Iron-oxidizing bacteria: an environmental and genomic perspective.

In the 1830s, iron bacteria were among the first groups of microbes to be recognized for carrying out a fundamental geological process, namely the oxidation of iron. Due to lingering questions about their metabolism, coupled with difficulties in culturing important community members, studies of Fe-oxidizing bacteria (FeOB) have lagged behind those of other important microbial lithotrophic metabolisms. Recently, research on lithotrophic, oxygen-dependent FeOB that grow at circumneutral pH has accelerated. This work is driven by several factors including the recognition by both microbiologists and geoscientists of the role FeOB play in the biogeochemistry of iron and other elements. The isolation of new strains of obligate FeOB allowed a better understanding of their physiology and phylogeny and the realization that FeOB are abundant at certain deep-sea hydrothermal vents. These ancient microorganisms offer new opportunities to learn about fundamental biological processes that can be of practical importance.

An Approach to Improve Laboratory Notebook Feedback and Decrease Instructor Fatigue from Grading Undergraduate Laboratory Notebooks.

Proper laboratory notebook maintenance is a critical skill for science, technology, engineering, and math (STEM) workers. Laboratory notebook grading can be time-consuming and lead to instructor fatigue. After many hours of grading laboratory notebooks, instructors can become biased or not provide detailed feedback to students. I developed a simple protocol to alleviate these problems. Students maintained a laboratory notebook typical for most STEM courses. Then, they were given a short quiz with laboratory-specific questions and could only use their notebooks to answer. The presence or absence of major notebook sections (date, introduction, etc.) were checked, and the laboratory notebook score was a combination of these two components. The learning gains were not assessed, but the instructor grading time decreased by 80%. This technique was applied to both in-person and concurrent online laboratories. With the ever-increasing demands on instructors, anything that decreases the instructor workload and the time for students to receive feedback will likely lead to a better classroom environment.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

High-Throughput Screening for Positive Allosteric Modulators Identified Potential Therapeutics against Acetylcholinesterase Inhibition.

The current standard of care for treatment of organophosphate (OP) poisoning includes pretreatment with the weak reversible acetylcholinesterase (AChE) inhibitor pyridostigmine bromide. Because this drug is an AChE inhibitor, similar side effects exist as with OP poisoning. In an attempt to provide a therapeutic capable of mitigating AChE inhibition without such side effects, high-throughput screening was performed to identify a compound capable of increasing the catalytic activity of AChE. Herein, two such novel positive allosteric modulators (PAMs) of AChE are presented. These PAMs increase AChE activity threefold, but they fail to upshift the apparent IC50 of a variety of OPs. Further development and optimization of these compounds may lead to pre- and/or postexposure therapeutics with broad-spectrum efficacy against pesticide and nerve agent poisoning. In addition, they could be used to complement the current therapeutic standard of care to increase the activity of uninhibited AChE, potentially increasing the efficacy of current therapeutics in addition to altering the therapeutic window.

Ecological succession among iron-oxidizing bacteria.

Despite over 125 years of study, the factors that dictate species dominance in neutrophilic iron-oxidizing bacterial (FeOB) communities remain unknown. In a freshwater wetland, we documented a clear ecological succession coupled with niche separation between the helical stalk-forming Gallionellales (for example, Gallionella ferruginea) and tubular sheath-forming Leptothrix ochracea. Changes in the iron-seep community were documented using microscopy and cultivation-independent methods. Quantification of Fe-oxyhydroxide morphotypes by light microscopy was coupled with species-specific fluorescent in situ hybridization (FISH) probes using a protocol that minimized background fluorescence caused by the Fe-oxyhydroxides. Together with scanning electron microscopy, these techniques all indicated that Gallionellales dominated during early spring, with L. ochracea becoming more abundant for the remainder of the year. Analysis of tagged pyrosequencing reads of the small subunit ribosomal RNA gene (SSU rRNA) collected during seasonal progression supported a clear Gallionellales to L. ochracea transition, and community structure grouped according to observed dominant FeOB forms. Axis of redundancy analysis of physicochemical parameters collected from iron mats during the season, plotted with FeOB abundance, corroborated several field and microscopy-based observations and uncovered several unanticipated relationships. On the basis of these relationships, we conclude that the ecological niche of the stalk-forming Gallionellales is in waters with low organic carbon and steep redoxclines, and the sheath-forming L. ochracea is abundant in waters that contain high concentrations of complex organic carbon, high Fe and Mn content and gentle redoxclines. Finally, these findings identify a largely unexplored relationship between FeOB and organic carbon.

The transition from freshwater to marine iron-oxidizing bacterial lineages along a salinity gradient on the Sheepscot River, Maine, USA.

