Extracellular electron transfer (EET) describes microbial bioelectrochemical processes in which electrons are transferred from the cytosol to the exterior of the cell. Mineral-respiring bacteria use elaborate haem-based electron transfer mechanisms but the existence and mechanistic basis of other EETs remain largely unknown. Here we show that the food-borne pathogen Listeria monocytogenes uses a distinctive flavin-based EET mechanism to deliver electrons to iron or an electrode. By performing a forward genetic screen to identify L. monocytogenes mutants with diminished extracellular ferric iron reductase activity, we identified an eight-gene locus that is responsible for EET. This locus encodes a specialized NADH dehydrogenase that segregates EET from aerobic respiration by channelling electrons to a discrete membrane-localized quinone pool. Other proteins facilitate the assembly of an abundant extracellular flavoprotein that, in conjunction with free-molecule flavin shuttles, mediates electron transfer to extracellular acceptors. This system thus establishes a simple electron conduit that is compatible with the single-membrane structure of the Gram-positive cell. Activation of EET supports growth on non-fermentable carbon sources, and an EET mutant exhibited a competitive defect within the mouse gastrointestinal tract. Orthologues of the genes responsible for EET are present in hundreds of species across the Firmicutes phylum, including multiple pathogens and commensal members of the intestinal microbiota, and correlate with EET activity in assayed strains. These findings suggest a greater prevalence of EET-based growth capabilities and establish a previously underappreciated relevance for electrogenic bacteria across diverse environments, including host-associated microbial communities and infectious disease.

BackgroundThe engineering of many-component, synthetic biological systems is being made easier by the development of collections of reusable, standard biological parts. However, the complexity of biology makes it difficult to predict the extent to which such efforts will succeed. As a first practical example, the Registry of Standard Biological Parts started at MIT now maintains and distributes thousands of BioBrick™ standard biological parts. However, BioBrick parts are only standardized in terms of how individual parts are physically assembled into multi-component systems, and most parts remain uncharacterized. Standardized tools, techniques, and units of measurement are needed to facilitate the characterization and reuse of parts by independent researchers across many laboratories.ResultsWe found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. We choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. We demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by ~50%. We defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured. We also characterized the relative activities of a reference collection of BioBrick promoters in order to further support adoption of RPU-based measurement standards.ConclusionRelative activity measurements based on an in vivoreference standard enables improved measurement of promoter activity given variation in measurement conditions and instruments. These improvements are sufficient to begin to support the measurement of promoter activities across many laboratories. Additional in vivo reference standards for other types of biological functions would seem likely to have similar utility, and could thus improve research on the design, production, and reuse of standard biological parts.

There is much interest in developing synthetic analogues of biological membrane channels with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up and top-down methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls, and short tubes have been forced into membranes to create sensors, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70-100 picosiemens under physiological conditions. Despite their structural simplicity, these 'CNT porins' transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors.

Engineering efficient, directional electronic communication between living and nonliving systems has the potential to combine the unique characteristics of both materials for advanced biotechnological applications. However, the cell membrane is designed by nature to be an insulator, restricting the flow of charged species; therefore, introducing a biocompatible pathway for transferring electrons across the membrane without disrupting the cell is a significant challenge. Here we describe a genetic strategy to move intracellular electrons to an inorganic extracellular acceptor along a molecularly defined route. To do so, we reconstitute a portion of the extracellular electron transfer chain of Shewanella oneidensis MR-1 into the model microbe Escherichia coli. This engineered E. coli can reduce metal ions and solid metal oxides ∼8× and ∼4× faster than its parental strain. We also find that metal oxide reduction is more efficient when the extracellular electron acceptor has nanoscale dimensions. This work demonstrates that a genetic cassette can create a conduit for electronic communication from living cells to inorganic materials, and it highlights the importance of matching the size scale of the protein donors to inorganic acceptors.cytochrome c | nanobioelectronics | synthetic biology | iron reduction | living-nonliving interfaces

