Sean M. McAllister

Biodiversity and emerging biogeography of the neutrophilic iron-oxidizing Zetaproteobacteria

ABSTRACT Members of the neutrophilic iron-oxidizing candidate class Zetaproteobacteria have predominantly been found at sites of microbially mediated iron oxidation in marine environments around the Pacific Ocean. Eighty-four full-length (>1,400-bp) and 48 partial-length Zetaproteobacteria small-subunit (SSU) rRNA gene sequences from five novel clone libraries, one novel Zetaproteobacteria isolate, and the GenBank database were analyzed to assess the biodiversity of this burgeoning class of the Proteobacteria and to investigate its biogeography between three major sampling regions in the Pacific Ocean: Loihi Seamount, the Southern Mariana Trough, and the Tonga Arc. Sequences were grouped into operational taxonomic units (OTUs) on the basis of a 97% minimum similarity. Of the 28 OTUs detected, 13 were found to be endemic to one of the three main sampling regions and 2 were ubiquitous throughout the Pacific Ocean. Additionally, two deeply rooted OTUs that potentially dominate communities of iron oxidizers originating in the deep subsurface were identified. Spatial autocorrelation analysis and analysis of molecular variance (AMOVA) showed that geographic distance played a significant role in the distribution of Zetaproteobacteria biodiversity, whereas environmental parameters, such as temperature, pH, or total Fe concentration, did not have a significant effect. These results, detected using the coarse resolution of the SSU rRNA gene, indicate that the Zetaproteobacteria have a strong biogeographic signal.

Hidden in plain sight: discovery of sheath-forming, iron-oxidizing Zetaproteobacteria at Loihi Seamount, Hawaii, USA

Lithotrophic iron-oxidizing bacteria (FeOB) form microbial mats at focused flow or diffuse flow vents in deep-sea hydrothermal systems where Fe(II) is a dominant electron donor. These mats composed of biogenically formed Fe(III)-oxyhydroxides include twisted stalks and tubular sheaths, with sheaths typically composing a minor component of bulk mats. The micron diameter Fe(III)-oxyhydroxide-containing tubular sheaths bear a strong resemblance to sheaths formed by the freshwater FeOB, Leptothrix ochracea. We discovered that veil-like surface layers present in iron-mats at the Loihi Seamount were dominated by sheaths (40-60% of total morphotypes present) compared with deeper (> 1 cm) mat samples (0-16% sheath). By light microscopy, these sheaths appeared nearly identical to those of L. ochracea. Clone libraries of the SSU rRNA gene from this top layer were dominated by Zetaproteobacteria, and lacked phylotypes related to L. ochracea. In mats with similar morphologies, terminal-restriction fragment length polymorphism (T-RFLP) data along with quantitative PCR (Q-PCR) analyses using a Zetaproteobacteria-specific primer confirmed the presence and abundance of Zetaproteobacteria. A Zetaproteobacteria fluorescence in situ hybridization (FISH) probe hybridized to ensheathed cells (4% of total cells), while a L. ochracea-specific probe and a Betaproteobacteria probe did not. Together, these results constitute the discovery of a novel group of marine sheath-forming FeOB bearing a striking morphological similarity to L. ochracea, but belonging to an entirely different class of Proteobacteria.

Zeta-Proteobacteria Dominate the Colonization and Formation of Microbial Mats in Low-Temperature Hydrothermal Vents at Loihi Seamount, Hawaii

In situ colonization experiments were performed to study the pioneer populations of bacteria at Loihi Seamount, Hawaii. Over a ten-year sampling period, 41 microbial growth chambers (MGCs) were deployed and recovered in Peles Pit and the surrounding area after short-term (4–10 days) and long-term (∼1–6 years) incubations in the flow of hydrothermal effluent. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the small subunit rRNA gene (SSU rDNA) revealed that the short-term MGC communities exhibited a low number of represented populations when compared to the long-term MGC communities and naturally occurring microbial mats. Cluster analysis of T-RFLP fingerprints showed the short-term MGC communities all had similar richness but were separated into three distinct groups with different arrays of colonizing populations. Clone library analysis showed that cooler vents (T ave = 40°C) were primarily colonized by Mariprofundus ferrooxydans, a neutrophilic Fe-oxidizing ζ −Proteobacteria while warmer vents (T ave = 71°C) were colonized by Sulfurimonas spp. and other sulfur-cycling members of the ϵ −Proteobacteria. Vents with an intermediate temperature (T ave = 51°C) were colonized by representatives of both ζ -Proteobacteria and ϵ -Proteobacteria. Long-term MGC communities did not cluster with any of the short-term communities and exhibited higher richness, indicating a greater number of bacterial populations were able to colonize and grow in the long-term growth chambers.

