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

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.