The field of synthetic biology aims to design biological systems to perform tasks to better understand analogous natural systems and for direct applications in research and medicine (e.g., see Andrianantoandro et al. 2006;Drubin et al. 2007). Currently our ability to design biological systems is limited by the difficulty of predicting the behavior of even simple genetic circuits because often a given network topology will show qualitatively different behavior depending on the quantitative features of the underlying components. While advances have been made with networks composed of well-studied modules in bacteria (Guido et al. 2006), this challenge is acute in more complex eukaryotic systems where the components rarely have measured characteristics. Therefore, to make rapid progress in designing eukaryotic systems, synthetic biologists require both a pool of quantitatively annotated biological parts and the knowledge that these parts can be logically engineered into more complex networks with predictable function.One such network, which carries intrinsic value and tests a bottom-up design approach, is a network that confers memory. In this study we describe the rational design and construction of a high fidelity, modular memory device in yeast based on transcriptionally controlled autoregulatory positive feedback. This device heritably retains an induced state in individual cells in response to a transient stimulus. The rational design approach used here employs an extensive in vivo quantitative characterization of a set of synthetic transcription factors and the prediction of system behavior via network models incorporating these measured parameters. By successfully constructing this memory device, we established the essential parameters for maintaining an autoregulatory positive feedback loop in a dividing cellular system. Most importantly, we demonstrated predictability of system behavior in eukaryotes when the system is built from well-understood components. Results and Discussion Functional activators based on a modular architectureTo rationally engineer a memory device, we designed a set of fluorescently labeled synthetic transcription factors and their corresponding reporter genes to serve as candidate components. Each activator gene consists of a DNA-binding domain (DBD), two tandem copies of the monomeric red fluorescent protein (RFP) mCherry (Shaner et al. 2004), the viral activation domain VP64 (Beerli et al. 1998), and the SV40 nuclear localization sequence (NLS) (Kalderon et al. 1984;Lanford and Butel 1984), all under control of the GAL1/10 promoter (Fig. 1A). Each reporter gene has multiple copies of the DNAbinding sites corresponding to its given transcription factor upstream of the minimal CYC1 promoter, and its protein coding region encodes two tandem copies of the yellow fluorescent protein variant (YFP) Venus ( Fig. 1A; Nagai et al. 2002). The DBDs used were the LexA DBD (Hurstel et al. 1986(Hurstel et al. , 1988, an engineered variant of the murine zinc-finger Zif268 (ZifH) (Hurt et al. 200...

Temperature is a key parameter in physiological processes, and probes able to detect small changes in local temperature are necessary for accurate and quantitative physical descriptions of cellular events. Several have recently emerged that offer excellent temperature sensitivity, spatial resolution, or cellular compatibility, but it has been challenging to realize all of these properties in a single construct. Here, we introduce a luminescent nanocrystal-based sensor that achieves this with a 2.4% change/°C ratiometric response over physiological temperatures in aqueous buffers, with a precision of at least 0.2 °C. Thermoresponsive dual emission is conferred by a Förster resonant energy transfer (FRET) process between CdSe-CdS quantum dot-quantum rods (QD-QRs) as donors and cyanine dyes as acceptors, which are conjugated to QD-QRs using an amphiphilic polymer coating. The nanothermometers were delivered to live cells using a pH-responsive cationic polymer colloid, which served to both improve uptake and release nanocrystals from endosomal confinement. Within cells, they showed an unexpected enhancement in their temperature response and sensitivity, highlighting the need to calibrate these and similar probes within the cell.

No abstract

Extracellular electron transfer pathways allow certain bacteria to transfer energy between intracellular chemical energy stores and extracellular solids through redox reactions. Microorganisms containing these pathways, exoelectrogens, are a critical part of microbial electrochemical technologies that aim to impact applications in bioenergy, biosensing, and biocomputing. However, there are not yet any examples of economically viable microbial electrochemical technologies due to the limitations of naturally occurring exoelectrogens. Here we first briefly summarize recent discoveries in understanding extracellular electron transfer pathways, then review in-depth the creation of customized and novel exoelectrogens for biotechnological applications. We analyze engineering efforts to increase current production in native exoelectrogens, which reveals that modulating certain processes within extracellular electron transfer are more effective than others. We also review efforts to create new exoelectrogens and highlight common challenges in this work. Lastly, we summarize work utilizing engineered exoelectrogens for biotechnological applications and the key obstacles to their future development. Fueled by the development of genetic tools, these approaches will continue to expand and genetically modified organisms will continue to improve the outlook for microbial electrochemical technologies.