The assembly and migration of SeqA-Gfp fusion in living cells of Escherichia coli

Abstract

SeqA protein, which binds to hemi-methylated GATC sequences of DNA, is localized to discrete fluorescent foci in wild-type Escherichia coli cells. In this work, we observed cellular localization of the SeqA-Gfp fusion in living cells. SeqA-Gfp was localized to a discrete focus or foci in wild-type and seqA null mutant cells, but the fusion was dispersed in the whole cell in dam null mutant cells lacking Dam methyltransferase. These results were consistent with the previous description of the localization of SeqA by immunofluorescence microscopy. Time-lapse experiments revealed that duplicated SeqA-Gfp foci migrated rapidly in opposite directions. Flow cytometry demonstrated that the fusion restored synchronous replication of chromosomal DNA from multiple origins in seqA null mutant cells, indicating that SeqA-Gfp is biologically active. Immunoprecipitation of the fusion from cell extracts using anti-Gfp antibody indicated that the fusion was assembled with the wild-type SeqA protein.