Direct analysis of the transcription of Escherichia coli rnpB gene harbored in a multicopy plasmid during bacterial growth

Abstract

To examine the growth-phase dependent control of Escherichia coli mpB gene we used a combination of Northern analysis for RNA determination and Southern analysis for plasmid DNA determination. The relative amounts of metabolically unstable transcript derived from the internally deleted mpB gene harbored on a multicopy plasmid as well as the relative plasmid contents were measured by Northern analysis and Southern analysis, respectively, of total nucleic acids from E. coli cells containing the plasmid. The relative transcription activity of the mpB was represented by a ratio of the relative amount of the transcript to that of the plasmid DNA during bacterial growth. The mpB transcription increased rapidly with time during exponential growth, but started to decrease before the transition period of an exponential growing cell culture into the stationary phase. Although the expression pattern was similar to the changes of beta-galactosidase activity expressed from the lysogenic strain carrying the chromosomal mpB-lacZ fusion which were shown in a previous work, the present data appears to represent a more actual growth-phase control of the mpB transcription than the previous data by the beta-galactosidase assay. In addition the present method described for a direct analysis of both RNA and plasmid DNA provides a rapid and efficient method that can applied to an examination of transcription control by using a multicopy plasmid.