Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Identification of a p-Cresol Degradation Pathway by a GFP-Based Transposon in Pseudomonas and Its Dominant Expression in Colonies

  • Cho, Ah-Ra (Department of Microbiology, Changwon National University) ;
  • Lim, Eun-Jin (Department of Microbiology, Changwon National University) ;
  • Veeranagouda, Yaligara (Department of Microbiology, Changwon National University) ;
  • Lee, Kyoung (Department of Microbiology, Changwon National University)
  • Received : 2011.04.08
  • Accepted : 2011.07.20
  • Published : 2011.11.28
Download PDF
⟨ Previous Next ⟩

Abstract

In this study, the chromosome-encoded pcuRCAXB genes that are required for p-cresol degradation have been identified by using a newly constructed green fluorescent protein (GFP)-based promoter probe transposon in the long-chain alkylphenol degrader Pseudomonas alkylphenolia. The deduced amino acid sequences of the genes showed the highest identities at the levels of 65-93% compared with those in the databases. The transposon was identified to be inserted in the pcuA gene, with the promoterless gfp gene being under the control of the pcu catabolic gene promoter. The expression of GFP was positively induced by p-cresol and was about 10 times higher by cells grown on agar than those in liquid culture. In addition, p-hydroxybenzoic acid was detected during p-cresol degradation. These results indicate that P. alkylphenolia additionally possesses a protocatechuate ortho-cleavage route for p-cresol degradation that is dominantly expressed in colonies.

Keywords

References

  1. Bayly, R. C., S. Dagley, and D. T. Gibson. 1966. The metabolism of cresols by species of Pseudomonas. Biochem. J. 101: 293-301. https://doi.org/10.1042/bj1010293
  2. 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
  3. Borrell, B. 2009. Why study pig odor? Sci. Am. Accessed at http:// www.scientificamerican.com/article.cfm?id=why-study-pig-odor.
  4. Cho, J. H., D. K. Jung, K. Lee, and S. Rhee. 2009. Crystal structure and functional analysis of the extradiol dioxygenase LapB from a long-chain alkylphenol degradation pathway in Pseudomonas. J. Biol. Chem. 284: 34321-34330. https://doi.org/10.1074/jbc.M109.031054
  5. 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
  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 ringfission hydrolase CmtE and induce the tod catabolic operon, respectively. Microbiology 149: 795-805. https://doi.org/10.1099/mic.0.26046-0
  7. Cunane, L. M., Z. W. Chen, N. Shamala, F. S. Mathews, C. N. Cronin, and W. S. McIntire. 2000. Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: Gated substrate entry and proton relays support the proposed catalytic mechanism. J. Mol. Biol. 295: 357-374. https://doi.org/10.1006/jmbi.1999.3290
  8. de Lorenzo, V., M. Herrero, U. Jakubzik, and K. N. Timmis. 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.
  9. Dennis, J. J. and G. J. Zylstra. 1998. Plasposons: Modular selfcloning minitransposon derivatives for rapid genetic analysis of Gram-negative bacterial genomes. Appl. Environ. Microbiol. 64: 2710-2715.
  10. Figurski, D. H. and D. R. Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76: 1648-1652. https://doi.org/10.1073/pnas.76.4.1648
  11. Harwood, C. S. and R. E. Parales. 1996. The beta-ketoadipate pathway and the biology of self-identity. Annu. Rev. Microbiol. 50: 553-590. https://doi.org/10.1146/annurev.micro.50.1.553
  12. Hopper, D. J. 1976. The hydroxylation of p-cresol and its conversion to p-hydroxybenzaldehyde in Pseudomonas putida. Biochem. Biophys. Res. Commun. 69: 462-468. https://doi.org/10.1016/0006-291X(76)90544-1
  13. 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
  14. Joesaar, M., E. Heinaru, S. Viggor, E. Vedler, and A. Heinaru. 2010. Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-cresol-degradative strains of Pseudomonas fluorescens. FEMS Microbiol. Ecol. 72: 464-475. https://doi.org/10.1111/j.1574-6941.2010.00858.x
  15. Kim, J., J. H. Fuller, G. Cecchini, and W. S. McIntire. 1994. Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida. J. Bacteriol. 176: 6349-6361. https://doi.org/10.1128/jb.176.20.6349-6361.1994
  16. 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.
  17. Kukor, J. J. and R. H. Olsen. 1992. Complete nucleotide sequence of tbuD, the gene encoding phenol/cresol hydroxylase from Pseudomonas pickettii PKO1, and functional analysis of the encoded enzyme. J. Bacteriol. 174: 6518-6526.
  18. Larsen, R. A., M. M. Wilson, A. M. Guss, and W. W. Metcalf. 2002. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch. Microbiol. 178: 193-201. https://doi.org/10.1007/s00203-002-0442-2
  19. Lee, K. and Y. Veeranagouda. 2009. Ultramicrocells form by reductive division in macroscopic Pseudomonas aerial structures. Environ. Microbiol. 11: 1117-1125. https://doi.org/10.1111/j.1462-2920.2008.01841.x
  20. Miller, V. L. and J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: Osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170: 2575-2583. https://doi.org/10.1128/jb.170.6.2575-2583.1988
  21. Schmidt, E. G. 1949. Urinary phenols; the simultaneous determination of phenol and p-cresol in urine. J. Biol. Chem. 179: 211-215.
  22. Shingler, V., J. Powlowski, and U. Marklund. 1992. Nucleotide sequence and functional analysis of the complete phenol/3,4- dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. J. Bacteriol. 174: 711-724. https://doi.org/10.1128/jb.174.3.711-724.1992
  23. 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
  24. Suarez, A., A. Guttler, M. Stratz, L. H. Staendner, K. N. Timmis, and C. A. Guzman. 1997. Green fluorescent proteinbased reporter systems for genetic analysis of bacteria including monocopy applications. Gene 196: 69-74. https://doi.org/10.1016/S0378-1119(97)00197-2
  25. Tang, X., B. F. Lu, and S. Q. Pan. 1999. A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens. FEMS Microbiol. Lett. 179: 37-42. https://doi.org/10.1111/j.1574-6968.1999.tb08704.x
  26. Thony, B. and H. Hennecke. 1989. The -24/-12 promoter comes of age. FEMS Microbiol. Rev. 5: 341-357.
  27. Wright, A. and R. H. Olsen. 1994. Self-mobilization and organization of the genes encoding the toluene metabolic pathway of Pseudomonas mendocina KR1. Appl. Environ. Microbiol. 60: 235-242.
  28. Yun, J. I., K. M. Cho, J. K. Kim, S. O. Lee, K. Cho, and K. Lee. 2007. Mutation of rpoS enhances Pseudomonas sp. KL28 growth at higher concentrations of m-cresol and changes its surface-related phenotypes. FEMS Microbiol. Lett. 269: 97- 103. https://doi.org/10.1111/j.1574-6968.2006.00610.x

Cited by

  1. Construction of Overexpression Vectors and Purification of the Oxygenase Component of Alkylphenol Hydroxylase of Pseudomonas alkylphenolia vol.49, pp.1, 2011, https://doi.org/10.7845/kjm.2013.008
  2. Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum vol.291, pp.12, 2011, https://doi.org/10.1074/jbc.m115.695320
  3. Pseudomonas alkylphenolica KL28에 존재하는 3종류의 p-cresol 분해 경로 및 유전자 발현 vol.52, pp.3, 2011, https://doi.org/10.7845/kjm.2016.6048
  4. Para -cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria vol.14, pp.9, 2011, https://doi.org/10.1371/journal.ppat.1007191