The assembly and migration of SeqA-Gfp fusion in living cells of Escherichia coli.
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.
Onogi
T
T
Department of Molecular Cell Biology, Kumamoto University School of Medicine, Japan.
Niki
H
H
Yamazoe
M
M
Hiraga
S
S
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000502:physiology; D002868:Chromosomal Proteins, Non-Histone; D004261:DNA Replication / Q000502:physiology; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000502:physiology*; D029968:Escherichia coli Proteins; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D008957:Models, Genetic; D016296:Mutagenesis; D010957:Plasmids; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; C067035:MukB protein, E coli; D011993:Recombinant Fusion Proteins; C089746:SeqA protein, E coli; D014157:Transcription Factors; D049452:Green Fluorescent Proteins