METHOD DEVELOPMENT AND EARLY TESTING OF MOLECULAR PROBES FOR THE MICROBIAL MERCURY METHYLATION GENE PAIR HGCAB

The identification of a gene pair responsible for microbial methylmercury (MeHg) production, hgcAB, provides a foundation for evaluating the distribution of Hg-methylating microbes in nature. To date, all hgcAB+ microbes tested are capable of methylating mercury, while organisms lacking hgcA and hgcB are not. This predictability, along with the highly conserved nature of the cobalamin-binding domain in hgcA and the ferrodoxin domain in hgcB means that hgcAB should serve as a biomarker for Hg methylation in nature.

Here we provide an overview of method development of qualitative and quantitative polymerase chain reaction (PCR) primers for these genes. Our approach has been to develop a universal set of DNA primers for qualitative evaluation of organismal diversity, plus a set of clade-specific qPCR primers.

A universal degenerate primer set was developed that binds the most highly conserved regions in hgcA and B. The primers amplify a long product that can be sequenced for organismal identification, and also provide confirmation that both genes are present and adjacent. The primers were designed using hgcAB sequences from the ~100 hgcAB+ organisms with published genomes including Deltaproteobacteria, Firmicutes, and methanogenic Archaea. Primers and reaction conditions were then evaluated (and modified) by testing against more than 30 pure cultures including methylators and non-methylators in all of the clades containing hgcAB+ organisms. Clade-specific qPCR (Deltaproteobacteria, Firmicutes, and methanogens) primers were developed using the same approach. Quantitative PCR requires amplification of shorter sections of DNA. Since the hgcA sequence outside the cobalamin-binding domain is more variable among clades, qPCR required the development of separate primers for each major group.

To ensure the primers and reaction conditions are robust and sensitive in complex environmental matrices, we tested the primers on natural samples spiked with mixtures of hgcAB+ cultures. Finally, copy number and diversity information obtained from hgcAB primers were compared to 16S rRNA diversity, and to hgcAB in metagenomic analysis in the same samples. We will show some early data on use of these primers to evaluate relationships between hgcA abundance, Hg-methylation, and terminal electron acceptor availability.