyruvate carboxylase activity. Specific activities were minimal when cells were grown on Casamino Acids, acetate, or succinate, but were three- to fourfold higher when cells were grown in glucose, gluconate, glycerol, lactate, or pyruvate minimal media. The reaction in crude cell extracts and in partially purified preparations was dependent on pyruvate, adenosine 5'-triphosphate, and Mg²⁺, but was not affected by either the presence or absence of acetyl coenzyme A. Activity was nearly totally inhibited by avidin and this inhibition was substantially blocked by free biotin in incubation mixtures. Cell extracts were shown to fix ¹⁴CO₂ in a reaction that had these same characteristics. Eight pleiotropic, carbohydrate-negative mutant strains of the organism were isolated after nitrosoguanidine mutagenesis. Each mutant strain grew normally in acetate, succinate, and citrate minimal media but failed to utilize glucose, gluconate, 2-ketogluconate, mannitol, glycerol, lactate, and pyruvate as sole sources of carbon and energy. These strains were found by quantitative transductional analysis with phage F116 to form a single linkage group. Cell extracts of each mutant strain were either lacking or severely deficient in pyruvate carboxylase activity. Spontaneous revertants of five of the eight strains were isolated and found to recover simultaneously both pyruvate carboxylase activity and the ability to utilize each of the C₆ and C₃ compounds. A second linkage group of similar mutant strains that grew on the C₃ compounds was found to contain normal levels of pyruvate carboxylase activity, but each strain was deficient in an enzyme of the Entner-Doudoroff pathway.

¹ Present address: Department of Botany and Microbiology, Auburn University, Auburn, Ala. 36830.

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