Strength and regulation of the different promoters for chromosomal beta-lactamases of Klebsiella oxytoca

The two groups of chromosomal P-lactamases from Klebsiella oxytoca (OXY-1 a nd OXY-2) can be overproduced 73- to 223-fold, due to point mutations in th e consensus sequences of their promoters. The different versions of promote rs from bla(OXY-1) and bla(OXY-2) were cloned upstream of the chloramphenic ol acetyltransferase (CAT) gene of pKK232-8, and their relative strengths w ere determined in Escherichia coli and in K, oxytoca, The three different m utations in the OXY beta-lactamase promoters resulted in a 4- to 31-fold in crease 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 g reater 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 th e -10 consensus sequence and a T-to-A transversion of the fourth base of th e -35 sequence). The strength of the promoter carrying a double mutation (t ransition in the Pribnow box and the transversion in the -35 hexamer) was i ncreased 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 resulte d in a ninefold decrease of the promoter strength, The expression of the bl a(OXY) promoter in E. coli differs from that in K. oxytoca, particularly fo r promoters carrying strong mutations. Furthermore, the bla(OXY) promoter a ppears not to be controlled by DNA supercoiling or an upstream curved DNA, but it is dependent on the gene copy number.