Regulatory mode of the pac gene expression.

1.65kb HindIII-BgIII DNA fragment containing pac operator was cloned from plasmid pPA4 to pBR322 at HindIII-BamHI sites, the resultant plasmid pPA41 was transformed into E. coli D816 carrying an intact pac operon on its chromosome, then the effect of operator titration was estimated. It was found that the penicillin acylase activity in E. coli D816 (pPA41) cells was higher than that of in E. coli D816 cells. The operators on high copy number plasmid pPA41 competed for the regulatory proteins with the single copy operator on the chromosomal pac operon, thus the expression of the pac gene was enhanced, because the free regulatory proteins were decreased by operator titration. The results of RNA-DNA hybridization showed that the cellular pac mRNA concentration was parallel to the penicillin acylase activity, and the pac mRNA in E. coli D816 (pPA41) cells was much higher than that of in E. coli D816 cells. These results indicated that the expression of pac gene was negatively controlled at transcriptional level.