RESUMEN
The accumulation of 2-keto-3-deoxy-6-phosphogluconate, the key intermediate of the Entner-Doudoroff pathway, has long been thought to inhibit growth of bacteria, but careful measurements of 2-keto-3-deoxy-6-phosphogluconate accumulation by growing cells and the correlation of intracellular 2-keto-3-deoxy-6-phosphogluconate levels to growth inhibition had not been made. A system designed for this purpose was developed in Escherichia coli strains, allowing 2-keto-3-deoxy-6-phosphogluconate accumulation to be experimentally induced and measured by extraction of the cell pool. Addition of gluconate to a strain which lacked 2-keto-3-deoxy-6-phosphogluconate aldolase and overproduced 6-phosphogluconate dehydratase resulted in an increase in the intracellular concentration of 2-keto-3-deoxy-6-phosphogluconate from undetectable levels to 2.0 mM within 15 s, as measured by anion-exchange HPLC. The accumulation of 2-keto-3-deoxy-6-phosphogluconate was correlated with an immediate and significant decrease in growth; this inhibition was determined to be bacteriostatic and not bactericidal. It had been proposed that the mechanism of 2-keto-3-deoxy-6-phosphogluconate toxicity involves competitive inhibition of 6-phosphogluconate dehydrogenase and the consequent block of the pentose phosphate pathway. An experiment addressing this hypothesis failed to provide any supporting data.
Asunto(s)
Aldehído-Liasas/fisiología , Escherichia coli/fisiología , Gluconatos/metabolismo , Vía de Pentosa Fosfato , Mutación , Fosfogluconato Deshidrogenasa/antagonistas & inhibidoresRESUMEN
Three pure bacterial cultures degrading methyl t-butyl ether (MTBE) were isolated from activated sludge and fruit of the Gingko tree. They have been classified as belonging to the genuses Methylobacterium, Rhodococcus, and Arthrobacter. These cultures degraded 60 ppm MTBE in 1-2 weeks of incubation at 23-25 degrees C. The growth of the isolates on MTBE as sole carbon source is very slow compared with growth on nutrient-rich medium. Uniformly-labeled [14C]MTBE was used to determine 14CO2 evolution. Within 7 days of incubation, about 8% of the initial radioactivity was evolved as 14CO2. These strains also grow on t-butanol, butyl formate, isopropanol, acetone and pyruvate as carbon sources. The presence of these compounds in combination with MTBE decreased the degradation of MTBE. The cultures pregrown on pyruvate resulted in a reduction in 14CO2 evolution from [14C]MTBE. The availability of pure cultures will allow the determination of the pathway intermediates and the rate-limiting steps in the degradation of MTBE.