Biomedical subjects

M E Watwood

Publications and source records attributed to M E Watwood.

6 recordsLinked to original sources

PubMed2002

A rapid fluorescence-based assay for detecting soluble methane monooxygenase.

A fluorescence-based assay was developed to estimate soluble methane monooxygenase (sMMO) activity in solution. Whole cells of Methylosinus trichosporium OB3b expressing sMMO were used to oxidize various compounds to screen for fluorescent products. Of the 12 compounds tested, only coumarin yielded a fluorescent product. The UV absorbance spectrum of the product matches that of 7-hydroxycoumarin, and this identification was confirmed by 13C-NMR spectroscopy. The dependence of the fluorescent reaction on sMMO activity was investigated by pre-incubation with acetylene, a known inhibitor of sMMO activity. Apparent kinetic parameters for whole cells were determined to be Km(app)=262 microM and Vmax(app)=821 nmol 7-hydroxycoumarin min(-1) mg protein(-1). The rate of coumarin oxidation by sMMO correlates well with those of trichloroethylene degradation and naphthalene oxidation. Advantages of the fluorescence-based coumarin oxidation assay over the naphthalene oxidation assay include a more stable product, direct detection of the product without additional reagents, and greater speed and convenience.

Coumarins↗

PubMed2001

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 alpha,alpha,alpha-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.

Acetylene↗

PubMed2000

BASIC program for reduction of data from community-level physiological profiling using biolog microplates: rationale and critical interpretation of data.

A BASIC program is offered that reduces data resulting from mixed-species inoculations into Biolog microplates. The procedures of the program are supported by a critical review of the literature relating to Biolog data reduction. The availability of standardized, accelerated data reduction protocols will facilitate study comparisons and allow efficient evaluation of new data reduction approaches.

Bacteria↗

PubMed1998

Activity-dependent fluorescent labeling of bacteria that degrade toluene via toluene 2,3-dioxygenase.

Alternative substrates for the toluene 2,3-dioxygenase pathway of several pseudomonads served as enzyme-activity-dependent fluorescent probes for the bacteria. Phenylacetylene and cinnamonitrile were transformed to fluorescent and brightly colored products by Pseudomonas putida F1, Pseudomonas fluorescens CFS215, and Burkholderia (Pseudomonas) strain JS150. Active bacteria transformed phenylacetylene, producing bright yellow solutions containing the putative product 2-hydroxy-6-oxo-7-octyn-2,4-dienoate. Transformation of cinnamonitrile resulted in bright orange solutions due to accumulation of the putative product 2-hydroxy-6-oxo-8-cyanoocta-2,4,7-trienoate. Chemical and physical properties of the products supported their identification, which indicated that the first three enzymes of the pathway catalyzed product formation. Phenylacetylene labeled bacteria with green fluorescence emission; bacteria were concentrated on black 0.2-micron-pore-size polycarbonate filters containing polyvinylpyrrolidone (PVP) as a wetting agent. Bacteria labeled with cinnamonitrile were fluorescent orange; labeling was effective with bacteria trapped on PVP-free polycarbonate filters. Production of the enzymes involved in labeling of P. putida F1 and P. fluorescens CFS215 was induced by growth (on arginine) in the presence of toluene; cells grown on arginine without toluene were not labeled. Labeling of P. putida F1 by phenylacetylene was inhibited by toluene, indicating that the same enzymatic pathway was required for transformations of both substrates. Bacteria expressing other toluene-degrading enzymatic pathways were not fluorescently labeled with phenylacetylene.

Biodegradation, Environmental↗

PubMed1998

Effects of hydrocarbon enrichment on trichloroethylene biodegradation and microbial populations in finished compost.

This study focused on the capacity of finished compost, often used as packing material in biofiltration units, to support microbial biodegradation of trichloroethylene (TCE). Finished compost was enriched with methane or propane (10% head space) to stimulate cometabolic biodegradation of gaseous TCE. Successful hydrocarbon enrichment, as indicated by rapid depletion of hydrocarbon gas and measurable growth of hydrocarbon-utilizing micro-organisms, occurred within a week. Within batch reactor flasks, approximately 75% of head space TCE (1-40 ppmv) was rapidly sorbed onto compost material. Up to 99% of the remaining head space TCE was removed via biodegradation in compost enriched with either hydrocarbon. Hydrocarbon enrichment with methane or propane corresponded to 10-fold increases in methanotrophic or propanotrophic populations, respectively. Based on growth assessment under different nutritional regimes, there appeared to be complex metabolic interactions within the microbial community in enriched compost. Five separate bacterial cultures were derived from the hydrocarbon-enriched compost and assayed for the ability to degrade TCE.

Actinomyces↗

PubMed1997

Removal of tetrachloroethylene in an anaerobic column bioreactor.

Removal of tetrachloroethylene (perchloroethylene; C2Cl4) by microbial consortia from two sites with different C2Cl4 exposure histories was examined in a bench-scale anaerobic column bioreactor. It was hypothesized that optimal removal would be observed in the reactor packed with sediments having an extensive exposure history. Microbial consortia were enriched from hyporheic-zone (HZ) sediments from the Portneuf aquifer near Pocatello, Idaho, and from industrial-zone (IZ) sediments from a highly contaminated aquifer in Portland, Oregon. Lactate and acetate were the electron donors during experiments conducted over 9 and 7 months for HZ and IZ sediments, respectively. In the HZ bioreactor, the retention time ranged from 31 h to 81 h, and inlet C2Cl4 concentrations ranged from 0.1 ppm to 1.0 ppm. Dechlorination of C2Cl4 averaged 60% and reached a maximum of 78%. An increase in C:N from 27:1 to 500:1 corresponded to an 18% increase in removal efficiency. Trichloroethylene production corresponded to decreased effluent C2Cl4; further intermediates were not detected. In the IZ bioreactor, the retention time varied from 34 h to 115 h; the inlet C2Cl4 concentration was 1.0 ppm. C2Cl4 removal averaged 70% with a maximum of 98%. Trichloroethylene and cis-dichloroethylene were detected in the effluent. Increases in C:N from 50:1 to 250:1 enhanced dechlorination activity.

Anaerobiosis↗