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BLAST searches were performed with the data-base available at http://www.ncbi.nlm.nih.gov/BLAST/ unfinishedgenome.html. Sequence analysis was performed with GCG software (Madison, WI) and MACBOXSHADE (Institute for Animal Health, Pirbright, UK).
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15
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0001284908
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2-terminal polyhistidine tag, and the recombinant protein was purified by nickel-chelate chromatography (Qiagen, Valencia, CA). Holo-DrBphP was assembled with PCB and PΦB as described for Cph1 (4, 6). Adducts were visualized by zinc-induced fluorescence of the chromoprotein following denaturing SDS-PAGE (11). Difference spectra were obtained after saturating R (660 nm) and FR (730 nm) irradiations (4).
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0343290080
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note
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We analyzed proteins and peptides using the API 365 LC/MS system (Perkin-Elmer, Norwalk, CT) with a C18 column (5 μm, 1.0 mm by 150 mm) (Vydac, Hesperia, CA) and a 0 to 95% water-acetonitrile gradient in 0.05% trifluoroacetic acid for LC. Peptide hydrolysis was accomplished by incubating the DrBphP holoprotein for 16 hours in the dark at ∼20°C in 70% formic acid with a 200-fold molar excess of CNBr over methionine.
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17
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0342855102
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note
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Site-directed mutations were introduced by Quikchange (Stratagene, La Jolla, CA) for DrBphP (M259A) and by overlap-extension PCR for DrBphP (M259C), DrBphP (C289A), and DrBphP (H260A).
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19
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0031012287
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L. Lemee, E. Peuchant, M. Clerc, M. Brunner, H. Pfander, Tetrahedron 53, 919 (1997).
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Brunner, M.4
Pfander, H.5
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0342855100
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note
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+ (Stratagene). Sequences flanking DrBphP, DrBphR, or both were amplified by PCR from D. radiodurans DNA and introduced into pFSI31 so that, after homologous integration, the respective loci would be exactly replaced by the chloroamphenicol-resistance marker. Transformation and mutant identification was according to (19), and mutants were identified and confirmed by Southern blotting (10). Cells were irradiated with varying fluences of white light provided by a sodium lamp supplemented with fluorescent lighting. Deinoxanthin was measured spectrophotometrically following methanol extraction, as described (19).
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22
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0027262177
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P. D. Gutman, P. Fuchs, L. Ouyang, K. W. Minton, J. Bacteriol. 175, 3581 (1993).
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Minton, K.W.4
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23
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0342855098
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note
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We thank K. Minton for supplying D. radiodurans R1 and the pI3 plasmid, O. White for discussions on the D. radiodurans sequence, The Institute for Genomic Research and the Pseudomonas Genome Project for distributing the genomic sequences described here, S. H. Bhoo and P.-S. Song for providing PCB and PΦB, and A. Harms for assistance with MS. We dedicate this paper to the memory of Rudy J. Wodzinski. This work was supported by an NIH predoctoral fellowship (S.J.D.) and U.S. Department of Energy grant DE-FG02-88ER-13968 (R.D.V.).
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