Ike Y, Hashimoto H, Motohashi K, Fujisawa N, Mitsuhashi S
J Bacteriol. 1980 Feb;141(2):577-83. doi: 10.1128/jb.141.2.577-583.1980.
A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37 degrees C. When Escherichia coli ML1410(Rms201ts14)(+) was grown at temperatures between 40 and 42 degrees C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)(+) in L-broth was shifted to 42 from 30 degrees C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42 degrees C. At 30 degrees C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42 degrees C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42 degrees C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42 degrees C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 x 10(6) and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 x 10(6). These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.
从复合质粒Rms201中分离出一株对R质粒复制温度敏感的突变体Rms201ts14，该突变体是通过用100 mM羟胺在37℃对P1转导裂解物诱变40小时后获得的。当大肠杆菌ML1410(Rms201ts14)(+)在L肉汤中于40至42℃之间的温度下生长时，会分离出抗生素敏感细胞。当ML1410(Rms201ts14)(+)在L肉汤中的培养温度从30℃转移到42℃时，温度转移90分钟后抗生素抗性细胞数量的增加停止。然而，细胞总数持续增加，在温度转移到42℃后5小时，只有3%的细胞保留了质粒。在30℃时，Rms201ts14和Rms201每条染色体的共价闭合环状脱氧核糖核酸的量分别为3.8%和6.3%。在42℃时，放射性胸苷不会掺入Rms201ts14的共价闭合环状脱氧核糖核酸中，而即使在42℃时，放射性胸苷也会以4%/染色体的速率掺入Rms201的共价闭合环状脱氧核糖核酸中。在含有Rms201ts14的细胞中，质粒共价闭合环状脱氧核糖核酸的合成在42℃时几乎完全被阻断。这些结果表明，负责质粒脱氧核糖核酸复制的基因在突变体Rms201ts14中受到了影响。从Rms201ts14中分离出了温度敏感型小质粒pMSts214，其分子量为5.3×10(6)，编码氨苄青霉素抗性。同样，从其亲本Rms201中分离出了编码单一氨苄青霉素抗性的小质粒pMS201，其分子量为4.7×10(6)。这些结果表明，导致Rms201复制温度敏感性的基因位于小质粒上。
