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 sel ective, irreversible inhibitors of monooxygenase enzymes in catabolic pathw ays: that permit growth of bacteria on toluene. 1-Pentyne selectively inhib ited growth of Burkholderia cepacia G4 (toluene 2-monooxygenase [T2MO] path way) and B. pickettii PKO1 (toluene 3-monooxygenase [T3MO] pathway) on tolu ene. but did not inhibit growth of bacteria expressing other pathways. In f urther studies with strain G4. chromogenic transformation of alpha,alpha,al pha -Trifluoro-m-cresol (TFC) was irreversibly inhibited by 1-pentyne. but the presence of phenol prevented this inhibition. Transformation of catecho l by G4 was unaffected by 1-pentyne. With respect to the various pathways and bacteria tested. phenylacetylene s electively inhibited growth of Pseudomonas mendocina KR1 (toluene 3-monooxy genase [T4MO] pathway) on toluene. but not on p-cresol. An Escherichia coli transformant expressing T4MO transformed indole or naphthalene in chromoge nic reactions. but not after exposure to phenylacetylene. The naphthalene r eaction remained diminished in phenylacetylene-treated cells relative to un treated cells after phenylacetylene was removed, indicating irreversible in hibition. 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. t he groundwater isolates were assigned to eight separate groups, some of whi ch apparently differ in their mode of toluene catabolism. (C) 2001 Elsevier Science B.V. All rights reserved.