Nucleotide sequence of an Rts1 fragment causing temperature-dependent instability

Abstract

Rts1 is a multiphenotypic drug resistance plasmid which is eliminated from host bacteria at 42 degrees C but not at 32 degrees C. This phenotype has been called temperature-dependent instability (Tdi). We determined the nucleotide sequence of the Rts1 DNA b' segment which causes this phenotype. Within this 786-base-pair segment, several open reading frames (ORFs) were found, including one which encodes a protein with a molecular weight of 16,000. A protein approximately corresponding to this protein is expressed in Escherichia coli minicells harboring plasmids containing the b' segment. In addition, we found the chi sequence at 112 bases proximal to this ORF. Temperature-dependent elimination due to this segment was not observed in the RecA strain of E. coli, but the RecB protein was not required for expression of this phenotype. We constructed various deletion derivatives and found that three portions, the region containing the chi (nucleotides 1 to 24), ORF (nucleotides 25 to 546), and tail (nucleotides 631 to 786) sequences are necessary for Tdi activity. Site-directed mutagenesis studies indicated that ORF I is required for Tdi expression.