|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1990 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1989 : ¥1,300,000 (Direct Cost : ¥1,300,000)
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
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.