Abstract
The toluate catabolic operon carried by the TOL plasmid pWWO of Pseudomonas putida is positively regulated by the benzoate-responsive XylS protein which, when activated, stimulates transcription from the operon promoter Pm. In this study, the mode in which XylS effects the activity of the Pm promoter was examined in vivo by genetic analysis of both protein and promoter variants. Substitution of His3lAsp/ Ser32Pro,Leu113Pro,Phe214Leu/G1u215Asp/Arg216Pro or Thr312Pro, all predicted to disrupt the secondary structure, renders XylS inactive and unable to compete with the native protein for activation of Pm. In contrast, Pro substitutions at Ser64 or GIy160 had no or only minor effects on XyIS activity, and a protein with residual capacity to activate Pm resulted when Glu11 was altered to Pro. None of a collection of truncated variants of XyIS protein deleted for more than 10 terminal amino acids retained activity or were able to negatively interact the wild-type XylS protein. These data indicate that amino- and carboxy-terminal portions of the protein sequence depend on each other for the maintenance of their functional structure, rather than being arranged as independent domains. Pm promoter derivatives, in which the two half-sites of the direct repeat sequence that confers responsiveness to XylS were separated and/or inverted by one-half or one complete DNA helix turn, lost responsiveness in vivo. These results favour a model for XyIS-mediated activation of Pm in which a tandem dimer is the protein form that binds the Pm promoter.
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Assinder SJ, De Marco P, Osborne DJ, Poh CL, Shaw LE, Winson MK, Williams PA (1993) A comparison of the multiple alleles of xylS carried by TOL plasmids pWW53 and pDK1 and its implications for their evolutionary relationship. J Gen Microbol 139:557–568
Brunelle A, Schleif R (1983) Determining residue-base interactions between AraC protein and araI DNA. J Mol Biol 209:607–622
Bustos SA, Schleif R (1993) Functional domains of the AraC protein. Proc Natl Acad Sci USA 90:5638–5642
Carra JH, Schleif R (1993) Variation of half-site organization and DNA looping by AraC protein. EMBO J 12:35–44
Chou PY, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45–148
Collado-Vides J, Magasanik B, Gralla JD (1991) Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev 55:371–394
de Lorenzo V, Herrero M, Jacubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172:6568–6572
de Lorenzo V, Eltis L, Kessler B, Timmis KN (1993) Analysis of Pseudomonas gene products using lacl q/Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 123:17–24
Fellay R, Frey J, Krisch, H (1987) Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram negative bacteria. Gene 52:147–154
Gallegos MT, Michan C, Ramos JL (1993) The Xy1S/AraC family of regulators. Nucleic Acids Res 21:807–810
Harayama S, Rekik M, Bairoch A, Neidle EL, Ornston LN (1991) Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoacenticus chromosomal benABC and Pseudomonas putida TOL pWWO plasmid xylXYZ, genes encoding benzoate dioxygenases. J Bacteriol 173:7540–7548
Herrero M, de Lorenzo V, Timmis KN (1990) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 172:6557–6567
Inouye S, Nakazawa A, Nakazawa T (1984) Nucleotide sequence of the promoter region of the xylDEFG operon on TOL plasmid of Pseudomonas putida. Gene 29:323–30
Inouye S, Nakazawa A, Nakazawa T (1987a) Overproduction of the xylS gene product and activation of the xylDLEGF operon of the TOL plasmid. Bacteriol 169:3587–3592
Inouye S, Nakazawa A, Nakazawa T (1987b) Expression of the regulatory gene xylS on the TOL plasmid is positively controlled by the xylR gene product. Proc Natl Acad Sci USA 84:5182–5186
Kessler B, de Lorenzo V, Timmis KN (1992) A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233:293–301
Kessler B, de Lorenzo V, Timmis KN (1993) Identification of a cisacting sequence within the Pm promoter of the TOL plasmid which confers XylS-mediated responsiveness to substituted benzoates. J Mol Biol 230:699–703
