title: Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase.
abstract: The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.
authors.affiliation: Environmental Biotechnology Institute, University of Idaho, PO Box 441052, Moscow, ID 83844-1052, USA. andrzej@uidaho.edu
authors.forename: Andrzej J
authors.initials: AJ
authors.lastname: Paszczynski
authors.forename: Ravindra
authors.initials: R
authors.lastname: Paidisetti
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Johnson
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Crawford
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Colwell
authors.forename: Tonia
authors.initials: T
authors.lastname: Green
authors.forename: Mark
authors.initials: M
authors.lastname: Delwiche
authors.forename: Hope
authors.initials: H
authors.lastname: Lee
authors.forename: Deborah
authors.initials: D
authors.lastname: Newby
authors.forename: Eoin L
authors.initials: EL
authors.lastname: Brodie
authors.forename: Mark
authors.initials: M
authors.lastname: Conrad
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D018441:Biofilms / Q000254:growth & development; D056148:Chromatography, Reverse-Phase; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004787:Environmental Pollution / Q000517:prevention & control*; D007061:Idaho; D013058:Mass Spectrometry; D008697:Methane / Q000378:metabolism*; D008772:Methylococcaceae / Q000201:enzymology* / Q000235:genetics; D059013:Microbial Consortia / Q000502:physiology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D010933:Plankton / Q000254:growth & development; D040901:Proteomics; D060888:Real-Time Polymerase Chain Reaction; D045483:Rivers; D014241:Trichloroethylene / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D014241:Trichloroethylene; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D010105:Oxygenases; C021555:methane monooxygenase; D008697:Methane