Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E, coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome, A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galaclosidase activity after maximal induction/specific beta-galactosidase activity before induction.) The following results were obtained from the experiments: induction of the nar promoter was optimal when E, coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate: the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD600 approximate to 1.3) before being transferred to the fermenter; the amount of beta-galactosidase per cell and per medium volume was maximal when E, coli was grown under aerobic conditions to OD600 = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD600 became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (C) 1996 John Wiley & Sons, Inc.