Localization of bacterial DNA polymerase: Evidence for a factory model of replication

Science

Volumn 282, Issue 5393, 1998, Pages 1516-1519

  Lemon, Katherine P ^a   Grossman, Alan D ^a  

^a MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL DNA; DNA POLYMERASE; GREEN FLUORESCENT PROTEIN; HYBRID PROTEIN; PROTEIN SUBUNIT;

ARTICLE; BACILLUS SUBTILIS; CELL DIVISION; DNA REPLICATION; DNA SYNTHESIS; DNA TEMPLATE; ENZYME ACTIVITY; ENZYME LOCALIZATION; MOLECULAR DYNAMICS; MOLECULAR MODEL; NONHUMAN; PRIORITY JOURNAL;

BACILLUS SUBTILIS; BACTERIA (MICROORGANISMS); UNIDENTIFIED BACTERIOPHAGE;

EID: 0032553470     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5393.1516     Document Type: Article

References (35)

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8. The GFP fusion proteins were visualized in living cells as in (16) and (20). About 2 μl of agarose (0.5 to 1%, in medium) was applied to a microscope slide, allowed to cool for ∼30 s, and then ∼10 μl of culture was added. After 2 to 5 min, excess liquid was aspirated, and a glass coverslip was placed on the slide. Cell membranes were stained by growing the cells for at least one doubling in the red membrane dye FM4-64 (50 to 70 ng/ml) (Molecular Probes, Eugene, OR). The cell outlines were visualized simultaneously with the GFP signal using Chroma filter set no. 41012. Microscopy was performed with a Zeiss Axioplan II. Images were captured with a cooled charge-coupled device (CCD) camera (Optronics Engineering, Goleta, CA) and a CG-7 frame grabber (Scion, Frederick, MD) with Scion Image 1.62 software.
9. Pspac-dnaA was constructed by cloning a 5′ fragment of dnaA [N. Ogasawara, S. Moriya, K. von Meyenburg, F. Hansen, H. Yoshikawa, EMBO J. 4, 3345 (1985)], including the ribosome binding site, into pDH88 downstream from Pspac [D. G. Yansura and D. J. Henner, Proc. Natl. Acad. Sci. U.S.A. 81, 439 (1984); D. J. Henner, Methods Enzymol. 185, 223 (1990)]. The Pspac-dnaA plasmid pKL98 was integrated into the B. subtilis chromosome by a single crossover at dnaA to produce strain KPL213. KPL311 contains polC-gfp and Pspac-dnaA and was constructed by transforming KPL304 (polC-gfp) with chromosomal DNA from KPL213. KPL311 was grown to midexponential phase at 30°C in defined minimal medium with glucose (1%), glutamate (0.1%), required amino acids (40 μg/ml), spectinomycin (40 μg/ml), chloramphenicol (2.5 μg/ml), and IPTG (1 mM). Cells were then centrifuged, washed, and resuspended at an optical density (600 nm) of 0.05 to 0.1 in the above medium in the presence or absence of IPTG. Live cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) at ∼0.5 μg/ml in 0.5-ml aliquots just before placement on agarose.
10. Pspac-dnaA was constructed by cloning a 5′ fragment of dnaA [N. Ogasawara, S. Moriya, K. von Meyenburg, F. Hansen, H. Yoshikawa, EMBO J. 4, 3345 (1985)], including the ribosome binding site, into pDH88 downstream from Pspac [D. G. Yansura and D. J. Henner, Proc. Natl. Acad. Sci. U.S.A. 81, 439 (1984); D. J. Henner, Methods Enzymol. 185, 223 (1990)]. The Pspac-dnaA plasmid pKL98 was integrated into the B. subtilis chromosome by a single crossover at dnaA to produce strain KPL213. KPL311 contains polC-gfp and Pspac-dnaA and was constructed by transforming KPL304 (polC-gfp) with chromosomal DNA from KPL213. KPL311 was grown to midexponential phase at 30°C in defined minimal medium with glucose (1%), glutamate (0.1%), required amino acids (40 μg/ml), spectinomycin (40 μg/ml), chloramphenicol (2.5 μg/ml), and IPTG (1 mM). Cells were then centrifuged, washed, and resuspended at an optical density (600 nm) of 0.05 to 0.1 in the above medium in the presence or absence of IPTG. Live cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) at ∼0.5 μg/ml in 0.5-ml aliquots just before placement on agarose.
11. Pspac-dnaA was constructed by cloning a 5′ fragment of dnaA [N. Ogasawara, S. Moriya, K. von Meyenburg, F. Hansen, H. Yoshikawa, EMBO J. 4, 3345 (1985)], including the ribosome binding site, into pDH88 downstream from Pspac [D. G. Yansura and D. J. Henner, Proc. Natl. Acad. Sci. U.S.A. 81, 439 (1984); D. J. Henner, Methods Enzymol. 185, 223 (1990)]. The Pspac-dnaA plasmid pKL98 was integrated into the B. subtilis chromosome by a single crossover at dnaA to produce strain KPL213. KPL311 contains polC-gfp and Pspac-dnaA and was constructed by transforming KPL304 (polC-gfp) with chromosomal DNA from KPL213. KPL311 was grown to midexponential phase at 30°C in defined minimal medium with glucose (1%), glutamate (0.1%), required amino acids (40 μg/ml), spectinomycin (40 μg/ml), chloramphenicol (2.5 μg/ml), and IPTG (1 mM). Cells were then centrifuged, washed, and resuspended at an optical density (600 nm) of 0.05 to 0.1 in the above medium in the presence or absence of IPTG. Live cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) at ∼0.5 μg/ml in 0.5-ml aliquots just before placement on agarose.
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34. Rabbit antibodies to GFP were from Clontech (Palo Alto, CA). Immunofluorescence microscopy (IFM) was done essentially as described [E. J. Harry, K. Pogliano, R. Losick, ibid. 177, 3386 (1995); K. Pogliano, E. Harry, R. Losick, Mol. Microbiol. 18, 459 (1995)], except that centrifugation steps were replaced by filtration. Centrifugation of cells (even after fixation) caused PolC-GFP foci to appear at or near the cell poles.
35. We thank S. Sanders, P. Levin, K. Pogliano, and J. Roberts for advice; B. Cormack for gfpmut2; R. Losick, A.L. Sonenshein, and S. Bell for comments on the manuscript; and members of our lab for discussions and comments on the manuscript. Supported in part by PHS grant GM41934 (A.D.G.).