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Mutational analysis of the inducer recognition sites of the LysR-type transcriptional regulator TfdT of Burkholderia sp. NK8

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Abstract

TfdT is a LysR-type transcriptional regulator that activates the transcription of the chlorocatechol degradative gene operon tfdCDEF of the chlorobenzoate-degrading bacterium Burkholderia sp. NK8. To identify the amino acids involved in the effector recognition by TfdT, a polymerase-chain-reaction-based random mutagenesis protocol was applied to introduce mutations into the tfdT gene. Nine types of TfdT mutant bearing a single-amino-acid substitution at positions, Lys-129, Arg-199, Val-226, Val-246, and Pro-267 were obtained on the basis of their altered effector profiles and enhanced responses particularly to 2-chlorobenzoate, 2-aminobenzoate, and 2,6-dichlorobenzoate. All the TfdT mutants showed enhanced response to the effectors with a chloro-group in C-2 of benzoic acid. A homology model of wild-type TfdT was built on the basis of the crystal structure of CbnR with SwissModel. In this model, residues corresponding to the mutation sites of isolated TfdT mutants were located at the interface between the domains RD-I and RD-II. The findings that these TfdT mutants expressed altered effector specificities and enhanced responses to specific effectors suggest that these five residues are involved in effector binding by TfdT.

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References

  • Akakura R, Winans SC (2002) Constitutive mutations of the OccR regulatory protein affect DNA bending in response to metabolites released from plant tumors. J Biol Chem 277:5866–5874

    Article  CAS  Google Scholar 

  • Aldrich TL, Frantz B, Gill JF, Kilbane JJ, Chakrabarty AM (1987) Cloning and complete nucleotide sequence determination of the catB gene encoding cis, cis-muconate lactonizing enzyme. Gene 52:185–195

    Article  CAS  Google Scholar 

  • Bagdasarian M, Lurz R, Rückert B, Franklin FCH, Bagdasarian MM, Frey J, Timmis KN (1981) Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host–vector system for gene cloning in Pseudomonas. Gene 16:237–247

    Article  CAS  Google Scholar 

  • Cebolla A, Sousa C, de Lorenzo V (1997) Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers. J Biol Chem 272:3986–3992

    Article  CAS  Google Scholar 

  • Coco WM, Rothmel RK, Henikoff S, Chakrabarty AM (1993) Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida. J Bacteriol 175:417–427

    Article  CAS  Google Scholar 

  • Dangel AW, Gibson JL, Janssen AP, Tabita FR (2005) Residues that influence in vivo and in vitro CbbR function in Rhodobacter sphaeroides and identification of a specific region critical for co-inducer recognition. Mol Microbiol 57:1397–1414

    Article  CAS  Google Scholar 

  • de Lorenzo V, Timmis KN (1994) Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235:386–405

    Article  CAS  Google Scholar 

  • Ezezika OC, Haddad S, Clark TJ, Neidle EL, Momany C (2007) Distinct effector-binding sites enable synergistic transcriptional activation by BenM, a LysR-type regulator. J Mol Biol 367:616–629

    Article  CAS  Google Scholar 

  • Farinha MA, Kropinski AM (1990) Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol 172:3496–3499

    Article  CAS  Google Scholar 

  • Filer K, Harker AR (1997) Identification of the inducing agent of the 2, 4-dichlorophenoxyacetic acid pathway encoded by plasmid pJP4. Appl Environ Microbiol 63:317–320

    Article  CAS  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723

    Article  CAS  Google Scholar 

  • Henikoff S, Haughn GW, Calvo JM, Wallace JC (1988) A large family of bacterial activator proteins. Proc Natl Acad Sci U S A 85:6602–6606

    Article  CAS  Google Scholar 

  • Horton RM (1995) PCR-mediated recombination and mutagenesis. SOEing together tailor-made genes. Mol Biotechnol 3:93–99

    Article  CAS  Google Scholar 

  • Horvath B, Bachem CWB, Schell J, Kondorosi A (1987) Host-specific regulation of nodulation genes in Rhizobium is mediated by a plant-signal, interacting with the nodD gene product. Embo J 6:841–848

    Article  CAS  Google Scholar 

  • Klemba M, Jakobs B, Witthich R-M, Pieper D (2000) Chromosomal integration of tcb chlorocatechol degradation pathway genes as a means of expanding the growth substrate range of bacteria to include haloaromatics. Appl Environ Microbiol 66:3255–3261

    Article  CAS  Google Scholar 

  • Kokotek W, Lotz W (1989) Construction of a lacZ-kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene 84:467–471

    Article  CAS  Google Scholar 

  • Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RMII, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176

    Article  CAS  Google Scholar 

  • Lessner DJ, Parales RE, Narayan S, Gibson DT (2003) Expression of the nitroarene dioxygenase genes in Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 is induced by multiple aromatic compounds. J Bacteriol 185:3895–3904

    Article  CAS  Google Scholar 

  • Leveau JHJ, van der Meer JR (1996) The tfdR gene product can successfully take over the role of the insertion element-inactivated TfdT protein as a transcriptional activator of the tfdCDEF gene cluster, which encodes chlorocatechol degradation in Ralstonia eutropha JMP134(pJP4). J Bacteriol 178:6824–6832

