Novel growth rate control of dam gene expression in Escherichia coli.

Abstract

Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical sigma 70 promoter with 18 bp spacing between the -10 and -35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of beta-galactosidase activity at all growth rates tested. In contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-4 confirming the different modes of regulation. The cde-4::miniTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene.