The electron transport chain of Shewanella oneidensis MR-1 can operate bidirectionally to enable microbial electrosynthesis.

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O2) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design.

Respiration is essential for aerobic growth of Zymomonas mobilis ZM4.

Zymomonas mobilis is an alpha-proteobacterium that is a promising platform for industrial scale production of biofuels due to its efficient ethanol fermentation and low biomass generation. Z. mobilis is aerotolerant and encodes a complete respiratory electron transport chain, but the benefit of respiration for growth in oxic conditions has never been confirmed, despite decades of research. Growth and ethanol production of wild-type Z. mobilis is poor in oxic conditions indicating that it does not benefit from oxidative phosphorylation. Additionally, in previous studies, aerobic growth improved significantly when respiratory genes were disrupted (ndh) or acquired point mutations (cydA and cydB), even if respiration was significantly reduced by these changes. Here, we obtained clean deletions of respiratory genes ndh and cydAB, individually and in combination, and showed, for the first time, that deletion of cydAB completely inhibited O2 respiration and dramatically reduced growth in oxic conditions. Both respiration and aerobic growth were restored by expressing a heterologous, water-forming NADH oxidase, noxE. Oxygen can have many negative effects, including formation of reactive oxygen species (ROS) or directly inactivating oxygen sensitive enzymes. Our results suggest that the effect of molecular oxygen on enzymes had a greater negative impact on Z. mobilis than formation of ROS. This result shows that the main role of the electron transport chain in Z. mobilis is reducing the intracellular concentration of molecular oxygen, helping to explain why it is beneficial for Z. mobilis to use electron transport chain complexes that have little capacity to contribute to oxidative phosphorylation. IMPORTANCE A key to producing next-generation biofuels is to engineer microbes that efficiently convert non-food materials into drop-in fuels, and to engineer microbes effectively, we must understand their metabolism thoroughly. Zymomonas mobilis is a bacterium that is a promising candidate biofuel producer, but its metabolism remains poorly understood, especially its metabolism when exposed to oxygen. Although Z. mobilis respires with oxygen, its aerobic growth is poor, and disruption of genes related to respiration counterintuitively improves aerobic growth. This unusual result has sparked decades of research and debate regarding the function of respiration in Z. mobilis. Here, we used a new set of mutants to determine that respiration is essential for aerobic growth and likely protects the cells from damage caused by oxygen. We conclude that the respiratory pathway of Z. mobilis should not be deleted from chassis strains for industrial production because this would yield a strain that is intolerant of oxygen, which is more difficult to manage in industrial settings.

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome.

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

A common inducer molecule enhances sugar utilization by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is an electroactive bacterium that is a promising host for bioelectrochemical technologies, which makes it a common target for genetic engineering, including gene deletions and expression of heterologous pathways. Expression of heterologous genes and gene knockdown via CRISPRi in S. oneidensis are both frequently induced by ß-D-1-thiogalactopyranoside (IPTG), a commonly used inducer molecule across many model organisms. Here, we report and characterize an unexpected phenotype; IPTG enhances the growth of wild-type S. oneidensis MR-1 on the sugar substrate N-acetylglucosamine (NAG). IPTG improves the carrying capacity of S. oneidensis growing on NAG while the growth rate remains similar to cultures without the inducer. Extracellular acetate accumulates faster and to a higher concentration in cultures without IPTG than those with it. IPTG appears to improve acetate metabolism, which combats the negative effect that acetate accumulation has on the growth of S. oneidensis with NAG. We recommend using extensive experimental controls and careful data interpretation when using both NAG and IPTG in S. oneidensis cultures.

NADH dehydrogenases drive inward electron transfer in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a promising chassis organism for microbial electrosynthesis because it has a well-defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH-dependent reaction in the cytoplasm, reduction of acetoin to 2,3-butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3-butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR-1 should focus on optimizing flux through NADH dehydrogenases.

Flux-Balance Analysis and Mobile CRISPRi-Guided Deletion of a Conditionally Essential Gene in Shewanella oneidensis MR-1.

