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 ultraperformance 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.

An active atmospheric methane sink in high Arctic mineral cryosols

“…The absence of pmoA and pmoC peptides in our samples may be partly due to the low extractability of these proteins as they are highly hydrophobic and embedded in the membrane lipid layer or partly because of their lower abundance relative to pmoB peptides. The more frequent detection of pmoB has also been reported in other CH 4 -oxidizing samples (Paszczynski et al, 2011). Among the three subunits of pMMO, β subunits may serve a regulatory role as they do for the soluble form of MMO (Murrell et al, 2000) and therefore show a higher expression level.…”

Microbial minorities modulate methane consumption through niche partitioning

“…The preferred detection of PmoB may be caused by the localization in the cytosol compared with PmoA, which is membrane bound and hard to solubilize during protein extraction. In a recent study (Paszczynski et al, 2011), the dominance of peptides belonging to PmoB of Methylosinus trichosporium OB3b was also described by the analyses of groundwater samples from a site contaminated with chlorinated ethenes. Peptides of other subunits were either not identified or identified in minor numbers.…”

Quantification of TCE Co‐Oxidation in Groundwater Using a 14C–Assay

“…Tools exist that can assist in providing tertiary evidence for MNA to document the aerobic co‐oxidation of TCE. Molecular tools to document the presence of oxygenases capable of degrading TCE are commercially available, including qPCR assays for soluble methane monooxygenase, ring hydroxylating toluene monooxygenase, and ethene monooxygenase (Baldwin et al ; Paszczynski et al ; Wiedemeier et al ). It is also possible to test for the expression of these enzymes by quantification of the associated mRNA.…”