Identification and Expression of the cym, cmt, and tod Catabolic Genes from Pseudomonas putida KL47: Expression of the Regulatory todST Genes as a Factor for Catabolic Adaptation

  • Lee Kyoung (Department of Microbiology, Changwon National University) ;
  • Ryu Eun-Kyeong (Department of Microbiology, Changwon National University) ;
  • Choi Kyung-Soon (Department of Microbiology, Changwon National University) ;
  • Cho Min-Chul (Department of Bioscience & Biotechnology, Konkuk University) ;
  • Jeong Jae-Jun (Department of Microbiology, Changwon National University) ;
  • Choi Eun-Na (Department of Microbiology, Changwon National University) ;
  • Lee Soo-O (Department of Microbiology, Changwon National University) ;
  • Yoon Do-Young (Department of Bioscience & Biotechnology, Konkuk University) ;
  • Hwang In-Gyu (School of Agricultural Biotechnology, Seoul National University) ;
  • Kim Chi-Kyung (Department of Microbiology, Chungbuk National University)
  • Published : 2006.04.01

Abstract

Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene $(C_1-C_4)$, biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99 %) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

Keywords

References

  1. Altschul, S.F., T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389- 3402 https://doi.org/10.1093/nar/25.17.3389
  2. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1990. Current Protocols in Molecular Biology. John Wiely and Sons, New York, NY
  3. Bertani, L.E. and G. Bertani. 1970. Preparation and characterization of temperate, non-inducible bacteriophage P2 (host: Escherichia coli). J. Gen. Virol. 6, 201-212 https://doi.org/10.1099/0022-1317-6-2-201
  4. Cho, M.C., D.-O. Kang, B.D. Yoon, and K. Lee. 2000. Toluene degradation pathway from Pseudomonas putida F1: substrate specificity and gene induction by 1-substituted benzenes. J. Ind. Microbiol. Biotechnol. 25, 163-170 https://doi.org/10.1038/sj.jim.7000048
  5. Cho, M.C., H.S. Lee, J.H. Kim, Y.K. Choe, J.T. Hong, S.G. Paik, and D.Y. Yoon. 2003. A simple ELISA for screening ligands of peroxisome proliferator-activated receptorgamma. J. Biochem. Mol. Biol. 36, 207-213 https://doi.org/10.5483/BMBRep.2003.36.2.207
  6. Choi, E.N., M.C. Cho, Y. Kim, C.K. Kim, and K. Lee. 2003. Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively. Microbiology 149, 795-805 https://doi.org/10.1099/mic.0.26046-0
  7. Dunn, N.W. and I.C. Gunsalus. 1973. Transmissible plasmid coding early enzymes of naphthalene oxidation in Pseudomonas putida. J. Bacteriol. 114, 974-979
  8. Eaton, R.W. 1996. p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon. J. Bacteriol. 178, 1351-1362 https://doi.org/10.1128/jb.178.5.1351-1362.1996
  9. Eaton, R.W. 1997. p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate. J. Bacteriol. 179, 3171-3180 https://doi.org/10.1128/jb.179.10.3171-3180.1997
  10. Faizal, I., K. Dozen, C.S. Hong, A. Kuroda, N. Takiguchi, H. Ohtake, K. Takeda, H. Tsunekawa, and J. Kato. 2005. Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system. J. Ind. Microbiol. Biotechnol. 32, 542-547 https://doi.org/10.1007/s10295-005-0253-y
  11. Finette, B.A., V. Subramanian, and D.T. Gibson. 1984. Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system. J. Bacteriol. 160, 1003-1009
  12. Furukawa, K., J. Hirose, A. Suyama, T. Zaiki, and S. Hayashida. 1993. Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon). J. Bacteriol. 175, 5224-5232 https://doi.org/10.1128/jb.175.16.5224-5232.1993
  13. Gibson, D.T., M. Hensley, H. Yoshioka, and T.J. Mabry. 1970. Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6- diene from toluene by Pseudomonas putida. Biochemistry 9, 1626-1630 https://doi.org/10.1021/bi00809a023
  14. Gibson, D.T., J.R. Koch, and R.E. Kallio. 1968. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene. Biochemistry 7, 2653-2662 https://doi.org/10.1021/bi00847a031
  15. Jeong, J.J., J.H. Kim, C.K. Kim, I. Hwang, and K. Lee. 2003. 3- and 4-alkylphenol degradation pathway in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase. Microbiology 149, 3265- 3277 https://doi.org/10.1099/mic.0.26628-0
  16. Johnson, J.L. 1994. Similarity analyses of rRNAs, p. 683-700. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C
  17. Kim, D., J.C. Chae, G.J. Zylstra, H.Y. Sohn, G.S. Kwon, and E. Kim. 2005. Identification of two-component regulatory genes involved in o-xylene degradation by Rhodococcus sp. strain DK17. J. Microbiol. 43, 49-53
