The copy number of Bacillus plasmid pRBH1 is negatively controlled by RepB protein

Abstract

The replication control mechanism of Bacillus plasmid pRBH1 was analysed; pRBH1 contains four promoters, P1 to P4, and a large inverted repeat (63 base pairs) upstream of the protein (RepB) coding sequence. The stem and loop structure is surrounded by two promoters, P1 and P3, with different directions of transcription. One base substitution in the loop structure caused a change in copy number. Since the P1 promoter is located upstream of the replication origin of pRBH1, the transcript from the P1 promoter might serve as the primer of DNA replication. In vivo transcription from the P1 promoter was repressed by a trans-acting plasmid gene product. Since the RepB protein is involved in copy number control and RepB contains the consensus amino acid sequence of DNA binding proteins, RepB was thought to be the repressor. It was concluded from these data that the inverted repeat is involved in the control of copy number of the plasmid pRBH1. The RepB protein also contains two regions highly homologous with the Rom protein encoded on Escherichia coli plasmid ColE1. The possible mechanism for the copy number control of the plasmid via RepB protein and/or RNAs is discussed.