Carbon-neutral production of valuable bioproducts is critical to sustainable development but remains limited by the slow engineering of photosynthetic organisms. Improving existing synthetic biology tools to engineer model organisms to fix carbon dioxide is one route to overcoming the limitations of photosynthetic organisms. In this work, we describe a pipeline that enabled the deletion of a conditionally essential gene from the Shewanella oneidensis MR-1 genome. S. oneidensis is a simple bacterial host that could be used for electricity-driven conversion of carbon dioxide in the future with further genetic engineering. We used Flux Balance Analysis (FBA) to model carbon and energy flows in central metabolism and assess the effects of single and double gene deletions. We modeled the growth of deletion strains under several alternative conditions to identify substrates that restore viability to an otherwise lethal gene knockout. These predictions were tested in vivo using a Mobile-CRISPRi gene knockdown system. The information learned from FBA and knockdown experiments informed our strategy for gene deletion, allowing us to successfully delete an "expected essential" gene, gpmA. FBA predicted, knockdown experiments supported, and deletion confirmed that the "essential" gene gpmA is not needed for survival, dependent on the medium used. Removal of gpmA is a first step toward driving electrode-powered CO2 fixation via RuBisCO. This work demonstrates the potential for broadening the scope of genetic engineering in S. oneidensis as a synthetic biology chassis. By combining computational analysis with a CRISPRi knockdown system in this way, one can systematically assess the impact of conditionally essential genes and use this knowledge to generate mutations previously thought unachievable.

Zymomonas mobilis ZM4 Utilizes an NADP+-Dependent Acetaldehyde Dehydrogenase To Produce Acetate.

Zymomonas mobilis is a promising bacterial host for biofuel production, but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main by-products generated by Z. mobilis is acetate, but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate, and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetate-forming acetaldehyde dehydrogenase in Z. mobilis, and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase. IMPORTANCE Biofuel production from nonfood crops is an important strategy for reducing carbon emissions from the transportation industry, but it has not yet become commercially viable. An important avenue to improve biofuel production is to enhance the characteristics of fermentation organisms by decreasing by-product formation via genetic engineering. Here, we identified and deleted a metabolic pathway and associated gene that lead to acetate formation in Zymomonas mobilis. Acetate is one of the major by-products generated during ethanol production by Z. mobilis, so this information may be used in the future to develop better strains for commercial biofuel production.

Zymomonas diversity and potential for biofuel production.

BACKGROUND: Zymomonas mobilis is an aerotolerant α-proteobacterium, which has been genetically engineered for industrial purposes for decades. However, a comprehensive comparison of existing strains on the genomic level in conjunction with phenotype analysis has yet to be carried out. We here performed whole-genome comparison of 17 strains including nine that were sequenced in this study. We then compared 15 available Zymomonas strains for their natural abilities to perform under conditions relevant to biofuel synthesis. We tested their growth in anaerobic rich media, as well as growth, ethanol production and xylose utilization in lignocellulosic hydrolysate. We additionally compared their tolerance to isobutanol, flocculation characteristics, and ability to uptake foreign DNA by electroporation and conjugation. RESULTS: Using clustering based on 99% average nucleotide identity (ANI), we classified 12 strains into four clusters based on sequence similarity, while five strains did not cluster with any other strain. Strains belonging to the same 99% ANI cluster showed similar performance while significant variation was observed between the clusters. Overall, conjugation and electroporation efficiencies were poor across all strains, which was consistent with our finding of coding potential for several DNA defense mechanisms, such as CRISPR and restriction-modification systems, across all genomes. We found that strain ATCC31821 (ZM4) had a more diverse plasmid profile than other strains, possibly leading to the unique phenotypes observed for this strain. ZM4 also showed the highest growth of any strain in both laboratory media and lignocellulosic hydrolysate and was among the top 3 strains for isobutanol tolerance and electroporation and conjugation efficiency. CONCLUSIONS: Our findings suggest that strain ZM4 has a unique combination of genetic and phenotypic traits that are beneficial for biofuel production and propose investing future efforts in further engineering of ZM4 for industrial purposes rather than exploring new Zymomonas isolates.

Large metabolic rewiring from small genomic changes between strains of Shigella flexneri.

The instability of Shigella genomes has been described, but how this instability causes phenotypic differences within the Shigella flexneri species is largely unknown and likely variable. We describe herein the genome of S. flexneri strain PE577, originally a clinical isolate, which exhibits several phenotypic differences compared to the model strain 2457T. Like many previously described strains of S. flexneri, PE577 lacks discernible, functional CRISPR and restriction-modification systems. Its phenotypic differences when compared to 2457T include lower transformation efficiency, higher oxygen sensitivity, altered carbon metabolism, and greater susceptibility to a wide variety of lytic bacteriophage isolates. Since relatively few Shigella phages have been isolated on 2457T or the previously characterized strain M90T, developing a more universal model strain for isolating and studying Shigella phages is critical to understanding both phages and phage-host interactions. In addition to phage biology, the genome sequence of PE577 was used to generate and test hypotheses of how pseudogenes in this strain-whether interrupted by degraded prophages, transposases, frameshifts, or point mutations-have led to metabolic rewiring compared to the model strain 2457T. Results indicate that PE577 can utilise the less-efficient pyruvate oxidase/acetyl-CoA synthetase (PoxB/Acs) pathway to produce acetyl-CoA, while strain 2457T cannot due to a nonsense mutation in acs, rendering it a pseudogene in this strain. Both strains also utilize pyruvate-formate lyase to oxidize formate but cannot survive with this pathway alone, possibly because a component of the formate-hydrogen lyase (fdhF) is a pseudogene in both strains.Importance Shigella causes millions of dysentery cases worldwide, primarily affecting children under five years old. Despite active research in developing vaccines and new antibiotics, relatively little is known about the variation of physiology or metabolism across multiple isolates. In this work, we investigate two strains of S. flexneri that share 98.9% genetic identity but exhibit drastic differences in metabolism, ultimately affecting the growth of the two strains. Results suggest additional strains within the S. flexneri species utilize different metabolic pathways to process pyruvate. Metabolic differences between these closely-related isolates suggest an even wider variety of differences in growth across S. flexneri and Shigella in general. Exploring this variation further may assist the development or application of vaccines and therapeutics to combat Shigella infections.

Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4.

Zymomonas mobilis is a bacterium that produces ethanol from glucose at up to 97% of theoretical efficiency on a carbon basis. One factor contributing to the high efficiency of ethanol production is that Z. mobilis has a low biomass yield. The low biomass yield may be caused partly by the low ATP yield of the Entner-Doudoroff (ED) glycolytic pathway used by Z. mobilis, which produces only one ATP per glucose consumed. To test the hypothesis that ATP yield limits biomass yield in Z. mobilis, we attempted to introduce the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (with double the ATP yield) by expressing phosphofructokinase (Pfk I) from Escherichia coli. Expression of Pfk I caused growth inhibition and resulted in accumulation of mutations in the pfkA gene. Co-expression of additional EMP enzymes, fructose bisphosphate aldolase (Fba) and triose phosphate isomerase (Tpi), with Pfk I did not enable EMP flux, and resulted in production of glycerol as a side product. Further analysis indicated that heterologous reactions may have operated in the reverse direction because of native metabolite concentrations. This study reveals how the metabolomic context of a chassis organism influences the range of pathways that can be added by heterologous expression.

NADH dehydrogenases Nuo and Nqr1 contribute to extracellular electron transfer by Shewanella oneidensis MR-1 in bioelectrochemical systems.

Shewanella oneidensis MR-1 is quickly becoming a synthetic biology workhorse for bioelectrochemical technologies due to a high level of understanding of its interaction with electrodes. Transmembrane electron transfer via the Mtr pathway has been well characterized, however, the role of NADH dehydrogenases in feeding electrons to Mtr has been only minimally studied in S. oneidensis MR-1. Four NADH dehydrogenases are encoded in the genome, suggesting significant metabolic flexibility in oxidizing NADH under a variety of conditions. A strain lacking the two dehydrogenases essential for aerobic growth exhibited a severe growth defect with an anode (+0.4 VSHE) or Fe(III)-NTA as the terminal electron acceptor. Our study reveals that the same NADH dehydrogenase complexes are utilized under oxic conditions or with a high potential anode. Our study also supports the previously indicated importance of pyruvate dehydrogenase activity in producing NADH during anerobic lactate metabolism. Understanding the role of NADH in extracellular electron transfer may help improve biosensors and give insight into other applications for bioelectrochemical systems.

Reversing an Extracellular Electron Transfer Pathway for Electrode-Driven Acetoin Reduction.

