PCR amplification, restriction fragment length polymorphism, and phylogenetic analysis of oxygenase genes
were used for the characterization of in situ methane- and ammonia-oxidizing bacteria from free-living and
attached communities in the Eastern Snake River Plain aquifer. The following three methane monooxygenase
(MMO) PCR primer sets were used: A189-A682, which amplifies an internal region of both the pmoA gene of
the MMO particulate form and the amoA gene of ammonia monooxygenase; A189-mb661, which specifically
targets the pmoA gene; and mmoXA-mmoXB, which amplifies the mmoX gene of the MMO soluble form
(sMMO). Whole-genome amplification (WGA) was used to amplify metagenomic DNA from each community
to assess its applicability for generating unbiased metagenomic template DNA. The majority of sequences in
each archive were related to oxygenases of type II-like methanotrophs of the genus Methylocystis. A small subset
of type I sequences found only in free-living communities possessed oxygenase genes that grouped nearest to
Methylobacter and Methylomonas spp. Sequences similar to that of the amoA gene associated with ammoniaoxidizing
bacteria (AOB) most closely matched a sequence from the uncultured bacterium BS870 but showed
no substantial alignment to known cultured AOB. Based on these functional gene analyses, bacteria related to
the type II methanotroph Methylocystis sp. were found to dominate both free-living and attached communities.
Metagenomic DNA amplified by WGA showed characteristics similar to those of unamplified samples. Overall,
numerous sMMO-like gene sequences that have been previously associated with high rates of trichloroethylene
cometabolism were observed in both free-living and attached communities in this basaltic aquifer.Copyrighted by American Society for Microbiology