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The xylS gene positive regulator of TOL plasmid pWWO: Identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon

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Summary

The Pseudomonas putida TOL plasmid pWWO carries an operon that specifies a meta-cleavage pathway for the catabolism of benzoate and toluates whose transcription is positively regulated by the xylS gene product. Stimulation of transcription of the operon is thought to result from activation of this protein by pathway substrates/effectors. In the present study, overexpression of the xylS gene has led to identification of the regulator as a 33 kDa protein. Overexpression of xylS also resulted in partially constitutive, i.e. effector-independent expression of the meta-cleavage operon. Determination of the polynucleotide sequence of the xylS gene revealed amino acid sequence homology with several DNA binding proteins, particularly with the araC products of Escherichia coli and Salmonella typhimurium and with the nifA and ntrC products of Klebsiella pneumoniae. Homologous sequences were mainly located in an α-helix-turn-α-helix domain of the polypeptide. Interestingly, amino acid sequence homology was also found with sigma factors of E. coli (ntrA and htpR products) and Bacillus subtilis (spoIIG and phage SPOI Gp34 products) and other RNA polymerase core-interacting proteins, such as the E. coli nusA product.

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References

  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterisation of new cloning vehicles. II. A multiple purpose cloning system. Gene 2:95–113

    Google Scholar 

  • Brosius J, Cate RL, Perlmotter AP (1982) Precise location of two promoters for the β-lactamase gene of pBR322. J Biol Chem 257:9205–9210

    Google Scholar 

  • Burton Z, Burgess RR, Lin J, Moore D, Holder S, Gross CA (1981) The sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E. coli K-12. Nucleic Acids Res 9:2889–2903

    Google Scholar 

  • Costanzo M, Brzustowicz L, Hannett N, Pero J (1984) Bacteriophages SPOI genes 33 and 34. Location and primary structure of genes encoding regulatory subunits of Bacillus subtilis RNA polymerase. J Mol Biol 180:533–547

    Google Scholar 

  • Chou PY, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45–148

    Google Scholar 

  • Dayhoff MO (1978) Atlas of Protein Sequence and Structure Vol 5B (suppl 3) Natl Biomed Res Found, Washington, DC pp 345–358

    Google Scholar 

  • Dente L, Cesarini G, Cortese R (1983) pEMBL: a new family of single stranded plasmids. Nucleic Acids Res 11:1645–1655

    Google Scholar 

  • Drumond N, Whitty P, Wooton J (1986) Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae homologies to other regulatory proteins. EMBO J 5:441–447

    Google Scholar 

  • Franklin FCH, Bagdasarian M, Bagdasarian MM, Timmis KN (1981) Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta-cleavage pathway. Proc Natl Acad Sci USA 78:7458–7462

    Google Scholar 

  • Franklin FCH, Lehrbach PR, Lurz R, Rückert B, Bagdasarian M, Timmis KN (1983) Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway. J Bacteriol 154:676–685

    Google Scholar 

  • Friedman DI, Olson ER (1983) Evidence that a nucleotide sequence “boxA” is involved in the action of the nusA protein. Cell 34:143–149

    Google Scholar 

  • Gitt MA, Wang L-F, Doi RH (1985) A strong sequence homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli. J Biol Chem 260:7178–7185

    Google Scholar 

  • Greenblatt J, Li J (1981) Interaction of the sigma factor and the nusA gene protein of E. coli with RNA polymerase in the initiation-termination cycle of transcription. Cell 24:421–428

    Google Scholar 

  • Guyer MS (1978) The γδ sequence of F is an insertion sequence. J Mol Biol 1126:347–365

    Google Scholar 

  • Harayama S, Leppik RA, Rekik M, Mermod N, Lehrbach PR, Reineke W, Timmis KN (1986a) Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzylalcohol by the xylA product. J Bacteriol 167:455–460

    Google Scholar 

  • Harayama S, Rekik M, Timmis KN (1986b) Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWWO of Pseudomonas putida. Mol Gen Genet 202:226–234

    Google Scholar 

  • Inouye S, Nakazawa A, Nakazawa T (1981) Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS. J Bacteriol 148:413–418

    Google Scholar 

  • Inouye S, Nakazawa A, Nakazawa T (1983) Molecular cloning of the regulatory gene xylR and the operator-promoter regions of xylABC and xylDEGF operons of TOL plasmid. J Bacteriol 155:1192–1199

