Instability of pUC19 in Escherichia coli transcription termination factor mutant, rho026

The higher copy number of pUC19, compared to its parent plasmid pBR322, is known to be due to deletion of rep, also known as rom, and to an ori mutation that impedes RNAI:RNAU interaction. pUC19, unlike pBR322, fails to transform E. coli rho mutant rho026 cells. Here we identify two features of pUC19 that contribute to this transformation defect. (1) The pUCori mutation is involved because replacing the pUCori with that of pBR322 restored transformation. (2) Transcription from the Inc promoter in pUC19 is important, since deletion or inversion of the promoter or insertion of a transcription terminator (lambda t0) downstream of it restored transformation. Host RNase E activity is responsible for the transformation defect because introduction of an rne-1 allele into rho026 cells suppressed this defect, indicating that RNAI instability due to RNase E is aggravated in the rho026 strain. We suggest that in rho026 cells pUC19 RNAI:RNAII interaction is more impeded than in rho(+) cells and Rop/Rom map confer stability by protecting RNAI against RNase E activity because expression of a rom gene inserted into pUC19 restored transformation. (C) 1999 Academic Press.