Molecular Assessment of the Role of Methanotrophs in Nitrogen Fixation in Lake Washington Sediment

Erin Ham and Megan Hockert with Dr. Ann J. Auman

Methanotrophs, or methane oxidizing bacteria, are microbes that utilize methane as their sole source of carbon and energy. These ubiquitous microorganisms have several constructive environmental capabilities. They oxidize methane, a harmful greenhouse gas, and can also degrade certain water pollutants such as trichloroethylene (TCE). There are two phylogenetic types of methanotrophs, Type I and Type II. Both types of methanotrophs have particulate methane mono-oxygenase (pMMO) and some have soluble methane mono-oxygenase (sMMO) enzymes as well. MMOs have the ability to co-metabolize compounds other than methane like TCE into TCE-epoxides, which spontaneously break down into innocuous organic compounds. TCE is a halogenated hydrocarbon that was used for many decades for many purposes including use as an industrial degreaser and as an anesthetic. Unfortunately, TCE has polluted many ground water systems and has since been shown to be a potential carcinogen.
Methanotrophs with sMMO are found to degrade TCE at much higher rates (100X-1000X faster) than those with pMMO only. Also methanotrophs that fix nitrogen have been shown to degrade TCE faster and are better able recover from the toxic effects of TCE exposure. Historically, it was believed that only Type II methanotrophs fixed nitrogen and were capable of sMMO production; however, recent studies including molecular analyses of pure cultures have shown that some Type I strains also exhibit one or both of these abilities. This is important because Type I strains are more abundant in many environments and can live in more diverse environments than Type II strains. Thus, research focusing on Type I methanotrophs, instead of the well-studied Type II strains, for the bioremediation of TCE-contaminated sites is essential to better understand these organisms' role in this process.
During the summer of 2004 we used molecular tools to examine natural microbial populations rich in methanotrophs. The research goal was to investigate the importance of Type I methanotrophs in the nitrogen fixing community in a known methanotroph rich environment. This involved utilizing the techniques of PCR, cloning, restriction analysis, and sequence analysis. Analyzing the significance of Type I nitrogen fixing bacteria in a variety of habitats is a step towards understanding the potential for using Type I methanotrophs in the bioremediation of TCE at contaminated sites.
Our research goal was to complete a PCR generated clone bank of nifH genes amplified from Lake Washington, an environment rich in methanotrophs. Sediment from Lake Washington was collected and the chromosomal DNA was extracted from it. The nifH fragments in this DNA were then amplified using the polymerase chain reaction (PCR), resulting in a mix of nifH fragments. The products were cloned and then PCR was used again to amplify the specific nifH fragments of the different clones. The clones were run on an agarose gel and those containing a nifH fragment were digested with restriction enzymes and then subjected to Restriction Fragment Length Polymorphism (RFLP) analysis. nifH fragments exhibiting unique restriction patterns were sequenced and analyzed phylogenetically against other prokaryotic nifH sequences, including those from Type I and Type II methanotrophs.
We obtained complete (double stranded) and single stranded sequences for 17 different nifH clones and 11 additional clones, respectively. We also obtained sequences for Methylomicrobium AMO-
1 and Methylomicrobium buyratense 5G-5, 2 members of a genus for which nifH had never before been sequenced. Phylogenetic analysis of our sequencing data revealed the following:
1. 11 out of 20 sequenced RFLP groups formed a distinct cluster closely related to Type I
Methylobacter nifH.
2. Sequences in groups V, XXIV, XXXVII, and XX are present in a divergent cluster most
closely related to Desulfovibrio, a d-proteobacterium, and Methanosarcina acetivorans, an
archaeal methanogen.
3. Group XXXIV nifH was closely related to Methylocystis, a Type II methanotroph, known to
be present in minor quantities in this sediment environment.