Summary
A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 by synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor.
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References
Anagnostopoulos C, Spizizen I (1960) Requirements for transformation in Bacillus subtilis. J Bacteriol 81:741–746
Batt CA, Bodies MS, Picataggio SK, Claps MC, James S, Sinskey AJ (1985) Analysis of xylose operon regulation by ud (Apr, lac) fusion: trans effect of plasmid coded xylose operon. Can J Microbiol 31:930–933
Birnboim HC, Doly I (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Boyer HW, Roulland-Dussiox D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472
Cariell HL, Glusker JP, Burger V, Manfie F, Tritsch D, Biellmann JF (1989) X-ray analysis of d-xylose isomerase at 1.9 Å: Native enzyme in complex with substrate and with a mechanismdesigned inactivator. Proc Natl Acad Sci USA 86:4440–4444
Cohen SN, Chang ACY, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation in Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
Dodd IB, Egan JB (1990) Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. Nucleic Acids Res 19:5019–5026
Gärtner D, Geissendörfer M, Hillen W (1988) Expression of the Bacillus subtilis xyl operon is repressed at the level of transcription and is induced by xylose. J Bacteriol 170:3102–3109
Hardy KG (1985) Bacillus cloning methods, In: Glover DM (ed) DNA cloning, a practical approach, vol 2. IRL Press, Oxford, Washington DC, pp 1–17
Harrison SC, Aggarwal AK (1990) DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem 59:933–969
Hillen W, Klein RD, Wells RD (1981) Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments. Biochemistry 20:3748–3756
Hochschild A, Ptashne M (1986) Cooperative binding of λ repressors to sites separated by integral turns of the DNA helix. Cell 44:681–687
Jacob S, Allmansberger R, Gartner D, Hillen W (1991) Catabolite repression of the operon for xylose utilization from Bacillus subtilis W23 is mediated at the level of transcription and depends on a cis site in the xylA reading frame. Mol Gen Genet 229:189–196
Klein RD, Selsing E, Wells RD (1980) A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid 3:88–91
Koudelka GB, Harrison SC, Ptashne M (1987) Effect of noncontacted bases on the affinity of 434 operator for 434 repressor and Cro. Nature 326:886–888
Krämer H, Niemöller N, Amouyal M, Revet B, von Wilcken-Bergmann B, Müller-Hüll B (1987) Lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J 6:1481–1491
Kreuzer P, Gartner D, Allmansberger R, Hillen W (1989) Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J Bacteriol 171:3840–3845
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-directed mutagenesis without phenotypic selection. Methods Enzymol 154:367–382
Kuwabara MD, Sigman DS (1987) Footprinting DNA-protein complexes in situ following gel retardation assays using, 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes. Biochemistry 26:7234–7238
Maniatis T, Fritsch EF, Sambrook I (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavage. Methods Enzymol 65:499–560
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 352–355
Shimotsu H, Henner D (1986) Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 43:85–94
Unger B, Klock G, Hillen W (1984) Nucleotide sequence of the repressor gene of the RA1 tetracycline resistance determinant: structural and functional comparison with three related Tet repressor genes. Nucleic Acids Res 12:7693–7703
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Gärtner, D., Degenkolb, J., Ripperger, J.A.E. et al. Regulation of the Bacillus subtilis W23 xylose utilization operon : interaction of the Xyl repressor with the xyl operator and the inducer xylose. Molec. Gen. Genet. 232, 415–422 (1992). https://doi.org/10.1007/BF00266245
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DOI: https://doi.org/10.1007/BF00266245