    Article  CAS  Google Scholar 

  • Liu S, Ogawa N, Miyashita K (2001) The chlorocatechol degradative genes, tfdT-CDEF, of Burkholderia sp. strain NK8 are involved in chlorobenzoate degradation and induced by chlorobenzoates and chlorocatechols. Gene 268:207–214

    Article  CAS  Google Scholar 

  • Lochowska A, Iwanicka-Nowicka R, Plochocka D, Hryniewicz MM (2001) Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control. J Biol Chem 276:2098–2107

    Article  CAS  Google Scholar 

  • Maddocks SE, Oyston PC (2008) Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154:3609–3623

    Article  CAS  Google Scholar 

  • McFall SM, Chugani SA, Chakrabarty AM (1998) Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme. Gene 223:257–267

    Article  CAS  Google Scholar 

  • McFall SM, Parsek MR, Chakrabarty AM (1997) 2-Chloromuconate and ClcR-mediated activation of the clcABD operon: in vitro transcriptional and DNase I footprint analyses. J Bacteriol 179:3655–3663

    Article  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 352–355

    Google Scholar 

  • Muraoka S, Okumura R, Ogawa N, Nonaka T, Miyashita K, Senda T (2003) Crystal structure of a full-length LysR-type transcriptional regulator, CbnR: unusual combination of two subunit forms and molecular bases for causing and changing DNA bend. J Mol Biol 328:555–566

    Article  CAS  Google Scholar 

  • Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, Martins dos Santos VAP, Fouts DE, Gill SR, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy RT, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Chris Lee P, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen J, Timmis KN, Düsterhöft A, Tümmler B, Fraser CM (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808

    Article  CAS  Google Scholar 

  • Ogawa N, Chakrabarty AM, Zaborina O (2004) Degradative plasmids. In: Funnell BE, Phillips GJ (eds) Plasmid biology. American Society for Microbiology, Washington, D. C., pp 341–376

    Google Scholar 

  • Ogawa N, McFall SM, Klem TJ, Miyashita K, Chakrabarty AM (1999) Transcriptional activation of the chlorocatechol degradative genes of Ralstonia eutropha NH9. J Bacteriol 181:6697–6705

    Article  CAS  Google Scholar 

  • Panke S, Sánchez-Romero JM, de Lorenzo V (1998) Engineering of quasi-natural Pseudomonas putida strains for toluene metabolism through an ortho-cleavage degradation pathway. Appl Environ Microbiol 64:748–751

    Article  CAS  Google Scholar 

  • Pieper DH, Reineke W (2004) Degradation of chloroaromatics by Pseudomona(d)s. In: Ramos J-L (ed) Pseudomonas vol. 3. Kluwer/Plenum, New York, pp 509–574

    Chapter  Google Scholar 

  • Ramos JL, Michan C, Rojo F, Dwyer D, Timmis K (1990) 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

    Article  CAS  Google Scholar 

  • Reineke W (1998) Development of hybrid strains for the mineralization of chloroaromatics by patchwork assembly. Annu Rev Microbiol 52:287–331

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Schell MA (1993) Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47:597–626

    Article  CAS  Google Scholar 

  • Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385

    Article  CAS  Google Scholar 

  • Smirnova IA, Dian C, Leonard GA, McSweeney S, Birse D, Brzezinski P (2004) Development of a bacterial biosensor for nitrotoluenes: the crystal structure of the transcriptional regulator DntR. J Mol Biol 340:405–418

    Article  CAS  Google Scholar 

  • Tropel D, van der Meer JR (2004) Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474–500

    Article  CAS  Google Scholar 

  • Tyrrell R, Verschueren KHG, Dodson EJ, Murshudov GN, Addy C, Wilkinson AJ (1997) The structure of the cofactor-binding fragment of the LysR family member, CysB: a familiar fold with a surprising subunit arrangement. Structure 5:1017–1032

    Article  CAS  Google Scholar 

  • van der Meer JR, Frijters ACJ, Leveau JHJ, Eggen RIL, Zehnder AJB, de Vos WM (1991) Characterization of the Pseudomonas sp. strain P51 gene tcbR, a LysR-type transcriptional activator of the tcbCDEF chlorocatechol oxidative operon, and analysis of the regulatory region. J Bacteriol 173:3700–3708

    Article  Google Scholar 

  • Verschueren KHG, Tyrrell R, Murshudov GN, Dodson EJ, Wilkinson AJ (1999) Solution of the structure of the cofactor-binding fragment of CysB: a struggle against non-isomorphism. Acta Crystallogr D Biol Crystallogr 55:369–378

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Drs. Yoshiyuki Ohtsubo and Masataka Tsuda for helpful advice on the experiments. This work was supported by a Grant-in-aid (Hazardous Chemicals) from the Ministry of Agriculture, Forestry, and Fisheries of Japan (HC-07-2324-1).

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Correspondence to Naoto Ogawa.

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Lang, Gh., Ogawa, N. Mutational analysis of the inducer recognition sites of the LysR-type transcriptional regulator TfdT of Burkholderia sp. NK8. Appl Microbiol Biotechnol 83, 1085–1094 (2009). https://doi.org/10.1007/s00253-009-1960-5

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  • DOI: https://doi.org/10.1007/s00253-009-1960-5

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