CELL-CYCLE REGULATION OF THE ESCHERICHIA-COLI NRD OPERON - REQUIREMENT FOR A CIS-ACTING UPSTREAM AT-RICH SEQUENCE

The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coil has been shown to be cell cycle regulated. To identif y the cis-acting elements required for the cell cycle regulation of th e nrd promoter, different 5' deletions as well as site-directed mutati ons were translationally fused to a lacZ reporter gene. The expression of beta-galactosidase from these nrd-lacZ fusions in single-copy plas mids was determined with synchronously growing cultures obtained by re peated phosphate starvation as well as with exponentially growing cult ures by flow cytometry analysis. Although Fis and DnaA, two regulatory proteins that bind at multiple sites on the E. coli chromosome, have been found to regulate the nrd promoter, the results in this study dem onstrated that neither Fis nor DnaA was required for nrd cell cycle re gulation. A cis-acting upstream AT-rich sequence was found to be requi red for the cell cycle regulation. This sequence could be replaced by a different sequence that maintained the AT richness. A flow cytometry analysis that combined specific immunofluorescent staining of beta-ga lactosidase with a DNA-specific stain was developed and employed to st udy the nrd promoter activity in cells at specific cell cycle position s. The results of the flow cytometry analysis confirmed the results ob tained from studies with synchronized cells.