Microbial electrosynthesis is an emerging technology with the potential to simultaneously store renewably generated energy, fix carbon dioxide, and produce high-value organic compounds. However, limited understanding of the route of electrons into the cell remains an obstacle to developing a robust microbial electrosynthesis platform. To address this challenge, we leveraged the native extracellular electron transfer pathway in Shewanella oneidensis MR-1 to connect an extracellular electrode with an intracellular reduction reaction. The system uses native Mtr proteins to transfer electrons from an electrode to the inner membrane quinone pool. Subsequently, electrons are transferred from quinones to NAD+ by native NADH dehydrogenases. This reverse functioning of NADH dehydrogenases is thermodynamically unfavorable; therefore, we added a light-driven proton pump (proteorhodopsin) to generate proton-motive force to drive this activity. Finally, we use reduction of acetoin to 2,3-butanediol via a heterologous butanediol dehydrogenase (Bdh) as an electron sink. Bdh is an NADH-dependent enzyme; therefore, observation of acetoin reduction supports our hypothesis that cathodic electrons are transferred to intracellular NAD+. Multiple lines of evidence indicate proper functioning of the engineered electrosynthesis system: electron flux from the cathode is influenced by both light and acetoin availability, and 2,3-butanediol production is highest when both light and a poised electrode are present. Using a hydrogenase-deficient S. oneidensis background strain resulted in a stronger correlation between electron transfer and 2,3-butanediol production, suggesting that hydrogen production is an off-target electron sink in the wild-type background. This system represents a promising step toward a genetically engineered microbial electrosynthesis platform and will enable a new focus on synthesis of specific compounds using electrical energy.

Shewanella oneidensis NADH dehydrogenase mutants exhibit an amino acid synthesis defect.

Shewanella oneidensis MR-1 is a dissimilatory metal reducing bacterium with a highly branched respiratory electron transport chain. The S. oneidensis MR-1 genome encodes four NADH dehydrogenases, any of which may be used during respiration. We previously determined that a double-knockout of two NADH dehydrogenases, Nuo and Nqr1, eliminated aerobic growth in minimal medium. However, the double-knockout strain was able to grow aerobically in rich medium. Here, we determined that amino acid supplementation rescued growth of the mutant strain in oxic minimal medium. To determine the mechanism of the growth defect, we monitored growth, metabolism, and total NAD(H) pools in S. oneidensis MR-1 and the NADH dehydrogenase knockout strain. We also used a genetically encoded redox sensing system and determined that NADH/NAD+ was higher in the mutant strain than in the wild-type. We observed that the double-knockout strain was able to metabolize d,l-lactate and N-acetylglucosamine when supplemented with tryptone, but excreted high concentrations of pyruvate and acetate. The requirement for amino acid supplementation, combined with an apparent inability of the mutant strain to oxidize pyruvate or acetate suggests that TCA cycle activity was inhibited in the mutant strain by a high NADH/NAD+.

Towards patterned bioelectronics: facilitated immobilization of exoelectrogenic Escherichia coli with heterologous pili.

Biosensors detect signals using biological sensing components such as redox enzymes and biological cells. Although cellular versatility can be beneficial for different applications, limited stability and efficiency in signal transduction at electrode surfaces represent a challenge. Recent studies have shown that the Mtr electron conduit from Shewanella oneidensis MR-1 can be produced in Escherichia coli to generate an exoelectrogenic model system with well-characterized genetic tools. However, means to specifically immobilize this organism at solid substrates as electroactive biofilms have not been tested previously. Here, we show that mannose-binding Fim pili can be produced in exoelectrogenic E. coli and can be used to selectively attach cells to a mannose-coated material. Importantly, cells expressing fim genes retained current production by the heterologous Mtr electron conduit. Our results demonstrate the versatility of the exoelectrogenic E. coli system and motivate future work that aims to produce patterned biofilms for bioelectronic devices that can respond to various biochemical signals.

Shewanella oneidensis MR-1 Utilizes both Sodium- and Proton-Pumping NADH Dehydrogenases during Aerobic Growth.