The first microbiological contamination assessment by deep-sea drilling and coring by the D/V Chikyu at the Iheya North hydrothermal field in the Mid-Okinawa Trough (IODP Expedition 331)

During the Integrated Ocean Drilling Program (IODP) Expedition 331 at the Iheya North hydrothermal system in the Mid-Okinawa Trough by the D/V Chikyu, we conducted microbiological contamination tests of the drilling and coring operations. The contamination from the drilling mud fluids was assessed using both perfluorocarbon tracers (PFT) and fluorescent microsphere beads. PFT infiltration was detected from the periphery of almost all whole round cores (WRCs). By contrast, fluorescent microspheres were not detected in hydrothermally active core samples, possibly due to thermal decomposition of the microspheres under high-temperature conditions. Microbial contamination from drilling mud fluids to the core interior subsamples was further characterized by molecular-based evaluation. The microbial 16S rRNA gene phylotype compositions in the drilling mud fluids were mainly composed of sequences of Beta- and Gammaproteobacteria, and Bacteroidetes and not archaeal sequences. The phylotypes that displayed more than 97% similarity to the sequences obtained from the drilling mud fluids were defined as possible contaminants in this study and were detected as minor components of the bacterial phylotype compositions in 13 of 37 core samples. The degree of microbiological contamination was consistent with that determined by the PFT and/or microsphere assessments. This study suggests a constructive approach for evaluation and eliminating microbial contamination during riser-less drilling and coring operations by the D/V Chikyu.

The Architecture of Iron Microbial Mats Reflects the Adaptation of Chemolithotrophic Iron Oxidation in Freshwater and Marine Environments

Microbes form mats with architectures that promote efficient metabolism within a particular physicochemical environment, thus studying mat structure helps us understand ecophysiology. Despite much research on chemolithotrophic Fe-oxidizing bacteria, Fe mat architecture has not been visualized because these delicate structures are easily disrupted. There are striking similarities between the biominerals that comprise freshwater and marine Fe mats, made by Beta- and Zetaproteobacteria, respectively. If these biominerals are assembled into mat structures with similar functional morphology, this would suggest that mat architecture is adapted to serve roles specific to Fe oxidation. To evaluate this, we combined light, confocal, and scanning electron microscopy of intact Fe microbial mats with experiments on sheath formation in culture, in order to understand mat developmental history and subsequently evaluate the connection between Fe oxidation and mat morphology. We sampled a freshwater sheath mat from Maine and marine stalk and sheath mats from Loihi Seamount hydrothermal vents, Hawaii. Mat morphology correlated to niche: stalks formed in steeper O2 gradients while sheaths were associated with low to undetectable O2 gradients. Fe-biomineralized filaments, twisted stalks or hollow sheaths, formed the highly porous framework of each mat. The mat-formers are keystone species, with nascent marine stalk-rich mats comprised of novel and uncommon Zetaproteobacteria. For all mats, filaments were locally highly parallel with similar morphologies, indicating that cells were synchronously tracking a chemical or physical cue. In the freshwater mat, cells inhabited sheath ends at the growing edge of the mat. Correspondingly, time lapse culture imaging showed that sheaths are made like stalks, with cells rapidly leaving behind an Fe oxide filament. The distinctive architecture common to all observed Fe mats appears to serve specific functions related to chemolithotrophic Fe oxidation, including (1) removing Fe oxyhydroxide waste without entombing cells or clogging flow paths through the mat and (2) colonizing niches where Fe(II) and O2 overlap. This work improves our understanding of Fe mat developmental history and how mat morphology links to metabolism. We can use these results to interpret biogenicity, metabolism, and paleoenvironmental conditions of Fe microfossil mats, which would give us insight into Earths Fe and O2 history.

