Research Accomplishment Reports 2007

Proteomic Analysis of Anaerobic Bacterial Metabolism

H.J. Strobel
Department of Animal and Food Sciences

 

Project Description

Microbial metabolism offers a platform for designing bio-based production technologies, and agricultural and forestry biomass are potential feedstocks for the production of bio-products. Clostridium thermocellum is an anaerobic, thermophilic bacterium that ferments cellulosic biomass to ethanol and organic acids. However, the organism is relatively sensitive to ethanol and so sophisticated bio-analytical approaches are being used to study the nature of this sensitivity.

Gel-based and liquid chromatography-based techniques were employed to separate protein fractions derived from wild-type and ethanol-adapted cultures. Mass spectrometry analysis was then used to identify proteins based on the recently completed C. thermocellum genome sequence. Our initial studies utilized traditional gel-based protein separations and showed that protein expression in the tolerant strain was dramatically different than in the sensitive wild-type. However, gel-based separations are limited by their ability to detect and resolve proteins that are high molecular weight, highly basic, or hydrophobic. With this in mind, we developed a two-dimensional liquid chromatography separation scheme coupled to tandem mass spectrometry (Multi-dimensional Protein Identification Technology, MudPIT).

Our most recent work has refined this non-gel approach to provide better quantification by using a metabolic labeling strategy. MudPIT analysis revealed that the expression of more than 60 proteins were significantly affected by adaptation to ethanol. These proteins take part in a wide variety of functional processes including regulation of transcription, translation, signal transduction, metabolism, transportation, post-translational modification. This is the first quantitative proteomic analysis of C. thermocellum based on the MudPIT approach. This knowledge will set the stage for developing rational schemes that lead to manipulation of ethanol-producing bacteria.

Impact

Continued dependence on fossil-based fuels has negative impacts on the environment, economy, and national security. The conversion of biomass by microorganisms to bio-based products and bio-energy is a sustainable alternative. Estimates are that nearly 11 million tons of agricultural, forestry and urban-waste fibrous biomass could be used each year for bio-fuel production in Kentucky. Bio-conversion of these feedstocks could yield 600 million gallons of ethanol, and this production could replace a significant quantity of the gasoline utilized in the state. However, there are still significant barriers that prevent the economical implementation of bio-based technologies. Our studies combine the information contained in genomic sequence databases with the emerging fields of proteomics and metabolomics to examine the physiology of anaerobic bacteria under industrially relevant conditions. The results of this work will be useful in designing economically relevant bio-based processes.

Publications

Fu, Y- J., Strobel, H. J., and Lynn, B. C. (2007). Proteomic Analysis of Clostridium thermocellum sub-cellular fractions using 15N-metabolic labeling strategy. American Society of Mass Spectrometry Meeting. Indianapolis, In.

Williams, T. I., Combs, J. C., Lynn, B. C., and Strobel, H. J. (2007). Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum. Applied Microbiology and Biotechnology 74:422-432.