Study of Two component signal transduction systems in Desulfovibrio vulgaris Hildenborough (Part of ESPP2)

Group Members:

• PI: Aindrila Mukhopadhyay
• Post Doctoral researcher: Jayashree Ray
• Staff Research Associate: Eric Luning
• Post Doctoral Researcher: Lara Rajeev

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 60 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 the recently developed methods (Laub et al 2007) for the in vitro confirmation of specific phosphotransfer from histidine kinases to their cognate response regulators. This is aided by in silico methods to predict candidate partners for histidine kinases or response regulators that are either ORFans or have no proximal genes that may be supposed to be their cognate partners. Such methods have been developed by several groups (e.g. Burger and van Nimwegen 2008) and can be checked for additional corroboration from microarray data in our VIMSS database. Further, using modified ChIP-chip workflows, we are elucidating the genes regulated by active response regulators. For the latter we use high density tiling arrays from Nimblegen designed by the Arkin lab at LBNL.

Relevant literature and links:

• Alm, E., K. Huang, and A. Arkin. 2006. The evolution of two-component systems in bacteria reveals different strategies for niche adaptation. PLoS Comput Biol 2:e143.

• Laub, M. T., E. G. Biondi, and J. M. Skerker. 2007. Phosphotransfer profiling: systematic mapping of two-component signal transduction pathways and phosphorelays. Methods Enzymol 423:531-48.

• Burger. L. and van Nimwegen, E. 2008 Accurate prediction of protein-protein interactions from sequence alignments using a Bayesian method. Molecular Systems Biology 4:165 (2008)

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Microbial Fuels Transport at JBEI: The Role of efflux pumps in Escherichia coli solvent resistance

Fuel Transport Team:

• Director: Aindrila Mukhopadhyay
• Post Doctoral researcher: Mary Dunlop

For microbial fuel production, the efficiency with which fuel can be exported from the cell is likely to have significant influence on production titer. Build-up of fuel molecules may directly reduce titer, and may also cause significant intracellular stress, leading to feedback inhibition of fuel production. Transport systems, such as efflux pumps and ABC-transport systems in bacteria and yeast, are documented to export a broad range of substrates, including solvents, and provide a valuable engineering route to relieve fuel accumulation-related stress and improve production titer.

We focus on investigating the role of native, as well as heterologously expressed, RND efflux pumps in E. coli. Targeted studies focus on the well-characterized E. coli AcrAB-TolC system, and efflux pumps from solvent resistant bacteria such as Pseudomonas putida S12. Because efflux pumps are likely to be specific to certain fuel molecules and stressors, a wider range of native and heterologous efflux pump systems must be tested against different fuel compound exposure, growth conditions, and in different engineered hosts. To address our broad goal of improving solvent resistance using efflux pumps, a high-throughput approach has been initiated to create a library of expression vectors representing all efflux pumps from E. coli as well from other organisms known to be naturally resistant to solvents.

Omics Research at JBEI

Group Members:

• Director: Aindrila Mukhopadhyay
• Scientist (Mass Spec Lead): Chirstopher J. Petzold
• Post Doctoral researcher (Proteomics): Alyssa M Redding
• Staff Research Associate (Microarray Lead): Mario Ouellet
• Staff Research Associate (Metabolite studies lead): Peter I. Benke
• Research Assistant (Proteomics): Tanveer S. Batth
• Research Assistant (Microarray): Pramila Tamrakar
• Research Assistant (Metabolite studies): Heather Szmidt

Image: Alyssa Redding

Omics research at JBEI is part of the Technologies division and provides enabling tools for a variety of cell wide and analytical measurements required for a Biofuels research program. For the conversion of sugars derived from deconstructed lingo-cellulosic biomass to fuel compounds, an important area of research is on engineered organisms that contain combinations of native and non-native biochemical pathways for the production of a target metabolite and also efforts to understand the causes of toxicity/ stress during such applications. For example, the incorporation of exogenous biochemical pathways into a host organism places unregulated strain on the cell by consuming metabolites, energy and critical cofactors creating an imbalance that will trigger a variety of stress response systems. While these stress responses may be useful to the cell in such an environment, they are unfavorable in an engineering context and reduce product yield or viability during production culturing. Systems biology, built on the foundations of omics studies (genomics, proteomics, metabolomics and fluxomics), enables a comprehensive view of the impact of an exogenous pathway on the host within the context of its full metabolism. To match the requirement of such high-throughput profiling of particular classes of cellular components, genes, proteins and metabolites, our capabilities now include microarray analysis and high resolution mass spectrometry (combined with LC, GC, and CE for shotgun proteomics, targeted protein studies, primary and secondary metabolite analysis and glycomics). In collaboration with the computational core we also have powerful data analysis and integration tools. These functional genomics workflows are now being applied to gather data for the effect of (1) exposure of deconstruction conditions (Ionic liquids, post saccharification mix from simple and complex cellulose sources), (2) accumulation of endogenous and exogenous target metabolites and (3) impact of different growth condition and expression of the biosynthetic pathway proteins in our model host microbes (E. coli, S. cerevisiae). Specifically the use of targeted proteomics using the MRM workflow has proved valuable in gauging the presence of a complete engineered pathway. Finally, the functional genomics and analytical tools described above are also being extensively used by the Feedstock division for a molecular characterization of cell wall and by the Deconstruction division in meta-genomic studies.

Relevant literature:

• Mukhopadhyay, A., A. M. Redding, B. J. Rutherford, and J. D. Keasling. 2008. Importance of systems biology in engineering microbes for biofuel production. Curr Opin Biotechnol 19:228-34.

• Fortman JL, C. S., Mukhopadhyay A, Chou H, Lee TS, Steen E, Keasling JD. 2008. Biofuel alternatives to ethanol: pumping the microbial well. Trends in Biotechnology.