ABSTRACT
Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.
Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Clostridium thermocellum/drug effects , Ethanol/pharmacology , Gene Expression Regulation, Bacterial , Proteomics , Bacterial Proteins/genetics , Clostridium thermocellum/genetics , Clostridium thermocellum/growth & development , Clostridium thermocellum/metabolism , Gene Expression Profiling , Industrial Microbiology/methods , Membrane Proteins/genetics , Membrane Proteins/metabolismABSTRACT
A novel, Bicine-based SDS-PAGE buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and running buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.
