Control of Partitioning of Chromosomal DNA and Cell Division by Sex Factor F in Escherichia coli : Participation of the gyrA Gene.

大肠杆菌F质粒含有letA和letD两个基因，其产物参与了F质粒DNA复制与宿主细菌细胞分裂的偶联。letD基因产物抑制宿主细菌染色体DNA的分裂和细胞分裂，而letA基因产物抑制letD基因产物的抑制活性。逃脱了letD产物生长抑制的细菌突变体被分为五组：携带groES、groEL、gyrA、tdiC和tdiD基因突变的突变体。在这些基因产物中，GyrA蛋白被认为是LetD蛋白的靶标，因为GyrA （ts）突变体产生的突变体GyrA蛋白和野生型GyrA蛋白通过多拷贝质粒产生过量的突变体GyrA蛋白，都克服了LetD产物的生长抑制。LetD过量产生细胞的质粒DNA随着LetD蛋白的诱导而广泛松弛，表明细胞的超卷曲活性大大降低。相应地，LetD过量菌株的无细胞提取物缺乏DNA超卷曲活性；超卷曲活性水平的降低依赖于letD基因的表达。提取物中的DNA放松活性未受影响。将纯化的LetA蛋白添加到细胞提取物中，使DNA超卷曲活性恢复到与亲本菌株提取物相当的水平。恢复的超卷曲活性将负超卷曲引入DNA，并对氧喹啉酸敏感。这些结果有力地表明，LetD蛋白失活了DNA回转酶，而LetA蛋白促进了酶的恢复。我们得出结论，LetA和LetD蛋白对染色体分离的抑制至少部分是由于DNA旋转酶的抑制，我们进一步推断LetA和LetD蛋白调节DNA旋转酶或分离机制的功能，以确保F质粒在宿主细菌的细胞周期中稳定维持。少

The F plasmid of Escherichia coli contains two genes, letA and letD, whose products are involved in the coupling between DNA replication of the F plasmid and cell division of the host bacteria. The letD gene product acts to inhibit partitioning of the chromosomal DNA and cell division of the host bacteria, whereas the letA gene product acts to suppress the inhibitory activity of the letD gene product. Bacterial mutants that escaped the letD product growth inhibition were classified into five groups : mutants carrying mutations in the groES, groEL, gyrA, tdiC or tdiD genes. Among these gene products, GyrA protein was supposed to be the target of LetD protein, since mutant GyrA proteins produced by gyrA (ts) mutants and wild type GyrA protein produced excess amount by means of multicopy plasmid, both, overcame the letD product growth inhibition.The plasmid DNA in the LetD overproducing cells was extensively relaxed along with the induction of LetD protein, indicative of a vastly reduced … More supercoiling activity in the cells. Correspondingly, a cell-free extract from the LetD overproducing strain lacked the DNA supercoiling activity ; the level of supercoiling activity was decreased in a manner dependent on the expression of letD gene. The DNA relaxing activity in the extract remained unaffected. Addition of purified LetA protein to the cell extract restored the DNA supercoiling activity to a level comparable to that observed with the extract from the parental strain. The restored supercoiling activity introduced negative supercoils into DNA and was sensitive to oxolinic acid. These results strongly suggest that the LetD protein inactivates the DNA gyrase, and the LetA protein facilitates rejuvenation of the enzyme. We conclude the t the inhibition of chromosome segregation by the LetD protein is due, at least in part, to the inhibition of DNA gyrase, and we further extrapolates that LetA and LetD proteins regulate the functions of DNA gyrase or a segregative machinery to ensure the stable maintenance of the F plasmid in the cell cycle of the host bacteria. Less