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

Biological Limitations Of Shewanella Oneidensis Mr-1 In Bioelectrochemical Systems

Shewanella oneidensis MR-1 is a model microbe for use in bioelectrochemical systems (BESs) for several reasons, including its ability to produce electric current in the presence of oxygen and its use of endogenous electron shuttles for electron transfer. I performed in-depth studies on the growth and physiology of S. oneidensis to gain insight into BES performance with this microbe. In the first study, I analyzed changes in current production when oxygen was added to batch- and continuously-fed BESs with S. oneidensis. These experiments revealed that oxygen is more beneficial under continuously-fed conditions because it allows S. oneidensis to grow and produce flavins at a faster rate, and therefore decreases flavin washout. In the second study I optimized poised electrode potentials, because previous research suggested that electrodes poised at oxidizing potentials may cause a stress response in S. oneidensis. I grew S. oneidensis in continuously-fed BESs with potentiostatically poised electrodes at 5 different redox potentials and concluded that oxidizing electrode potentials do not cause a general stress response, but decrease current production by direct damage of biofilm cells at the electrode surface. In the third study, I compared the transcriptomes of S. oneidensis grown in a wide variety of conditions and used machine learning to discover genes important to anode- and iron-respiration in this organism. This meta-analysis revealed that some putative members of the electron transport chain, including an NADH dehydrogenase and a cytochrome oxidase, were important under anode- or Fe(III)-respiring conditions. Knockouts strains with these genes deleted confirmed their role in the anaerobic electron transport chain of S. oneidensis. Future work is needed to better characterize the efficiency of the anaerobic electron transport chain in S. oneidensis. The overall finding of this work is that S. oneidensis is not appropriate for use in BES applications that require strict anaerobic conditions or quick exchange of medium (e.g., wastewater treatment), because it performs better when mediators and planktonic cells are not washed out of the system

A study of atmosphere-forest exchange of reactive nitrogen via measurements of nitrate in canopy top leaves

REUNitric acid deposited on leaf surfaces may be an important player in the atmospheric nitrogen balance in the deciduous forest environment near UMBS. Levels of nitrate deposited on leaf surfaces at the top of the canopy were measured at different times of day to determine when the canopy was a source and when it was a sink of atmospheric nitrogen. Data was also analyzed in conjunction with precipitation and tree species recordings to determine whether these have an effect on deposited nitrogen and, in turn, on atmospheric nitrogen. A significant trend in nitrate concentrations throughout the day was found and involved decrease of leaf nitrate in the morning, changing to accumulation in the afternoon. This is suspected to be due to photolysis rates and the converion of nitric acid to nitrous acid and subsequent volatilization of nitrous acid.http://deepblue.lib.umich.edu/bitstream/2027.42/62028/1/TerAvest_Michaela_2008_REU.pd

Zymomonas diversity and potential for biofuel production.

BackgroundZymomonas 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.ResultsUsing 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.ConclusionsOur 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

Zymomonas diversity and potential for biofuel production.

BackgroundZymomonas 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.ResultsUsing 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.ConclusionsOur 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

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

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

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

Shewanella oneidensis (S. 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 bioelectrochemical systems 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