Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Abstract

Previous publications showed that a covalently closed circular (CCC) Rtsl plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 420C but not at 32°C. At 42°C, the CCC Rtsl DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rtsl on host bacteria, Rtsl was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rtsl did not exert the thermosensitive effect on these cells, and CCC Rtsl DNA was formed even at 42°C. Upon addition of cAMP to E. coli CA7902(Rtsl), cell growth and formation of CCC Rtsl DNA were inhibited at 42°C. The addition of cAMP to E. coli PP47(Rtsl) did not cause inhibitory effects on either cell growth or CCC Rtsl DNA formation at 420C. The inhibitory effect of cAMP on E. coli CA7902(Rtsl) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rtsl DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [3H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rtsl DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rtsl DNA formation and cell growth at 420C. 80 on July 16, 2020 by guest