Shewanella oneidensis MR-1 is a metal-reducing bacterium with the ability to utilize many different terminal electron acceptors, including oxygen and solid-metal oxides. Both metal oxide reduction and aerobic respiration have been studied extensively in this organism. However, electron transport chain processes upstream of the terminal oxidoreductases have been relatively understudied in this organism, especially electron transfer from NADH to respiratory quinones. Genome annotation indicates that S. oneidensis MR-1 encodes four NADH dehydrogenases, a proton-translocating dehydrogenase (Nuo), two sodium ion-translocating dehydrogenases (Nqr1 and Nqr2), and an "uncoupling" dehydrogenase (Ndh), but none of these complexes have been studied. Therefore, we conducted a study specifically focused on the effects of individual NADH dehydrogenase knockouts in S. oneidensis MR-1. We observed that two of the single-mutant strains, the ΔnuoN and ΔnqrF1 mutants, exhibited significant growth defects compared with the wild type. However, the defects were minor and only apparent under certain growth conditions. Further testing of the ΔnuoN ΔnqrF1 double-mutant strain yielded no growth in minimal medium under oxic conditions, indicating that Nuo and Nqr1 have overlapping functions, but at least one is necessary for aerobic growth. Coutilization of proton- and sodium ion-dependent energetics has important implications for the growth of this organism in environments with varied pH and salinity, including microbial electrochemical systems.IMPORTANCE Bacteria utilize a wide variety of metabolic pathways that allow them to take advantage of different energy sources, and to do so with varied efficiency. The efficiency of a metabolic process determines the growth yield of an organism, or the amount of biomass it produces per amount of substrate consumed. This parameter has important implications in biotechnology and wastewater treatment, where low growth yields are often preferred to minimize the production of microbial biomass. In this study, we investigated respiratory pathways containing NADH dehydrogenases with varied efficiency (i.e., the number of ions translocated per NADH oxidized) in the metal-reducing bacterium Shewanella oneidensis MR-1. We observed that two different respiratory pathways are used concurrently, and at least one pathway must be functional for growth under oxic conditions.

Oxygen Tension and Riboflavin Gradients Cooperatively Regulate the Migration of Shewanella oneidensis MR-1 Revealed by a Hydrogel-Based Microfluidic Device.

Shewanella oneidensis is a model bacterial strain for studies of bioelectrochemical systems (BESs). It has two extracellular electron transfer pathways: (1) shuttling electrons via an excreted mediator riboflavin; and (2) direct contact between the c-type cytochromes at the cell membrane and the electrode. Despite the extensive use of S. oneidensis in BESs such as microbial fuel cells and biosensors, many basic microbiology questions about S. oneidensis in the context of BES remain unanswered. Here, we present studies of motility and chemotaxis of S. oneidensis under well controlled concentration gradients of two electron acceptors, oxygen and oxidized form of riboflavin (flavin+), using a newly developed microfluidic platform. Experimental results demonstrate that either oxygen or flavin+ is a chemoattractant to S. oneidensis. The chemotactic tendency of S. oneidensis in a flavin+ concentration gradient is significantly enhanced in an anaerobic in contrast to an aerobic condition. Furthermore, either a low oxygen tension or a high flavin+ concentration considerably enhances the speed of S. oneidensis. This work presents a robust microfluidic platform for generating oxygen and/or flavin+ gradients in an aqueous environment, and demonstrates that two important electron acceptors, oxygen and oxidized riboflavin, cooperatively regulate S. oneidensis migration patterns. The microfluidic tools presented as well as the knowledge gained in this work can be used to guide the future design of BESs for efficient electron production.

CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli.

Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. Here we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits, the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe2O3 (s) reducing conditions. Overall, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.

Transforming exoelectrogens for biotechnology using synthetic biology.

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.

A miniaturized monitoring system for electrochemical biosensing using Shewanella oneidensis in environmental applications.

We present a miniaturized, free-floating monitoring system which makes use of electron transfer in Shewanella oneidensis sequestered behind a permeable membrane while maintaining diffusive contact with the environment, allowing for sensing environmental conditions. The system makes use of a commercial off-the-shelf (COTS) integrated circuit (IC) which sets a potential between a working electrode and a Ag/AgCl reference electrode while recording the resulting current from the electroactive cells. We successfully sensed both pyruvate and the environmental presence of E. coli via changes in the currents sensed. This work will enable the development of mobile aquatic sensing systems which make use of bacterial electron transfer as a transduction method. Further miniaturization of the recording mote, electrodes, packaging, and system is discussed.

An arsenic-specific biosensor with genetically engineered Shewanella oneidensis in a bioelectrochemical system.

Genetically engineered microbial biosensors have yet to realize commercial success in environmental applications due, in part, to difficulties associated with transducing and transmitting traditional bioluminescent information. Bioelectrochemical systems (BESs) output a direct electric signal that can be incorporated into devices for remote environmental monitoring. Here, we describe a BES-based biosensor with genetically encoded specificity for a toxic metal. By placing an essential component of the metal reduction (Mtr) pathway of Shewanella oneidensis under the control of an arsenic-sensitive promoter, we have genetically engineered a strain that produces increased current in response to arsenic when inoculated into a BES. Our BES-based biosensor has a detection limit of ~40 µM arsenite with a linear range up to 100 µM arsenite. Because our transcriptional circuit relies on the activation of a single promoter, similar sensing systems may be developed to detect other analytes by the swap of a single genetic part.