Regulated expression of the Escherichia coli dam gene

Abstract

Regulated expression of the Escherichia coli dam gene has been achieved with the araBAD promoter lacking a ribosome binding site. Cultures of dam mutants containing plasmid pMQ430 show no detectable methylation in the absence of arabinose and complete methylation in its presence. Dam methyltransferase is a substrate for the Lon protease.

FIG. 1.

Effect of adding inducer to log-phase cultures. Arabinose was added to a culture of pMQ430/GM3819 growing in L broth to a final concentration of 0.2% for 20 and 40 min. Cells were harvested, and total DNA was extracted and digested with Sau3A (lanes S) (cuts independently of methylation status), DpnI (lanes I) (cuts methylated DNA), and DpnII (lanes II) (cuts unmethylated DNA). First lane, 100-bp ladder.

FIG. 2.

Loss of methylation after removal of inducer. Log-phase cells of pMQ430/GM3819 were exposed to arabinose, harvested, and resuspended in L medium with glucose. The cells were kept in the logarithmic phase of growth by dilution into fresh medium. Samples were removed at 0, 2.5, 3.5, and 4.5 h. For each time point the order of digestion was Sau3A (lanes S), DpnI (lanes I), and DpnII (lanes II). Fourth lane, 100-bp ladder.