Detection and Isolation of Methanotrophic Bacteria Possessing Soluble Methane Monooxygenase (sMMO) Genes Using the Polymerase Chain Reaction (PCR)

Abstract

Methanotrophic bacteria possessing sMMO activity have gained notoriety in recent years due to their ability to oxidize a wide variety of halogenated aliphatic compounds, including trichloroethylene (TCE), and are being used as the basis for developing new bioremediation processes. PCR primers were designed from DNA sequences of the alpha and beta subunits of the hydroxylase component of the sMMO from Methylococcus capsulatus (Bath). The mmoY1-mmoY2 primer set was derived from the beta subunit and was specific for the mmoY gene from M. capsulatus, but failed to produce the expected 395-nucleotide (nt) fragment from Methylosinus sporium (ATCC 35069) or from Methylosinus trichosporium (ATCC 36070), even at low stringency. A second primer set, primers mmoX1-mmoX2, was derived from the alpha subunit and produced the expected 369-nt fragment from all three methanotrophic cultures tested at the highest stringency used (72°C). Soil and groundwater samples were tested for the presence of sMMO-containing bacteria using these two primer sets. One diesel-contaminated soil sample and one TCE-contaminated groundwater sample gave positive results after amplification of total extracted DNA using the mmoX1-mmoX2 primers. Culture enrichment in small chemostats inoculated with the same positive samples led to the isolation of 13 cultures possessing sMMO activity and containing DNA amplifiable by the mmoX1-mmoX2 primers. Our results indicated that attempts to directly cultivate sMMO-positive bacteria may give false negative results with some environmental samples. We recommend that primers and/or gene probes based on the sMMO be used in parallel with the naphthalene oxidation test for any environmental assessment of the methanotrophic population. RAPD-PCR analysis revealed that half of these isolates appeared to be different from each other and from M. capsulatus, M. sporium, or M. trichosporium.