title: QPCR analysis of functional genes in iron-rich microbial mats at an active hydrothermal vent system Loihi Seamount, Hawaii
Creators: name: Jesser, Kelsey J.
Publisher: name: Western Washington University
Year: 2014

description: <p>The recently discovered Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III), driving CO2 fixation at hydrothermal vents. These chemolithoautotrophs are the dominant bacterial population in iron-rich microbial mats, and represent a unique opportunity to investigate the connection between deep-sea geochemical processes and the dark microbial world. Zetaproteobacteria were first discovered at Loihi Seamount, located 35 km southeast off the big island of Hawaii and characterized by low-temperature diffuse hydrothermal venting. These vents are surrounded by luxuriant, iron-rich microbial mats that are colonized and often dominated by Zetaproteobacteria. Five novel non-degenerate QPCR assays were designed using sequence data derived from microbial iron-mat samples collected at Loihi in March 2013. Genes of interest were nifH, nirK, and arsC, associated with microbial nitrogen fixation, denitrification and arsenic detoxification, respectively. We also examined carbon fixation genes cbbM and aclB, which are indicators for the Calvin Benson Bassham (CBB) and reductive tricarboxylic acid (rTCA) cycles, respectively. All functional genes were found to be present at Loihi Seamount with the exception of nifH, which was undetectable with our method. Functional genes arsC and nirK were detected in all samples assayed, indicating that both arsenic detoxification and denitrification processes are likely occurring across all hydrothermal mat habitats. cbbM and aclB were also detectable in all samples assayed, indicating the co-occurence of these two modes of carbon fixation. T-RFLP analysis indicates that the communities in iron-rch mat samples collected in 2013 are very similar to one another. T-RFLP Group 1 had high Zetaproteobacteria abundance and low aclB relative to cbbM, indicating that the CBB cycle is the major mode of carbon fixation in Zetaproteobacteria-rich mat communities. T-RFLP Group 2 had low Zetaproteobacteria abundance and high aclB gene copy numbers, suggesting that the rTCA cycle is operating in non-Zetaproteobacteria taxa and plays an important role in carbon fixation in these communities. Based on these results, we conclude that aclB may be an important functional gene indicator of community composition. QPCR variance was explained by mat morphology but not temperature or sample site. Gene aclB was significantly associated with mat morphology, and may contribute to the significant relationship between the QPCR data and mat type. Fe(II) was significant with mat morphology. Geochemistry data was significantly associated with sample site and mat morphology, indicating that there is a range of chemistries in which these iron-rich microbial communities can thrive, and/or that the abundance of functional genes in these mat communities changes gradually in response to more dynamic chemical variation over time. Together, these QPCR assays constitute a ‘functional gene signature’ for iron mat samples across a broad array of temperatures, mat types, chemistries, and sampling sites in and around Pele’s Pit at Loihi Seamount.</p>