The overall aim of this project is to probe the mechanisms regulating cell division in Escherichia coli. In virtually all cell types, cell division is strictly controlled; periods of cell growth, DNA duplication and chromosome partitioning always precede cytokinesis. Yet the mechanisms by which these processes are coordinated are poorly understood. E. coli provides a relatively simple model system for studying cell division. An understanding of how division is regulated should eventually lead to an explanation of how DNA replication, chromosome partitioning and cell division are coordinated. This information could prove valuable in the search for ways of blocking uncontrolled growth and division in eukaryotic cells. The sfiD gene appears to play a role in regulating cell division in E. coli. Insertions of the IS1 transposon into an adjacent, upstream site confer mutant phenotypes that can be complemented by the sfiD+ gene. These include normal division in the presence of the inducible division inhibitors SulA and SfiC, cold sensitivity for growth and sensitivity to beta-lactam antibiotics. A number of the proposed experiments are directed toward analysis of the transcription patterns in the sfiD region. These include the use of RT-PCR and primer extension to define the sfiD mRNA(s) in normal and mutant cells. The effect of deleting potential regulatory sites on transcription initiated at the sfiD promoter will also be examined. In other experiments the SfiD protein will be purified and its structure, cellular location and stability compared in normal and mutant cells. As a means of understanding how upstream IS1 insertions alter sfiD, the sequence changes in revertants carrying second site mutations linked to sfiD will be determined. Other unlinked second site mutations will be mapped with a view toward identifying proteins that interact with SfiD. Placement of the division complex will be examined in revertants exhibiting altered morphology. Finally, the penicillin binding proteins will be compared in normal and mutant cells.
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