Point mutations that reduce the expression of malPQ, a positively controlled operon of Escherichia coli

Abstract

malPQ is one of three operons controlled by the positive regulator gene malT. With the objective of defining DNA sequences essential for malPQ transcription, we looked for cis-dominant mutations that reduced the level of expression of this operon. We first constructed malP-lac fusion strains, selected from one of them a series of mutants resistant to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease), and retained the clones that contained a mutation reducing the expression of the hybrid operon in a cis-dominant fashion. Nineteen such mutations were sequenced, and their effect on an otherwise wild type malPQ operon was studied. Three of them mapped in a transcribed portion of the operon, and are believed to exert their effect at the translation level. The others map upstream from the transcription startpoint (co-ordinate +1) and help define three DNA segments that must play a predominant role in transcription initiation: the Pribnow box (from positions -7 to -12); and two inverted repeats, extending from position -32 to -36, and -59 to -63, respectively, which are proposed to constitute part of the binding site for MalT protein.