STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE SFID REGION OF ESCHERICHIA COLI

本项目的总体目标是探索调节细胞的机制

The overall aim of this project is to probe the mechanisms regulating cell division in Escherichia coli. The proposed work focuses on the cellular role of the sfiD gene. Mutations at this locus prevent the action of two division inhibitors, Su1A and SfiC, whose synthesis is stimulated as part of the recA-dependent response to DNA damage. Su1A prevents "FtsZ ring formation," i.e. aggregation of the FtsZ cell division initiator protein on the cell envelope just prior to division. An sfiD mutation allows FtsZ ring formation in the presence of Su1A. The sfiD gene is located between kilobase pair 3,102 and 3,110 on the E. coli physical map, or roughly 63.7 minutes on the genetic map. A second phenotype associated with the sfiD mutations is extremely poor growth at temperatures below 29 degree. This "cold sensitivity" may be attributable to partial cell lysis at low temperature. In complementation studies, the mutant sfiD alleles are recessive to sfid plus. A number of the proposed experiments are directed toward structural analysis of the sfiD region. These include subcloning of complete and partial sfiD gene segments into phagemid vectors and DNA sequence determination. The nucleotide positions of mutant sfiD25 and sfiD27 alleles will be established using a combination of PCR, gene cloning and sequencing. In other experiments the effect of mutations in sfiD on the integrity of the cell wall will be examined, either directly or by response to antibiotic inhibitors of peptidoglycan synthesis. As a means of identifying genes whose products may interact with sfiD, the map positions of second site suppressor mutations that reverse the cold sensitivity phenotype will be determined. Finally, additional experiments designed to explore the physiological role of SfiD will include monitoring the effect of SfiD overproduction on division frequency and Su1A stability measurements in sfiD plus versus sfiD mutant strains.