• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Nov 1996; 178(22): 6427–6434.
PMCID: PMC178527

The TACAN4TGCA motif upstream from the -35 region in the sigma70-sigmaS-dependent Pm promoter of the TOL plasmid is the minimum DNA segment required for transcription stimulation by XylS regulators.

Abstract

Transcription from the TOL plasmid meta-cleavage pathway operon promoter Pm is dependent on the XylS regulator activated by benzoate effectors or after XylS overproduction. We have generated 5' deletions in Pm and have analyzed expression from wild-type and mutant promoters with the wild-type XylS regulator and XylS mutant regulators that stimulated transcription constitutively. We have found that the motifs T(C or A)CAN4TGCA located between -46 and -57 and -67 and -78 with respect to the main transcription initiation point are required for maximal stimulation of transcription from Pm with effector-activated wild-type XylS. Deletion of the farthest TCCA submotif decreased but did not abolish transcription mediated by the pair XylS with 3-methylbenzoate; however, removal of the motif between -67 and -78 resulted in the loss of stimulation by the wild-type regulator. XylSG44S and XylSS229I stimulated high levels of transcription in the absence of effectors from the wild-type promoter and from a mutant promoter exhibiting only the -46 to -57 motif only when an effector was present. The point mutation Pm5U (with C-47 replaced by G [C-47-->G]) and Pm4 (C-68-->G), located in each 3' TGCA submotif of each motif, resulted in a 90% decrease in transcription stimulation with wild-type XylS; however, the mutant XylSS229I stimulated high levels of transcription from the point mutation promoters both in the presence and in the absence of effectors, while mutant XylSG44S suppressed the two point mutations only with 3-methylbenzoate. Overexpression of XylS and XylSG44S allowed the two regulators to stimulate high levels of transcription from the wild-type promoter, the point mutation Pm4 and Pm5U promoters, and deltaPm promoters exhibiting at least the -46 to -57 motif. Therefore the TACAN4TGCA motif between -46 and -57 represents the minimal DNA segment required for stimulation of transcription from Pm.

Full Text

The Full Text of this article is available as a PDF (298K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Brunelle A, Schleif R. Determining residue-base interactions between AraC protein and araI DNA. J Mol Biol. 1989 Oct 20;209(4):607–622. [PubMed]
  • Collado-Vides J, Magasanik B, Gralla JD. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. [PMC free article] [PubMed]
  • Egan SM, Schleif RF. A regulatory cascade in the induction of rhaBAD. J Mol Biol. 1993 Nov 5;234(1):87–98. [PubMed]
  • Franklin FC, Bagdasarian M, Bagdasarian MM, Timmis KN. 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 U S A. 1981 Dec;78(12):7458–7462. [PMC free article] [PubMed]
  • Gallegos MT, Michán C, Ramos JL. The XylS/AraC family of regulators. Nucleic Acids Res. 1993 Feb 25;21(4):807–810. [PMC free article] [PubMed]
  • Harayama S, Rekik M. The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes. Mol Gen Genet. 1990 Mar;221(1):113–120. [PubMed]
  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. [PubMed]
  • Inouye S, Nakazawa A, Nakazawa T. Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS. J Bacteriol. 1981 Nov;148(2):413–418. [PMC free article] [PubMed]
  • Inouye S, Nakazawa A, Nakazawa T. Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida. Gene. 1984 Sep;29(3):323–330. [PubMed]
  • Inouye S, Nakazawa A, Nakazawa T. Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product. Gene. 1986;44(2-3):235–242. [PubMed]
  • Inouye S, Nakazawa A, Nakazawa T. Overproduction of the xylS gene product and activation of the xylDLEGF operon on the TOL plasmid. J Bacteriol. 1987 Aug;169(8):3587–3592. [PMC free article] [PubMed]
  • Kessler B, de Lorenzo V, Timmis KN. Identification of a cis-acting sequence within the Pm promoter of the TOL plasmid which confers XylS-mediated responsiveness to substituted benzoates. J Mol Biol. 1993 Apr 5;230(3):699–703. [PubMed]
  • Lange R, Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol. 1991 Jan;5(1):49–59. [PubMed]
  • Marqués S, Gallegos MT, Ramos JL. Role of sigma S in transcription from the positively controlled Pm promoter of the TOL plasmid of Pseudomonas putida. Mol Microbiol. 1995 Dec;18(5):851–857. [PubMed]
  • Marqués S, Holtel A, Timmis KN, Ramos JL. Transcriptional induction kinetics from the promoters of the catabolic pathways of TOL plasmid pWW0 of Pseudomonas putida for metabolism of aromatics. J Bacteriol. 1994 May;176(9):2517–2524. [PMC free article] [PubMed]
  • Marqués S, Ramos JL. Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways. Mol Microbiol. 1993 Sep;9(5):923–929. [PubMed]
  • Marqués S, Ramos JL, Timmis KN. Analysis of the mRNA structure of the Pseudomonas putida TOL meta fission pathway operon around the transcription initiation point, the xylTE and the xylFJ regions. Biochim Biophys Acta. 1993 Nov 16;1216(2):227–236. [PubMed]
  • Mermod N, Lehrbach PR, Reineke W, Timmis KN. 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. 1984 Nov;3(11):2461–2466. [PMC free article] [PubMed]
  • Mermod N, Ramos JL, Bairoch A, Timmis KN. 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. 1987 May;207(2-3):349–354. [PubMed]
  • Michan C, Zhou L, Gallegos MT, Timmis KN, Ramos JL. Identification of critical amino-terminal regions of XylS. The positive regulator encoded by the TOL plasmid. J Biol Chem. 1992 Nov 15;267(32):22897–22901. [PubMed]
  • Ramos JL, Mermod N, Timmis KN. Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas. Mol Microbiol. 1987 Nov;1(3):293–300. [PubMed]
  • Ramos JL, Stolz A, Reineke W, Timmis KN. Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8467–8471. [PMC free article] [PubMed]
  • Spooner RA, Lindsay K, Franklin FC. Genetic, functional and sequence analysis of the xylR and xylS regulatory genes of the TOL plasmid pWW0. J Gen Microbiol. 1986 May;132(5):1347–1358. [PubMed]
  • Tobin JF, Schleif RF. Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes. J Mol Biol. 1987 Aug 20;196(4):789–799. [PubMed]
  • Webster C, Kempsell K, Booth I, Busby S. Organisation of the regulatory region of the Escherichia coli melibiose operon. Gene. 1987;59(2-3):253–263. [PubMed]
  • Worsey MJ, Williams PA. Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol. 1975 Oct;124(1):7–13. [PMC free article] [PubMed]
  • Zhou LM, Timmis KN, Ramos JL. 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. 1990 Jul;172(7):3707–3710. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...