Hidden diversity revealed by genome-resolved metagenomics of iron-oxidizing microbial mats from L|[omacr]||[rsquo]|ihi Seamount, Hawai|[rsquo]|i

The Zetaproteobacteria are ubiquitous in marine environments, yet this class of Proteobacteria is only represented by a few closely-related cultured isolates. In high-iron environments, such as diffuse hydrothermal vents, the Zetaproteobacteria are important members of the community driving its structure. Biogeography of Zetaproteobacteria has shown two ubiquitous operational taxonomic units (OTUs), yet much is unknown about their genomic diversity. Genome-resolved metagenomics allows for the specific binning of microbial genomes based on genomic signatures present in composite metagenome assemblies. This resulted in the recovery of 93 genome bins, of which 34 were classified as Zetaproteobacteria. Form II ribulose 1,5-bisphosphate carboxylase genes were recovered from nearly all the Zetaproteobacteria genome bins. In addition, the Zetaproteobacteria genome bins contain genes for uptake and utilization of bioavailable nitrogen, detoxification of arsenic, and a terminal electron acceptor adapted for low oxygen concentration. Our results also support the hypothesis of a Cyc2-like protein as the site for iron oxidation, now detected across a majority of the Zetaproteobacteria genome bins. Whole genome comparisons showed a high genomic diversity across the Zetaproteobacteria OTUs and genome bins that were previously unidentified by SSU rRNA gene analysis. A single lineage of cosmopolitan Zetaproteobacteria (zOTU 2) was found to be monophyletic, based on cluster analysis of average nucleotide identity and average amino acid identity comparisons. From these data, we can begin to pinpoint genomic adaptations of the more ecologically ubiquitous Zetaproteobacteria, and further understand their environmental constraints and metabolic potential.

Novel Pelagic Iron-Oxidizing Zetaproteobacteria from the Chesapeake Bay Oxic–Anoxic Transition Zone

Chemolithotrophic iron-oxidizing bacteria (FeOB) could theoretically inhabit any environment where Fe(II) and O2 (or nitrate) coexist. Until recently, marine Fe-oxidizing Zetaproteobacteria had primarily been observed in benthic and subsurface settings, but not redox-stratified water columns. This may be due to the challenges that a pelagic lifestyle would pose for Zetaproteobacteria, given low Fe(II) concentrations in modern marine waters and the possibility that Fe oxyhydroxide biominerals could cause cells to sink. However, we recently cultivated Zetaproteobacteria from the Chesapeake Bay oxic–anoxic transition zone, suggesting that they can survive and contribute to biogeochemical cycling in a stratified estuary. Here we describe the isolation, characterization, and genomes of two new species, Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8, which are the first Zetaproteobacteria isolates from a pelagic environment. We looked for adaptations enabling strains CP-5 and CP-8 to overcome the challenges of living in a low Fe redoxcline with frequent O2 fluctuations due to tidal mixing. We found that the CP strains produce distinctive dreadlock-like Fe oxyhydroxide structures that are easily shed, which would help cells maintain suspension in the water column. These oxides are by-products of Fe(II) oxidation, likely catalyzed by the putative Fe(II) oxidase encoded by the cyc2 gene, present in both CP-5 and CP-8 genomes; the consistent presence of cyc2 in all microaerophilic FeOB and other FeOB genomes supports its putative role in Fe(II) oxidation. The CP strains also have two gene clusters associated with biofilm formation (Wsp system and the Widespread Colonization Island) that are absent or rare in other Zetaproteobacteria. We propose that biofilm formation enables the CP strains to attach to FeS particles and form flocs, an advantageous strategy for scavenging Fe(II) and developing low [O2] microenvironments within more oxygenated waters. However, the CP strains appear to be adapted to somewhat higher concentrations of O2, as indicated by the presence of genes encoding aa3-type cytochrome c oxidases, but not the cbb3-type found in all other Zetaproteobacteria isolate genomes. Overall, our results reveal adaptations for life in a physically dynamic, low Fe(II) water column, suggesting that niche-specific strategies can enable Zetaproteobacteria to live in any environment with Fe(II).

