qPCR analysis of carbon, nitrogen, and arsenic cycling in Zetaproteobacteria-dominated microbial mats

The recently discovered Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) to fix CO2 at hydrothermal vents. Zetaproteobacteria were first discovered at Lo'ihi Seamount, located 35 km southeast of the big island of Hawai'i and characterized by low-temperature diffuse hydrothermal vents. The hydrothermal vents at Lo'ihi are surrounded by luxuriant iron-rich microbial mats dominated by Zetaproteobacteria. We aim to use real-time quantitative PCR (qPCR) to quantify functional genes associated with the microbial carbon, nitrogen, and arsenic cycles in complex Zetaproteobacteria- dominated iron mat communities. Unique qPCR primer sets have been developed based on Illumina next-generation sequence data from an iron mat collected in 2009 at Lo'ihi. These primers target the sequences for arsenate reductase and nitrite reductase, genes associated with arsenic detoxification and denitrification, respectively. Additionally, we are utilizing published primer sets to quantify genes associated with autotrophic carbon and nitrogen fixation pathways. Genomic DNA was isolated from microbial mats at multiple vent sites with varying temperatures and fluid flow during our 2013 expedition to Lo'ihi. The qPCR data for these samples can be used to draw correlations among fine scale mat structures and nutrient cycling processes across diverse mat morphologies, as previous research has identified unique microbial communities and metabolic strategies associated with distinct mat morphologies. This work will enable us to better identify samples for further molecular analysis, and may provide insights into the evolutionary history and metabolic functionality of various mat morphotypes. We hypothesize that Zetaproteobacteria act as ecosystem engineers, driving the structure and function of iron mat ecosystems.