质粒RP4CFIts突变体筛选及Kil表型分析

用羟胺体外诱变质粒RP4后再转化受体菌,我们选择到一抹温度敏感的菌落形成抑制突变体。其表型为在非允许温度时培养,不能由单个菌体繁殖为集落,类似于Kor基因失活引起的Kil表型。生化分析表明:在非允许条件下,突变体的生长逐步受到抑制,大约繁殖两代后即全面抑制,此时宿主菌的DNA、RNA、蛋白质等大分子合成几乎全部下降,但β-半乳糖苷酶的合成却并未停止,只是由于细胞通透性改变而大部分排出胞外,另外噬菌体感染后的产率也明显降低,并且在非允许温度处理20h内,此种状态是完全可逆的,即仍可因放回允许温度培养而形成集落,据此抑制现象的逐步发生性,大分子合成的全面抑制性和完全可逆性判断,很可能和宿主细胞在紧急状态下的整体调节机制——严紧型反应有关。

Isolation of CFIts Mutant of RP_4 and Analysis of Kil Phenotype

A temperature sensitive colony-formation inhibition mutant, RP4ts872, was obtained by hydroxylamine mutagenesis in vitro. It failed to form colony at nonpermissive temperature and had the similar characteristics as the Kil phenotype of plasmid RP4 reported previously. We proposed that this mutant was actually a temperature-sensitive mutant of Kor gene. The biochemical analysis indicated that at nonpermissive condition the growth of host cell was gradually inhibited and the doubling time was extended. After about two doublings, the total number of viable cell started to remain constant, during this time the macromolecular syntheses of host cell was nearly completely blocked and the yields of bacteriophages declined steadily. These inhibition phenomena were reversible if the temperature was shifted down from 42℃, to 30℃, even already incubation at 42℃ for 22h. From the properties described above, it seems to be the stringent response, which is a kind of whole regulatory mechnisms of host cell under urgent states.