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382
Lauble H, Georgalis D, Heinemann U (1989) Studies on the domain structure of the Salmonella typhimurium AraC protein. Eur J Biochem 185:319–325
Laudet V, Stehelin D (1992) Flexible friends. Curr Biol 2:293–295
Lee DH, Schleif R (1989) In vivo DNA loops in araBAD: size limits and helical repeat. Proc Natl Acad Sci USA 86:476–480
Lobell RB, Schleif R (1990) DNA looping and unlooping by AraC protein. Science. 250:528–532
Lu Y, Flaherty C, Hendrickson W (1992) AraC protein contacts asymmetric sites in the Escherichia coli araFGH promoter. J Biol Chem 267:24848–24857
Maeda S, Mizuno T (1990) Evidence for multiple OmpR-binding sites in the upstream activation sequence of the ompC promoter in Escherichia coli: a single OmpR binding site is capable of activating the promoter. J Bacteriol 172:501–503
Manoil C, Beckwith J (1985) TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci USA 82:8129–8133
Menon KP, Lee NL (1990) Activation of ara operons by a truncated AraC protein does not require inducer. Proc Natl Acad Sci USA 87:3708–3712
Mermod N, Ramos JL, Lehrbach PR, Timmis KN (1986) Vector for regulated expression of cloned genes in a wide range of Gram-negative bacteria. J Bacteriol 167:447–454
Michan C, Kessler B, de Lorenzo V, Timmis KN, Ramos JL (1992a) XylS domain interactions can be deduced from intra-allelic domainance in double mutants of Pseudomonas putida. Mol Gen Genet 235:406–412
Michan C, Zhou L, Gallegos MT, Timmis KN, Ramos JL (1992b) Identification of critical amino terminal regions of XylS. J Biol Chem 267:22897–22901
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Myers RM, Lerman LS, Maniatis T (1985) A general method for saturation mutagenesis of cloned DNA fragments. Science 229:242–247
Pabo CO, Sauer RT (1984) Protein-DNA recognition. Annu Rev Biochem 53:293–321
Raibaud O (1989) Nucleoprotein structures at positively regulated bacterial promoters: homology with replication origins and some hypotheses on the quaternary structure of the activator proteins in these complexes. Mol Microbiol 3:455–458
Ramos JL, Mermod N, Timmis KN (1987) Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas. Mol Microbiol 1:293–300
Ramos JL, Rojo F, Timmis KN (1990a) A family of positive regulators related to the Pseudomonas putida TOL plasmid XylS and the Escherichia coli AraC activators. Nucleic Acids Res 18:2149–2152
Ramos JL, Michan C, Rojo F, Dwyer D, Timmis KN (1990b) Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon. J Mol Biol 211:373–382
Russel M, Model P (1984) Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol 159:1034–1039
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Schleif R (1988) DNA binding proteins. Science 241:1182–1187
Simon R, Priefer U, Pühler A (1983) A broad hot range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784–791
Su GF, Brahmbhatt HN, Wehland J, Rohde M, Timmis KN (1992) Construction of stable LamB-shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-specific mucosal and serum antibody responses. Infect Immun 60:3345–3359
Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078
Travers AA (1989) DNA conformation and protein binding. Annu Rev Biochem 58:427–452
Zaballos A, Salas M, Mellado RP (1987) A set of expression plasmids for the synthesis of fused and unfused polypeptides in Escherichia coli. Gene 58:67–76
Zhou L, Timmis KN, Ramos JL (1990) Mutations leading to constitutive expression from the TOL plasmid meta-cleavage pathway operon are located at the C-terminal end of the positive regulator protein XylS. J Bacteriol 172:3707–3710
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Communicated by W. Goebel
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Kessler, B., Timmis, K.N. & de Lorenzo, V. The organization of the Pm promoter of the TOL plasmid reflects the structure of its cognate activator protein XylS. Molec. Gen. Genet. 244, 596–605 (1994). https://doi.org/10.1007/BF00282749
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DOI: https://doi.org/10.1007/BF00282749