  18. Kim, J.S., J.H. Kim, E.K. Ryu, J.-K. Kim, C.K. Kim, I. Hwang, and K. Lee. 2004. Versatile catabolic properties of the Tn4371-encoded bph pathway in Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643. J. Microbiol. Biotechnol. 14, 302-311
  19. Lau, P.C., Y. Wang, A. Patel, D. Labbe, H. Bergeron, R. Brousseau, Y. Konishi, and M. Rawlings. 1997. A bacterial basic region leucine zipper histidine kinase regulating toluene degradation. Proc. Natl. Acad. Sci. USA 94, 1453-1458
  20. Mosqueda, G. and J.L. Ramos. 2000. A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism. J. Bacteriol. 182, 937-943 https://doi.org/10.1128/JB.182.4.937-943.2000
  21. Mosqueda, G., M.I. Ramos-Gonzalez, and J.L. Ramos. 1999. Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization. Gene 232, 69-76 https://doi.org/10.1016/S0378-1119(99)00113-4
  22. Ngai, K.L., M. Schlomann, H.J. Knackmuss, and L.N. Ornston. 1987. Dienelactone hydrolase from Pseudomonas sp. strain B13. J. Bacteriol. 169, 699-703 https://doi.org/10.1128/jb.169.2.699-703.1987
  23. Ohta, Y., M. Maeda, and T. Kudo. 2001. Pseudomonas putida CE2010 can degrade biphenyl by a mosaic pathway encoded by the tod operon and cmtE, which are identical to those of P. putida F1 except for a single base difference in the operator-promoter region of the cmt operon. Microbiology 147, 31-41 https://doi.org/10.1099/00221287-147-1-31
  24. O'Leary, N.D., W.A. Duetz, A.D. Dobson, and K.E. O'Connor. 2002. Induction and repression of the sty operon in Pseudomonas putida CA-3 during growth on phenylacetic acid under organic and inorganic nutrient-limiting continuous culture conditions. FEMS Microbiol. Lett. 208, 263-268 https://doi.org/10.1111/j.1574-6968.2002.tb11092.x
  25. Phoenix, P., A. Keane, A. Patel, H. Bergeron, S. Ghoshal, and P.C. Lau. 2003. Characterization of a new solventresponsive gene locus in Pseudomonas putida F1 and its functionalization as a versatile biosensor. Environ. Microbiol. 5, 1309-1327 https://doi.org/10.1111/j.1462-2920.2003.00426.x
  26. Puskas, L.G., M. Inui, Z. Kele, and H. Yukawa. 2000. Cloning of genes participating in aerobic biodegradation of p-cumate from Rhodopseudomonas palustris. DNA Seq. 11, 9-20 https://doi.org/10.3109/10425170009033965
  27. Ramos-Gonzalez, M.I., M. Olson, A.A. Gatenby, G. Mosqueda, M. Manzanera, M.J. Campos, S. Vichez, and J.L. Ramos. 2002. Cross-regulation between a novel two-component signal transduction system for catabolism of toluene in Pseudomonas mendocina and the TodST system from Pseudomonas putida. J. Bacteriol. 184, 7062-7067 https://doi.org/10.1128/JB.184.24.7062-7067.2002
  28. Song, J.S., D.H. Lee, K. Lee, and C.K. Kim. 2004. Genetic organization of the dhlA gene encoding 1,2-dichloroethane dechlorinase from Xanthobacter flavus UE15. J. Microbiol. 42, 188-193
  29. Stanier, R.Y., N.J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxomonic study. J. Gen. Microbiol. 43, 159-271 https://doi.org/10.1099/00221287-43-2-159
  30. Staskawicz, B., D. Dahlbeck, N. Keen, and C. Napoli. 1987. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacteriol. 169, 5789-5794 https://doi.org/10.1128/jb.169.12.5789-5794.1987
  31. Swoboda-colberg, N.G. 1995. Chemical contamination of the environment: sources, types, and fate of synthetic organic chemicals, p. 27-74. In L. Y. Young and C. E. Cerniglia (ed.), Microbial transformation and degradation of toxic organic chemicals. John Wiely and Sons, New York
  32. Tropel, D. and J.R. van der Meer. 2004. Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol. Mol. Biol. Rev. 68, 474-500 https://doi.org/10.1128/MMBR.68.3.474-500.2004
  33. van der Meer, J.R., W.M. de Vos, S. Harayama, and A.J.B. Zehnder. 1992. Molecular mechanisms of genetic adaptation to xenobiotic compounds. Microbiol. Rev. 56, 677-694
  34. van Hamme, J.D., A. Singh, and O.P. Ward. 2003. Recent advances in petroleum microbiology. Microbiol. Mol. Biol. Rev. 67, 503-549 https://doi.org/10.1128/MMBR.67.4.503-549.2003
  35. Velazquez, F., V. Parro, and V. de Lorenzo. 2005. Inferring the genetic network of m-xylene metabolism through expression profiling of the xyl genes of Pseudomonas putida mt-2. Mol. Microbiol. 57, 1557-1569 https://doi.org/10.1111/j.1365-2958.2005.04787.x
  36. Zylstra, G.J., and D.T. Gibson. 1989. Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli. J. Biol. Chem. 264, 14940-14946
  37. Zylstra, G.J., W.R. McCombie, D.T. Gibson, and B.A. Finette. 1988. Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon. Appl. Environ. Microbiol. 54, 1498-1503