Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “ Pseudomonas butanovora ”

Abstract

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ϳ20 nmol mg protein ؊1 of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein ؊1 . Oxidation of similar amounts of each DCE isomer (ϳ20 nmol mg protein ؊1 ) produced different negative effects on lactic aciddependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.Chlorinated ethenes (CEs), such as perchloroethene (PCE) and trichloroethene (TCE), are common groundwater contaminants that have been linked to liver and kidney damage and are suspected carcinogens (1). Although the microbially driven process of reductive dechlorination can effectively reduce the concentrations of both PCE and TCE under anaerobic groundwater conditions (5,23,27,28), partially dechlorinated products, such as dichloroethenes (DCEs), often persist and disperse in groundwater plumes (35). Recent evidence has emerged for the existence of bacteria that will grow aerobically on 1,2-cis DCE as a sole C source, yet their enrichment is difficult and growth is extremely slow (8). In contrast, it is well documented that hydrocarbon-oxidizing microorganisms are able to degrade DCEs via reductant-driven cooxidative mechanisms (4,6,9,15,17,19,38,39). Unfortunately, the need for the natural hydrocarbon to serve both as enzyme inducer and source of reductant results in competition between the natural substrate and CEs, and cooxidation efficiency is compromised (6,11,21,24,25,37).In this connection, several studies have shown that a combination of TCE and nonhydrocarbon substrates simultaneously induce the genes for toluene oxygenases and provide reductant for TCE oxidation by some toluene-degrading bacterial strains (26,30,36,41). Conceptually, this observation has some appeal for bioremediation because it implies that ...

Product Repression of Alkane Monooxygenase Expression in Pseudomonas butanovora

“…Liquid cultures were grown and harvested as previously described (11). BMO activity was routinely measured using ethene-dependent ethene oxide production as described elsewhere (11). The specific activity of BMO in whole cells is routinely expressed in nmol ethene oxide · min Ϫ1 · mg protein Ϫ1 .…”

“…P. butanovora (ATCC 43655) is a gram-negative bacterium that grows on n-alkanes (C 2 to C 9 ) (31). Liquid cultures were grown and harvested as previously described (11). BMO activity was routinely measured using ethene-dependent ethene oxide production as described elsewhere (11).…”

“…Unlike the alkane-responsive system regulating monooxygenase expression in P. putida GPo1, the transcriptional activity of the BMO promoter in P. butanovora is up-regulated in response to the products of monooxygenase activity, butyraldehyde and 1-butanol. In contrast, neither the substrate, butane, nor the immediate downstream product of butyraldehyde oxidation, butyric acid, was found to be an inducer (11,28). A constitutive albeit low level of BMO activity allows cells to respond to alkanes by transforming them into products which then induce higher levels of BMO (28).…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…Induction of the alkane monooxygenase in P. butanovora is well studied. Alkane monooxygenase is not expressed during growth of P. butanovora on organic acids, but is induced during growth on C 2 -C 5 n-alkanes [137][138][139] and during the growth on alcohols and aldehydes that result from the oxidation of C 2 -C 9 n-alkanes [25,137,140,141]. The BMO structural genes are transcribed as a single polycistronic mRNA and the transcription of the cluster is controlled by a σ 54 promoter [131], identical to the consensus sequence of mmo genes in Methylosinus trichosporium OB3b, Methylocystis sp.…”

“…The six structural genes cluster, Induction of the alkane monooxygenase in P. butanovora is well studied. Alkane monooxygenase is not expressed during growth of P. butanovora on organic acids, but is induced during growth on C 2 -C 5 n-alkanes [137][138][139] and during the growth on alcohols and aldehydes that result from the oxidation of C 2 -C 9 n-alkanes [25,137,140,141]. In a variety of other microorganisms short chain alkane monooxygenase enzymes were described to be induced during growth on alkanes, but not during growth on non-alkane substrates [62,66,137].…”