Quantitative PCR Analysis of Functional Genes in Iron-Rich Microbial Mats at an Active Hydrothermal Vent System (Lō'ihi Seamount, Hawai'i)

Abstract

The chemolithotrophic Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) and are the dominant bacterial population in iron-rich microbial mats. Zetaproteobacteria were first discovered at Lo 'ihi Seamount, located 35 km southeast off the big island of Hawai'i, which is characterized by low-temperature diffuse hydrothermal venting. Novel nondegenerate quantitative PCR (qPCR) assays for genes associated with microbial nitrogen fixation, denitrification, arsenic detoxification, Calvin-Benson-Bassham (CBB), and reductive tricarboxylic acid (rTCA) cycles were developed using selected microbial mat community-derived metagenomes. Nitrogen fixation genes were not detected, but all other functional genes were present. This suggests that arsenic detoxification and denitrification processes are likely cooccurring in addition to two modes of carbon fixation. Two groups of microbial mat community types were identified by terminal restriction fragment length polymorphism (T-RFLP) and were further described based on qPCR data for zetaproteobacterial abundance and carbon fixation mode preference. qPCR variance was associated with mat morphology but not with temperature or sample site. Geochemistry data were significantly associated with sample site and mat morphology. Together, these qPCR assays constitute a functional gene signature for iron microbial mat communities across a broad array of temperatures, mat types, chemistries, and sampling sites at Lo 'ihi Seamount. Deep-sea hydrothermal vents are dynamic and extremely productive biological ecosystems supported by chemosynthetic microbial primary production. In the absence of photosynthesis, microorganisms derive energy via the oxidation of reduced chemicals [e.g., H 2 , H 2 S, Fe(II), and CH 4 ] emitted in hydrothermal fluids (1). In contrast to other strategies for microbial chemosynthesis at hydrothermal vents, iron oxidation has only more recently been studied (2). By weight, iron is the most abundant element in the earth, and it has vast potential as an energy source for microbes via chemolithoautotrophy coupled to Fe(II) oxidation (3). However, iron's ability to act as an electron donor for the biotic fixation of CO 2 in neutrophilic environments is limited by the rapid abiotic oxidation of Fe(II) to Fe(III) in the presence of oxygen (4, 5). Despite the ephemeral nature of iron as an energy source, iron-oxidizing bacteria (FeOB) have been identified in a wide array of freshwater and marine habitats and can flourish at circumneutral deep-sea vents with sharp redox gradients and hydrothermal fluids high in CO 2 and reduced iron (6-9). The recently described Zetaproteobacteria represent a novel class of marine Proteobacteria that are diverse and abundant contributors to deep-sea FeOB communities (10).Zetaproteobacteria were first discovered at iron-rich, low-temperature hydrothermal vents at Lo 'ihi Seamount, HI (2, 11), and have been demonstrated to be significant microbial colonizers of seamounts (10, 12). Lo 'ihi Seamount is a s...

Bringing microbial diversity into focus: high‐resolution analysis of iron mats from the Lō‘ihi Seamount

“…Interestingly two of these Gammaproteobacteria nodes (0621 and 0616) appear to be the closest relatives of the veil associate described above (node 1668). Our analysis supports earlier findings showing that streamers are likely the product of sulfur-oxidizing bacteria (Moyer et al, 1995;Emerson and Moyer, 2010;Jesser et al, 2015). Consistent with this explanation, vent fluids at North Hiolo Ridge tend to carry higher concentrations of hydrogen sulfide (5-45 mM) compared with other L o'ihi sites where sulfide concentrations are <10 mM (Glazer and Rouxel, 2009).…”

“…Some L o'ihi samples contained abundant helical stalks, while others had a variety of thin, filamentous forms that were more challenging to associate with a unified structure. Our analysis supports earlier findings showing that streamers are likely the product of sulfur-oxidizing bacteria (Moyer et al, 1995;Emerson and Moyer, 2010;Jesser et al, 2015). An exception to this may be the two samples identified as a white streamer morphotype that proliferated at vent orifices along the North Hiolo Ridge (Supporting Information Figs S3 and S4).…”

“…Previous studies at L o'ihi have shown the general predominance of Zetaproteobacteria in bulk mats (Rassa et al, 2009;Jesser et al, 2015) as well as other microbial taxa. These studies also demonstrated that the sheathforming bacteria responsible for the production of veil-like mats are also Zetaproteobacteria and are not related to freshwater, sheath-forming iron-oxidizer, Leptothrix ochracea, as was originally thought (Fleming et al, 2013).…”

“…Based on our analyses ZetaOtu6 appeared restricted to mats in close proximity of active venting. Recent work by Jesser et al showed marked differences in arsenic resistance genes between samples collected only a few centimeters apart in L o'ihi mats (Jesser et al, 2015), suggesting that toxic metals/metalloids resistance may also be important drivers shaping local community diversity. Similar patterns were observed for ZetaOtu26 at Marker 38 on the South Hiolo Ridge -abundant near the vent and then decreasing to near extinction in the other two samples (Supporting Information Fig.…”

“…Factors such as flow velocity over the face of the mat, or dynamic changes in overall vent chemistry may also play a role. Recent work by Jesser et al showed marked differences in arsenic resistance genes between samples collected only a few centimeters apart in L o'ihi mats (Jesser et al, 2015), suggesting that toxic metals/metalloids resistance may also be important drivers shaping local community diversity. Based on these findings we also expected to see differences in community composition with mat depth, however no substantial changes in community composition from depth profiles were observed from three different sites at the North Hiolo Ridge and Pohaku (data not shown).…”