Strength and Regulation of the Different Promoters for Chromosomal β-Lactamases of Klebsiella oxytoca

Abstract

ABSTRACT The two groups of chromosomal β-lactamases from Klebsiella oxytoca (OXY-1 and OXY-2) can be overproduced 73- to 223-fold, due to point mutations in the consensus sequences of their promoters. The different versions of promoters from bla OXY-1 and bla OXY-2 were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene of pKK232-8, and their relative strengths were determined in Escherichia coli and in K. oxytoca . The three different mutations in the OXY β-lactamase promoters resulted in a 4- to 31-fold increase in CAT activity compared to that of the wild-type promoter. The G→T transversion in the first base of the −10 consensus sequence caused a greater increase in the promoter strength of the wild-type promoter than the two other principal mutations (a G-to-A transition of the fifth base of the −10 consensus sequence and a T-to-A transversion of the fourth base of the −35 sequence). The strength of the promoter carrying a double mutation (transition in the Pribnow box and the transversion in the −35 hexamer) was increased 15- to 61-fold in comparison to that of the wild-type promoter. A change from 17 to 16 bp between the −35 and −10 consensus sequences resulted in a ninefold decrease of the promoter strength. The expression of the bla OXY promoter in E. coli differs from that in K. oxytoca , particularly for promoters carrying strong mutations. Furthermore, the bla OXY promoter appears not to be controlled by DNA supercoiling or an upstream curved DNA, but it is dependent on the gene copy number.