Oxygen-dependent, neutrophilic iron-oxidizing bacteria (FeOB) are important drivers of iron transformations in marine and freshwater environments. Despite remarkable similarities in physiology and morphotype, known freshwater and marine FeOB are clustered in different classes of Proteobacteria; freshwater FeOB in the Betaproteobacteria and marine FeOB in the Zetaproteobacteria. To determine effects of salinity on these microbes, we examined the mineral biosignatures and molecular ecology of bacteria in FeOB mats collected along an estuarine salinity gradient. Light microscopy and scanning electron microscopy analyses showed the presence of iron oxide stalk and sheath structures in both freshwater and saline iron mats. Results of tagged pyrosequencing, quantitative PCR and fluorescent in situ hybridization, all based on the small subunit rRNA gene, confirmed Zetaproteobacteria were not present in freshwater mats, but were in saline mats at salinities down to 5‰. Among the Betaproteobacteria, Leptothrix spp. were only found in the freshwater mat. Gallionella spp. were limited to freshwater and low salinity mats (< 5‰). Sideroxydans sp. were salt tolerant; however, their relative abundance decreased with increasing salinity. These results suggest salinity is important in shaping the population biology of iron mat communities, and some coexistence between marine and freshwater populations occurs in brackish waters.

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.

The Microbial Ferrous Wheel in a Neutral pH Groundwater Seep.

Evidence for microbial Fe redox cycling was documented in a circumneutral pH groundwater seep near Bloomington, Indiana. Geochemical and microbiological analyses were conducted at two sites, a semi-consolidated microbial mat and a floating puffball structure. In situ voltammetric microelectrode measurements revealed steep opposing gradients of O(2) and Fe(II) at both sites, similar to other groundwater seep and sedimentary environments known to support microbial Fe redox cycling. The puffball structure showed an abrupt increase in dissolved Fe(II) just at its surface (∼5 cm depth), suggesting an internal Fe(II) source coupled to active Fe(III) reduction. Most probable number enumerations detected microaerophilic Fe(II)-oxidizing bacteria (FeOB) and dissimilatory Fe(III)-reducing bacteria (FeRB) at densities of 10(2) to 10(5) cells mL(-1) in samples from both sites. In vitro Fe(III) reduction experiments revealed the potential for immediate reduction (no lag period) of native Fe(III) oxides. Conventional full-length 16S rRNA gene clone libraries were compared with high throughput barcode sequencing of the V1, V4, or V6 variable regions of 16S rRNA genes in order to evaluate the extent to which new sequencing approaches could provide enhanced insight into the composition of Fe redox cycling microbial community structure. The composition of the clone libraries suggested a lithotroph-dominated microbial community centered around taxa related to known FeOB (e.g., Gallionella, Sideroxydans, Aquabacterium). Sequences related to recognized FeRB (e.g., Rhodoferax, Aeromonas, Geobacter, Desulfovibrio) were also well-represented. Overall, sequences related to known FeOB and FeRB accounted for 88 and 59% of total clone sequences in the mat and puffball libraries, respectively. Taxa identified in the barcode libraries showed partial overlap with the clone libraries, but were not always consistent across different variable regions and sequencing platforms. However, the barcode libraries provided confirmation of key clone library results (e.g., the predominance of Betaproteobacteria) and an expanded view of lithotrophic microbial community composition.

Lithotrophic iron-oxidizing bacteria produce organic stalks to control mineral growth: implications for biosignature formation.

Neutrophilic Fe-oxidizing bacteria (FeOB) are often identified by their distinctive morphologies, such as the extracellular twisted ribbon-like stalks formed by Gallionella ferruginea or Mariprofundus ferrooxydans. Similar filaments preserved in silica are often identified as FeOB fossils in rocks. Although it is assumed that twisted iron stalks are indicative of FeOB, the stalk's metabolic role has not been established. To this end, we studied the marine FeOB M. ferrooxydans by light, X-ray and electron microscopy. Using time-lapse light microscopy, we observed cells excreting stalks during growth (averaging 2.2 µm h(-1)). Scanning transmission X-ray microscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy show that stalks are Fe(III)-rich, whereas cells are low in Fe. Transmission electron microscopy reveals that stalks are composed of several fibrils, which contain few-nanometer-sized iron oxyhydroxide crystals. Lepidocrocite crystals that nucleated on the fibril surface are much larger (∼100 nm), suggesting that mineral growth within fibrils is retarded, relative to sites surrounding fibrils. C and N 1s NEXAFS spectroscopy and fluorescence probing show that stalks primarily contain carboxyl-rich polysaccharides. On the basis of these results, we suggest a physiological model for Fe oxidation in which cells excrete oxidized Fe bound to organic polymers. These organic molecules retard mineral growth, preventing cell encrustation. This model describes an essential role for stalk formation in FeOB growth. We suggest that stalk-like morphologies observed in modern and ancient samples may be correlated confidently with the Fe-oxidizing metabolism as a robust biosignature.

What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH.

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.

Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium.

Methylmercury has been thought to be produced predominantly by sulfate-reducing bacteria in anoxic sediments. Here we show that in circumneutral pH sediments (Clear Lake, CA) application of a specific inhibitor of sulfate-reducing bacteria at appropriate concentrations typically inhibited less than one-half of all anaerobic methylation of added divalent mercury. This suggests that one or more additional groups of microbes are active methylators in these sediments impacted by a nearby abandoned mercury mine. From Clear Lake sediments, we isolated the iron-reducing bacterium Geobacter sp. strain CLFeRB, which can methylate mercury at a rate comparable to Desulfobulbus propionicus strain 1pr3, a sulfate-reducing bacterium known to be an active methylator. This is the first time that an iron-reducing bacterium has been shown to methylate mercury at environmentally significant rates. We suggest that mercury methylation by iron-reducing bacteria represents a previously unidentified and potentially significant source of this environmental toxin in iron-rich freshwater sediments.