Mukhopadhyay:Research

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Host Engineering for solvent tolerant microbes Efflux Pumps provide a direct mechanism to alleviate product toxicity (Image prepared by Everett Kaplan (JBEI)) For efficient fuel and chemicals production in microbes, the efficiency with which the final product is exported from the cell is likely to have significant influence on production titer. Build-up of the product may directly reduce titer, and when toxic (as is often the case), also cause significant intracellular stress, leading to feedback inhibition of fuel production. Additional sources of inhibition arise from the byproducts of biomass deconstruction and from toxic intermediates. We take both targeted and systems biology approaches to identifying candidates that both alleviate this growth inhibition and improve production. Of these, transport systems, such as efflux pumps in bacteria (and and ABC-transport systems yeast), are documented to export a broad range of substrates including solvents and provide a direct engineering route to relieve fuel accumulation-related stress and improve production titer. We have used a high-throughput approaches to identify efflux pumps that, in E. coli, confer tolerance to many desirable candidate fuels and chemicals. We have successfully used systems biology to identify tolerance genes that not only alleviate toxicity but also improve production. We have also used directed evolution, and improved regulation, to further enhance the fitness and tolerance provided by efflux pumps. Signal Transduction and survival in metal reducing bacteria Map of genes regulated by Response Regulators in D. vulgaris (From Rajeev et al 2011) Signaling systems are critical to bacteria in enabling them to continually monitor their environment and respond appropriately to any changes. The numbers and types of signaling systems a microbe possesses is an indication both of the variability of its environment as well as its ability to perceive and fine-tune its response to diverse signals. As part of the ENIGMA Scientific Focus Area, we are studying signaling systems in microbes present in DOE-relevant sites. Desulfovibrio vulgaris is a model sulfate-reducing bacterium with a vast array of uncharacterized signaling and regulatory systems. Our group has developed and optimized an in vitro microarray-based DAP-chip (or seq) method to determine gene targets for bacterial response regulators and used this method to reveal regulatory networks by determining the gene targets for almost all (twenty-four) D. vulgaris two component response regulators that function via transcriptional control. Our study led to the discovery of a complex regulatory network around the central carbon metabolic pathway of lactate uptake and oxidation, which is under the control of lactate-sensing, nitrite-sensing, and phosphate-sensing two-component systems. Currently, we are characterizing cyclic-di-GMP based signaling pathways, the role of which has not been examined in sulfate-reducing bacteria. To this end, we have identified one cyclic-di-GMP-modulating response regulator that impacts biofilm formation, and one that impacts planktonic growth. In collaboration with other ENIGMA researchers, we are also examining sigma54-dependent one-component systems, and unique tungstate-responsive transcription factors. Other ongoing experiments involve using transposon mutant pools to determine genes required for fitness in limiting nutrient conditions as often found in the environment, as well as genes that are required for motility and chemotaxis Signaling and gene regulation in dominant cyanobacteria in Desert soil crusts A gift of Microcoleus culture from the Garcia-Pichel group to start off our project Desert soil crusts are living systems, primarily microbial, that cover large areas of our planet. Complex enough to survive extreme conditions and simple enough to be studied using state of the art technologies, these microbial communities provide invaluable systems to evaluate the impact of climate change on carbon flux. The cyanobacterium that is the dominant organism in these crusts is being sequenced at JGI as is the entire desert soil crusts community. These organisms encode elegant signal transduction and response regulatory systems that are at the core of the ability of these microbes to respond and survive in their ecosystems. The study of these signaling mechanisms and the corresponding response provides the molecular level assessment of important biogeochemical activities that will be utilized for improved climate models.