Yuri A. Gorby

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

Significance Bacterial nanowires from Shewanella oneidensis MR-1 were previously shown to be conductive under nonphysiological conditions. Intense debate still surrounds the molecular makeup, identity of the charge carriers, and cellular respiratory impact of bacterial nanowires. In this work, using in vivo fluorescence measurements, immunolabeling, and quantitative gene expression analysis, we demonstrate that S. oneidensis MR-1 nanowires are extensions of the outer membrane and periplasm, rather than pilin-based structures, as previously thought. We also demonstrate that the outer membrane multiheme cytochromes MtrC and OmcA localize to these membrane extensions, directly supporting one of the two models of electron transport through the nanowires; consistent with this, production of bacterial nanowires correlates with an increase in cellular reductase activity. Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Functionally Stable and Phylogenetically Diverse Microbial Enrichments from Microbial Fuel Cells during Wastewater Treatment

Microbial fuel cells (MFCs) are devices that exploit microorganisms as biocatalysts to recover energy from organic matter in the form of electricity. One of the goals of MFC research is to develop the technology for cost-effective wastewater treatment. However, before practical MFC applications are implemented it is important to gain fundamental knowledge about long-term system performance, reproducibility, and the formation and maintenance of functionally-stable microbial communities. Here we report findings from a MFC operated for over 300 days using only primary clarifier effluent collected from a municipal wastewater treatment plant as the microbial resource and substrate. The system was operated in a repeat-batch mode, where the reactor solution was replaced once every two weeks with new primary effluent that consisted of different microbial and chemical compositions with every batch exchange. The turbidity of the primary clarifier effluent solution notably decreased, and 97% of biological oxygen demand (BOD) was removed after an 8–13 day residence time for each batch cycle. On average, the limiting current density was 1000 mA/m2, the maximum power density was 13 mW/m2, and coulombic efficiency was 25%. Interestingly, the electrochemical performance and BOD removal rates were very reproducible throughout MFC operation regardless of the sample variability associated with each wastewater exchange. While MFC performance was very reproducible, the phylogenetic analyses of anode-associated electricity-generating biofilms showed that the microbial populations temporally fluctuated and maintained a high biodiversity throughout the year-long experiment. These results suggest that MFC communities are both self-selecting and self-optimizing, thereby able to develop and maintain functional stability regardless of fluctuations in carbon source(s) and regular introduction of microbial competitors. These results contribute significantly toward the practical application of MFC systems for long-term wastewater treatment as well as demonstrating MFC technology as a useful device to enrich for functionally stable microbial populations.

Electrically conductive bacterial nanowires in bisphosphonate- related osteonecrosis of the jaw biofilms

OBJECTIVE Bacterial biofilms play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this preliminary study was to test the hypothesis that the extracellular filaments observed in biofilms associated with BRONJ contain electrically conductive nanowires. STUDY DESIGN Bone samples of patients affected by BRONJ were evaluated for conductive nanowires by scanning electron microscopy (SEM) and conductive probe atomic force microscopy (CP-AFM). We created nanofabricated electrodes to measure electrical transport along putative nanowires. RESULTS SEM revealed large-scale multispecies biofilms containing numerous filamentous structures throughout necrotic bone. CP-AFM analysis revealed that these structures were electrically conductive nanowires with resistivities on the order of 20 Ω·cm. Nanofabricated electrodes spaced along the nanowires confirmed their ability to transfer electrons over micron-scale lengths. CONCLUSIONS Electrically conductive bacterial nanowires to date have been described only in environmental isolates. This study shows for the first time that these nanowires can also be found in clinically relevant biofilm-mediated diseases, such as BRONJ, and may represent an important target for therapy.

Syntrophic growth of Desulfovibrio alaskensis requires genes for H2 and formate metabolism as well as those for flagellum and biofilm formation.

ABSTRACT In anaerobic environments, mutually beneficial metabolic interactions between microorganisms (syntrophy) are essential for oxidation of organic matter to carbon dioxide and methane. Syntrophic interactions typically involve a microorganism degrading an organic compound to primary fermentation by-products and sources of electrons (i.e., formate, hydrogen, or nanowires) and a partner producing methane or respiring the electrons via alternative electron accepting processes. Using a transposon gene mutant library of the sulfate-reducing Desulfovibrio alaskensis G20, we screened for mutants incapable of serving as the electron-accepting partner of the butyrate-oxidizing bacterium, Syntrophomonas wolfei. A total of 17 gene mutants of D. alaskensis were identified as incapable of serving as the electron-accepting partner. The genes identified predominantly fell into three categories: membrane surface assembly, flagellum-pilus synthesis, and energy metabolism. Among these genes required to serve as the electron-accepting partner, the glycosyltransferase, pilus assembly protein (tadC), and flagellar biosynthesis protein showed reduced biofilm formation, suggesting that each of these components is involved in cell-to-cell interactions. Energy metabolism genes encoded proteins primarily involved in H2 uptake and electron cycling, including a rhodanese-containing complex that is phylogenetically conserved among sulfate-reducing Deltaproteobacteria. Utilizing an mRNA sequencing approach, analysis of transcript abundance in wild-type axenic and cocultures confirmed that genes identified as important for serving as the electron-accepting partner were more highly expressed under syntrophic conditions. The results imply that sulfate-reducing microorganisms require flagellar and outer membrane components to effectively couple to their syntrophic partners; furthermore, H2 metabolism is essential for syntrophic growth of D. alaskensis G20.

Geomicrobiology of sublacustrine thermal vents in Yellowstone Lake: geochemical controls on microbial community structure and function

Yellowstone Lake (Yellowstone National Park, WY, USA) is a large high-altitude (2200 m), fresh-water lake, which straddles an extensive caldera and is the center of significant geothermal activity. The primary goal of this interdisciplinary study was to evaluate the microbial populations inhabiting thermal vent communities in Yellowstone Lake using 16S rRNA gene and random metagenome sequencing, and to determine how geochemical attributes of vent waters influence the distribution of specific microorganisms and their metabolic potential. Thermal vent waters and associated microbial biomass were sampled during two field seasons (2007–2008) using a remotely operated vehicle (ROV). Sublacustrine thermal vent waters (circa 50–90°C) contained elevated concentrations of numerous constituents associated with geothermal activity including dissolved hydrogen, sulfide, methane and carbon dioxide. Microorganisms associated with sulfur-rich filamentous “streamer” communities of Inflated Plain and West Thumb (pH range 5–6) were dominated by bacteria from the Aquificales, but also contained thermophilic archaea from the Crenarchaeota and Euryarchaeota. Novel groups of methanogens and members of the Korarchaeota were observed in vents from West Thumb and Elliots Crater (pH 5–6). Conversely, metagenome sequence from Mary Bay vent sediments did not yield large assemblies, and contained diverse thermophilic and nonthermophilic bacterial relatives. Analysis of functional genes associated with the major vent populations indicated a direct linkage to high concentrations of carbon dioxide, reduced sulfur (sulfide and/or elemental S), hydrogen and methane in the deep thermal ecosystems. Our observations show that sublacustrine thermal vents in Yellowstone Lake support novel thermophilic communities, which contain microorganisms with functional attributes not found to date in terrestrial geothermal systems of YNP.