Fe oxidation by a fused cytochrome-porin common to diverse Fe-oxidizing bacteria

Fe oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our ability to track the contributions of Fe-oxidizing microbes is limited by our relatively incomplete knowledge of microbial Fe oxidation mechanisms, particularly in neutrophilic Fe-oxidizers. The genomes of many Fe-oxidizers encode homologs to an outer-membrane cytochrome (Cyc2) that has been shown to oxidize Fe in two acidophiles. Here, we demonstrate the Fe oxidase function of a heterologously expressed Cyc2 homolog derived from a neutrophilic Fe oxidizer. Phylogenetic analyses show that Cyc2 from neutrophiles cluster together, suggesting a common function. Sequence analysis and modeling reveal the entire Cyc2 family is defined by a unique structure, a fused cytochromeporin, consistent with Fe oxidation on the outer membrane, preventing internal Fe oxide encrustation. Metatranscriptomes from Fe-oxidizing environments show exceptionally high expression of cyc2, supporting its environmental role in Fe oxidation. Together, these results provide evidence that cyc2 encodes Fe oxidases in diverse Fe-oxidizers and therefore can be used to recognize microbial Fe oxidation. The presence of cyc2 in 897 genomes suggests that microbial Fe oxidation may be a widespread metabolism.

Iroki: automatic customization for phylogenetic trees

Background Phylogenetic trees are an important analytical tool for examining species and community diversity and the evolutionary history of species. In the case of microorganisms, decreasing sequencing costs have enabled researchers to generate ever-larger sequence datasets, which in turn have begun to fill gaps in the evolutionary history of microbial groups. However, phylogenetic analyses of large sequence datasets present challenges to extracting meaningful trends from complex trees. Scientific inferences made by visual inspection of phylogenetic trees can be simplified and enhanced by customizing various parts of the tree, including label color, branch color, and other features. Yet, manual customization is time-consuming and error prone, and programs designed to assist in batch tree customization often require programming experience. To address these limitations, we developed Iroki, a program for fast, automatic customization of phylogenetic trees. Iroki allows the user to incorporate information on a broad range of metadata for each experimental unit represented in the tree. Results Iroki was applied to four existing microbial sequence datasets to demonstrate its utility in data exploration and presentation. Iroki was used to highlight connections between viral phylogeny and host taxonomy, to explore the abundance of microbial groups across samples of cattle hide, to examine short-term temporal dynamics of virioplankton communities, and to search for trends in the biogeography of Zetaproteobacteria. Conclusions Iroki is an easy-to-use web app and command line application for fast, automatic customization of phylogenetic trees based on user-provided categorical or continuous metadata. Iroki allows for rapid hypothesis testing through visualizing custom phylogenetic trees, streamlining the process of phylogenetic data exploration and presentation. Availability Iroki can be accessed through a web app or via installation through RubyGems, from source, or through the Iroki Docker image. All source code and documentation is available under the GPLv3 license at https://github.com/mooreryan/iroki. The Iroki web-app is accessible at www.iroki.net or through the Virome portal (http://virome.dbi.udel.edu), and its source code is released under GPLv3 license at https://github.com/mooreryan/iroki_web. The Docker image can be found here: https://hub.docker.com/r/mooreryan/iroki.

Silica Biomineralization of Calothrix-Dominated Biofacies from Queen's Laundry Hot-Spring, Yellowstone National Park, USA

Experiments on microorganisms capable of surviving silicification are often conducted to gain a better understanding of the process of silica biomineralization and to gain insights into microbially influenced rock formations and biofabrics like those found in ancient deposits such as the Early Archean Apex Chert formation (House et al., 2000; Schopf, 1993). An ideal microorganism for studying silicification is the large sheathed cyanobacterium Calothrix, which form distinctive organo-sedimentary structures in the low to moderate temperature regions of hydrothermal springs or columnar stromatolitic structures in aquatic systems. Our ability to identify and characterize microfossils from ancient deposits allows us to gain a better understanding of environmental conditions on early Earth. Here we characterized Calothrix-dominated biofacies along the outflow apron of Queen’s Laundry Hot-Spring in Yellowstone National Park using microscopy and molecular techniques to examine biofacies morphology and phylogenetic diversity. We found that flow regime and temperature had a profound effect on community composition as identified by the observation of five distinct Calothrix-dominated communities and on biofacies architecture along the outflow apron.