Role of Pyruvate Oxidase in Escherichia coli Strains Lacking the Phosphoenolpyruvate:Carbohydrate Phosphotransferase SystemFlores N.a · de Anda R.a · Flores S.a · Escalante A.a · Hernández G.a · Martínez A.a · Ramírez O.T.b · Gosset G.a · Bolívar F.a
aDepartamento de Ingeniería Celular y Biocatálisis and bDepartamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología/UNAM, Cuernavaca, Morelos, Mexico
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We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (µ) in glucose minimal medium of 0.1 h–1 is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a µ of 0.70 h–1). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a µ of 0.40 h–1. In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB–derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS– strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.
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