Multicopy plasmids affect replisome positioning in Bacillus subtilis.
The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.
Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Jue D
JD
Wang
Megan E
ME
Rokop
Melanie M
MM
Barker
Nathaniel R
NR
Hanson
Alan D
AD
Grossman
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D056968:Lac Repressors; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D056968:Lac Repressors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins