Abstract
Full Text
Role of Bacillus subtilis SpoIIIE in DNA Transport Across the Mother Cell-Prespore Division Septum
Jonathan Bath, Ling Juan Wu, Jeffery Errington, and James C. Wang

Supplementary Material

Supplemental Figure 1. Targeting of SpoIIIE to the division septum by the NH2-terminal domain of the protein. B. subtilis strain 1257 cells expressing the NH2-terminal portion of SpoIIIE fused to the green fluorescent protein (1) were induced to sporulate (2, 3) and viewed directly under the fluorescence microscope. The fusion protein was seen to localize to the central division septa in vegetative cells (arrowheads) and to the asymmetric division septa in sporulating cells (arrows). Fluorescence and phase-contrast images of the same cells are shown in (A) and (B), respectively.


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Overexpression and purification of the cytoplasmic part of SpoIIIE.
DNA corresponding to residues 177 to 787 of SpoIIIE was first amplified from B. subtilis strain SG38 DNA by the polymerase chain reaction, using a pair of primers 5'-GATCGGAATGACGGCCGTAACAGGACGCTCGC-3' and 5'-CTCCCTTCCTGCAGAAGAGAGCTCATC-3'. The PCR product was partially digested with Eag I, filled with the Klenow fragment of Escherichia coli DNA polymerase I, and then digested with Pst I. Insertion of the resulting fragment between the Bsa I and Pst I sites of pASK75 (4) yielded plasmid pSG4909. In pSG4909, a SpoIIIE fusion protein, consisting of an OmpA tag at the NH2-terminus, SpoIIIE residues 177 to 787 in the middle, and a 10-residue peptide that specifically binds to streptavidin at the COOH-terminus, is expressed from a tetracycline-inducible promoter of the tetA gene. A similar strategy was used to generate pSG4910, in which the SpoIIIE segment carried the mutation K473A. To obtain pJB103, complementary oligonucleotides 5'-GGTCACCACCACCACCACCACTAATAAA-3' and 5'-AGCTTTTATTAGTGGTGGTGGTGGTGGTGACCTGCA-3' were annealed and introduced between the Pst I and Hind III sites of pSG4909. In pJB103, the streptavidin affinity tag was replaced by a hexahistidine tag. E. coli DH5α cells harboring pJB103 were grown at 37°C to midlog phase and were then induced by addition of anhydrotetracycline to 4 μg/ml. Cells were harvested after 2 hours, and the tagged protein was purified by affinity chromatography on Ni(II)-agarose resin. Approximately 0.1 mg of the recombinant protein was obtained from a liter of culture.

Assay of SpoIIIE-mediated supercoiling of DNA.
A 3.7-kb pUC18 derivative was used throughout this study. The plasmid was first relaxed by treatment with vaccinia virus topoisomerase, and assays were performed in 20 mM Tris-HCl, pH 7.4, 30 mM NaCl, 10 mM MgCl2, 1 mM ATP, and 10 μg/ml of bovine serum albumin. Each 80 μl of reaction mixture contained approximately 1 μg of relaxed plasmid DNA, 0.4 μg of SpoIIIE, and 1.0 μg of E. coli DNA topoisomerase I. The NaCl concentration in the assay buffer was kept at a minimum to improve the processivity of E. coli DNA topoisomerase I (5).

Measurements of ATPase activity.

The ATPase activity was measured at room temperature in a buffer containing 20 mM Tris-HCl, pH 7.4, 8 mM MgCl2, 2.5 mM dithiothreitol, 0.5 mg/ml bovine serum albumin, 0.1 mM NADH, 2 mM phosphoenolpyruvate, 3 units of pyruvate kinase and 4 units of lactate dehydrogenase, using a spectrophotometric assay in which the oxidation of NADH was coupled to the regeneration of ATP [see, for example (6)].

Two-dimensional gel electrophoresis.
Two-dimensional gel electrophoresis was first done in 90 mM Tris base, 90 mM boric acid, and 1.8 mM EDTA at 1.2 V/cm for 16 hours; the gel was then equilibrated with the same buffer plus 1.3 μM chloroquine and run in the second dimension at 1.0 V/cm for 20 hours. As a reference, a topoisomer ladder was generated by mixing samples of the 3.7-kb plasmid substrate treated with E. coli DNA topoisomerase I in the presence of varying amounts of ethidium bromide.

REFERENCES AND NOTES

1. A DNA fragment encoding the NH2-terminal portion of SpoIIIE (residues 2 to 183) was amplified by PCR from strain SG38 DNA, using primers 5'-CAGTAAGGTGATGCTCGAGGCAAAGAAAAAACG-3' and 5'-CCACTTTTTAAGCTTTTCTTGCAGCGAGCG-3'. The PCR product was inserted between the Xho I and Hind III sites of pSG1154 to give pSG4912. In pSG4912, expression of the fusion protein, comprised of residues 2 to 183 of SpoIIIE on the NH2-terminal side and the green fluorescence protein on the COOH-terminal side, is from the inducible Pxyl promoter. This plasmid was used to transform a spoIIIE null mutant to give strain 1257.

2. S. R. Partridge, J. Errington, Mol. Micriobiol.8, 945 (1993)

3. Strain 1257 was grown in medium containing 0.5 % xylose to midlog phase, and was induced to sporulate in a medium lacking xylose. After 30 min, xylose was added to 0.1 %. Untreated cells were viewed directly under the fluorescence microscope.

4. A. Skerra, Gene151, 131 (1994).

5. J. C. Wang, J. Mol. Biol.55, 523 (1971).

6. S. W. Morrical, J. Lee, M. M. Cox, Biochemistry8, 1482 (1986).