Use of selective inhibitors and chromogenic substrates to differentiate bacteria based on toluene oxygenase activity

W. K. Keener, Maribeth E Watwood, K. D. Schaller, M. R. Walton, J. K. Partin, W. A. Smith, S. R. Clingenpeel

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

In whole-cell studies, two alkynes, 1-pentyne and phenylacetylene, were selective, irreversible inhibitors of monooxygenase enzymes in catabolic pathways that permit growth of bacteria on toluene. 1-Pentyne selectively inhibited growth of Burkholderia cepacia G4 (toluene 2-monooxygenase [T2MO] pathway) and B. pickettii PKO1 (toluene 3-monooxygenase [T3MO] pathway) on toluene, but did not inhibit growth of bacteria expressing other pathways. In further studies with strain G4, chromogenic transformation of α,α,α-Trifluoro-m-cresol (TFC) was irreversibly inhibited by 1-pentyne, but the presence of phenol prevented this inhibition. Transformation of catechol by G4 was unaffected by 1-pentyne. With respect to the various pathways and bacteria tested, phenylacetylene selectively inhibited growth of Pseudomonas mendocina KR1 (toluene 4-monooxygenase [T4MO] pathway) on toluene, but not on p-cresol. An Escherichia coli transformant expressing T4MO transformed indole or naphthalene in chromogenic reactions, but not after exposure to phenylacetylene. The naphthalene reaction remained diminished in phenylacetylene-treated cells relative to untreated cells after phenylacetylene was removed, indicating irreversible inhibition. These techniques were used to differentiate toluene-degrading isolates from an aquifer. Based on data generated with these indicators and inhibitors, along with results from Biolog analysis for sole carbon source oxidation, the groundwater isolates were assigned to eight separate groups, some of which apparently differ in their mode of toluene catabolism.

Original languageEnglish (US)
Pages (from-to)171-185
Number of pages15
JournalJournal of Microbiological Methods
Volume46
Issue number3
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Chromogenic Compounds
Oxygenases
Toluene
Bacteria
Groundwater
Growth
Pseudomonas mendocina
Burkholderia cepacia
Alkynes
Enzyme Inhibitors
Mixed Function Oxygenases
Phenol
Carbon
phenylacetylene
Escherichia coli

Keywords

  • Aquifer bacteria
  • Enzyme indicators
  • Enzyme inhibitors
  • Toluene
  • Toluene oxygenases

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

Use of selective inhibitors and chromogenic substrates to differentiate bacteria based on toluene oxygenase activity. / Keener, W. K.; Watwood, Maribeth E; Schaller, K. D.; Walton, M. R.; Partin, J. K.; Smith, W. A.; Clingenpeel, S. R.

In: Journal of Microbiological Methods, Vol. 46, No. 3, 2001, p. 171-185.

Research output: Contribution to journalArticle

Keener, W. K. ; Watwood, Maribeth E ; Schaller, K. D. ; Walton, M. R. ; Partin, J. K. ; Smith, W. A. ; Clingenpeel, S. R. / Use of selective inhibitors and chromogenic substrates to differentiate bacteria based on toluene oxygenase activity. In: Journal of Microbiological Methods. 2001 ; Vol. 46, No. 3. pp. 171-185.
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AU - Keener, W. K.

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AU - Schaller, K. D.

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AU - Partin, J. K.

AU - Smith, W. A.

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AB - In whole-cell studies, two alkynes, 1-pentyne and phenylacetylene, were selective, irreversible inhibitors of monooxygenase enzymes in catabolic pathways that permit growth of bacteria on toluene. 1-Pentyne selectively inhibited growth of Burkholderia cepacia G4 (toluene 2-monooxygenase [T2MO] pathway) and B. pickettii PKO1 (toluene 3-monooxygenase [T3MO] pathway) on toluene, but did not inhibit growth of bacteria expressing other pathways. In further studies with strain G4, chromogenic transformation of α,α,α-Trifluoro-m-cresol (TFC) was irreversibly inhibited by 1-pentyne, but the presence of phenol prevented this inhibition. Transformation of catechol by G4 was unaffected by 1-pentyne. With respect to the various pathways and bacteria tested, phenylacetylene selectively inhibited growth of Pseudomonas mendocina KR1 (toluene 4-monooxygenase [T4MO] pathway) on toluene, but not on p-cresol. An Escherichia coli transformant expressing T4MO transformed indole or naphthalene in chromogenic reactions, but not after exposure to phenylacetylene. The naphthalene reaction remained diminished in phenylacetylene-treated cells relative to untreated cells after phenylacetylene was removed, indicating irreversible inhibition. These techniques were used to differentiate toluene-degrading isolates from an aquifer. Based on data generated with these indicators and inhibitors, along with results from Biolog analysis for sole carbon source oxidation, the groundwater isolates were assigned to eight separate groups, some of which apparently differ in their mode of toluene catabolism.

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