The activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. A mutant strain producing a glycerol kinase resistant to inhibition by fructose-I , 6-diphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon and energy. The amount of intracellular fructose-1 ,6-diphosphate was determined for wild-type cells growing exponentially on glycerol. The water content of such cells was also determined, allowing calculation of the intracellular concentration of fructose-1 ,6-diphosphate. This value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type glycerol kinase. The desensitization of glycerol kinase to feedback inhibition also enhances the power of glycerol to exert catabolite repression, both on the enzymes of the glycerol system itself and on those of the lactose system. However, desensitization of glycerol kinase alone does not eliminate the phenomenon of diauxic growth in a glucose-glycerol medium. Biphasic growth in such a medium is abolished if the altered enzyme is produced constitutively. The constitutive production of the mutant kinase at high levels, however, renders the cells vulnerable to glycerol. Thus, when the cells have been grown on a carbon source with a low power for catabolite repression, e.g., succinate, sudden exposure to glycerol leads to overconsumption of the nutrient and cell death.for sensitivity of the enzyme to FDP at pH 7.5 (29). Protein was determined by the biuret reagent (8).Specific activities are expressed as micromoles of substrate converted per minute per milligram of protein at 25 C.Determination of intracellular FDP levels. Cells of strain 7 were grown in 35 ml of glycerol mineral me-753 on August 5, 2020 by guest

Fructose-1 ,6-diphosphate is a feedback inhibitor of the catabolic enzyme, glycerol kinase, in Escherichia coli. A mutant was isolated which produced a desensitized enzyme. Glucose was no longer as effective in preventing the utilization of exogenous glycerol by cells which synthesized constitutively such an altered enzyme, even though the usual degree of catabolite repression still operated.

A spontaneously arising regulatory mutant of the gluconate system in Escherichia coli was isolated. This mutant became constitutive, probably in one step, for gluconate high-affinity transport, gluconokinase, and gluconate-6-P dehydrase. The mutation involved ( gntR18 ) is cotransducible with asd . Pseudorevertants, derived from a mutant (M2) that shows a long lag for growth on gluconate mineral medium, were also isolated and characterized. They give constitutive levels of gluconokinase and gluconate-6-P dehydrase but lack high-affinity transport function. Genetic experiments performed with one of these pseudorevertants (M4) indicate that it carries a secondary mutation in the gntR gene. The M4 phenotype is thus the result of the interaction of expression of a constitutive mutation ( gntR4 ) with the mutation of strain M2 ( gntM2 ).

A lethal product (BPG) produced by a glycerol kinase mutant of Escherichia coli was purified, and its mode of action on E. coli was studied. At concentrations where BPG strongly inhibits in vivo deoxyribonucleic acid, ribonucleic acid, and protein synthesis, it produces small effects on other functions: slight inhibition of respiration and small changes in intracellular pools of substrates, nucleic acids degradation, and adenosine triphosphate levels. BPG also inhibits in vitro protein synthesis and produces inactivation of bacteriophage T4. The bactericidal product has been identified in another laboratory as methylglyoxal (MG). By comparing BPG and MG, we confirmed this observation and concluded that the activity found in our BPG preparation is due to its MG content. We also observed that MG is able to react with guanosine triphosphate. According to these results, it is interpreted that MG could act directly on macromolecular synthesis by reacting with the guanine residues of nucleic acids and its precursors. All the other chemicals employed were of the highest purity commercially available.

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