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) Two component systems, comprised typically of Histidine Kinase and Response regulator proteins, represent the primary and ubiquitous mechanism in bacteria for initiating cellular response towards a wide variety of environmental conditions. In D. vulgaris Hildenborough, more than 70 such systems have been predicted, but remain mostly uncharacterized. The ability of D. vulgaris to survive in its environment is no doubt linked with the activity of genes modulated by these two component signal transduction systems. These genes in D. vulgaris also present a fascinating set for detailed study. The large number of Histidine kinases are predicted to have arisen from extensive gene duplication (Alm et al 2006), rather than HGT as is predicted to be the case with other microbes such as E. coli and B. subtilis. As a result the Histidine Kinases in D. vulgaris often contain multiple similar domains in a variety of configurations. Further, the majority of both Histidine Kinases and Response regulators in D. vulgaris are mostly encoded in monocistronic operons providing little clue as to the signal they respond to. In order to map Histidine Kinases to their cognate Response regulators and the Response regulators to the genes they may regulate, our project uses a library of purified Histidine Kinase and Response regulator proteins. We use biochemical and array based methods to map histidine kinases to their cognate response regulators and down stream functions. 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.