    Google Scholar 

  • Inouye S, Nakazawa A, Nakazawa T (1984a) Nucleotide sequence surrounding the transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida. Proc Natl Acad Sci USA 81:1688–1691

    Google Scholar 

  • Inouye S, Nakazawa A, Nakazawa T (1984b) Nucleotide sequence of the promoter region of the xylDEFG operon on TOL plasmid of Pseudomonas putida. Gene 29:323–330

    Google Scholar 

  • Inouye S, Nakazawa A, Nakazawa T (1986) Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product. Gene 44:235–242

    Google Scholar 

  • Ishii S, Ihara M, Maekawa T, Nakamura Y, Uchida H, Imamoto F (1984) The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia coli. Nucleic Acids Res 12:3333–3342

    Google Scholar 

  • Kieny MP, Lathe R, Lecocq JP (1983) New versatile cloning and sequencing vectors based on bacteriophage M13. Gene 26:91–99

    Google Scholar 

  • Kunz DA, Chapman PJ (1981) Catabolism of pseudocumene and 3-ethyltoluene by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL (pWWO) plasmid. J Bacteriol 146:179–191

    Google Scholar 

  • Landick R, Vaughn V, Lau ET, Van Bogelen RA, Erickson JW, Neidhardt FC (1984) Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor. Cell 38:175–182

    Google Scholar 

  • Lorence MC, Alcorn JL, Rupert CS (1984) Construction of an improved maxicell strain for the identification of recombinant plasmid encoded proteins. In: Helinski DR, Cohen SN, Clewell DW, Jackson DA, Hollaender A (eds) Plasmids in Bacteria, Plenum Press, New York, p 955

    Google Scholar 

  • Mermod N, Lehrach PR, Reineke W, Timmis KN (1984) Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters. EMBO J 3:2461–2466

    Google Scholar 

  • Mermod N, Harayama S, Timmis KN (1986a) New route to bacterial indigo production. Bio/Technology 4:321–324

    Google Scholar 

  • Mermod N, Lehrbach PR, Don R, Timmis KN (1986b) Gene cloning and manipulation in Pseudomonads. In: Sokatch JR (ed) The Bacteria, Vol X. Acad Press, New York, pp 325–355

    Google Scholar 

  • Mermod N, Ramos JL, Lehrbach PR, Timmis KN (1986c) Vector for regulated expression of cloned genes in a wide range of Gram negative bacteria. J Bacteriol 167:447–454

    Google Scholar 

  • Merrick MJ, Gibbins, JR (1985) The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. Nucleic Acids Res 13:6707–7620

    Google Scholar 

  • Miyada CG, Horwitz AH, Cass LG, Timko HJ, Wilcox G (1980) DNA sequence of the araC regulatory gene from Escherichia coli B/r. Nucleic Acids Res 8:5267–5274

    Google Scholar 

  • Needleman SB, Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48:443–453

    Google Scholar 

  • Pabo CO, Sauer RT (1984) Protein-DNA recognition. Annu Rev Biochem 53:293–321

    Google Scholar 

  • Prentki P, Krisch HM (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313

    Google Scholar 

  • Raibaud O, Schwarz M (1984) Positive control of transcription initiation in bacteria. Annu Rev Genet 18:173–206

    Google Scholar 

  • Remaut E, Stanssens P, Fiers W (1983a) Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli. Nucleic Acids Res 11:4677–4688

    Google Scholar 

  • Remaut E, Tsao H, Fiers W (1983b) Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene 22:103–113

    Google Scholar 

  • Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693

    Google Scholar 

  • Spooner AR, Lindsay K, Franklin FCH (1986) Genetic, functional and sequence analysis of the xylR and xylS regulatory genes of the TOL plasmid pWWO. J Gen Microbiol 132:1347–1358

    Google Scholar 

  • Stragier P, Bouvier J, Bonamy C, Szulmajster J (1984) A developmental gene product of Bacillus subtilis homologous to the sigma factor of Escherichia coli. Nature 312:367–378

    Google Scholar 

  • Viera J, Messing J (1982) The pUC plasmids, an M13 mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

  • Worsey MJ, Williams PA (1975) Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol 124:7–13

    Google Scholar 

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Communicated by W. Arber

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Mermod, N., Ramos, J.L., Bairoch, A. et al. The xylS gene positive regulator of TOL plasmid pWWO: Identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon. Mol Gen Genet 207, 349–354 (1987). https://doi.org/10.1007/BF00331600

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