title: Quantitative proteomic analysis of the cellulolytic system of Clostridium termitidis CT1112 reveals distinct protein expression profiles upon growth on α-cellulose and cellobiose.
abstract: Clostridium termitidis CT1112 is an anaerobic, mesophilic, cellulolytic bacterium with potential applications in consolidated bioprocessing of lignocellulosic biomass. To understand how C. termitidis degrades lignocellulose, iTRAQ-based 2D HPLC-MS/MS proteomics was used to measure protein expression in cell lysates and extracellular (secretome) fractions of C. termitidis grown on α-cellulose and cellobiose at both exponential and stationary growth phases. Exoglucanases (GH48, GH9), endoglucanases (GH5, GH8, GH9), hemicellulases including xylanases (GH8, GH10, GH11, GH30) and mannanase (GH26) as well as extracellular adhesion proteins and cellulosome associated proteins, exhibited higher expression on cellulose-grown cells. The expression of these proteins increased with a decrease in growth rate. Non-cellulosomal proteins however did not change significantly between substrate conditions, although there were a few exceptions. Collectively, these would contribute to hydrolysis of lignocellulosic material for uptake through ABC sugar transport proteins. On cellobiose, chitinases (GH18) were expressed abundantly. Although a large number of proteins were shared between the fractions analyzed, some proteins were detected exclusively in the cellular fraction, while others were detected in the secretome. This study reports for the first time on the cellulolytic machinery employed by C. termitidis to hydrolyze cellulosic substrate and provides an understanding of how this microbe deconstructs biomass.
  
  
  CONCLUSIONS
  The genome of C. termitidis CT1112 contains genes for a wide variety of carbohydrate active enzymes. Based on bioinformatics analyses, many of these genes appear to encode cellulosome-associated proteins, while others may be secreted extracellularly. To understand how C. termitidis degrades and depolymerizes cellulosic substrates, cells were grown on simple and complex carbohydrates, and quantitative 4-plex iTRAQ-based 2D HPLC-MS/MS proteomics was applied to measure protein expression levels in biological replicates of both cell lysates and extracellular protein (secretome) fractions, at exponential and stationary phases of growth. The resulting data have provided insight into the range of substrates that may be hydrolyzed by C. termitidis, and may be useful in determining potential industrial applications of C. termitidis in biomass to bioenergy production via consolidated bioprocessing.
authors.affiliation: Department of Biosystems Engineering, University of Manitoba, Winnipeg R3T 2N2, MB, Canada.
authors.forename: Riffat I
authors.initials: RI
authors.lastname: Munir
authors.affiliation: Department of Physics and Astronomy, University of Manitoba, Winnipeg R3T 2N2, MB, Canada; Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, Winnipeg R3T 2N2, MB, Canada.
authors.forename: Victor
authors.initials: V
authors.lastname: Spicer
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, Winnipeg R3T 2N2, MB, Canada.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Shamshurin
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, Winnipeg R3T 2N2, MB, Canada.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Krokhin
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, Winnipeg R3T 2N2, MB, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Wilkins
authors.affiliation: Department of Biosystems Engineering, University of Manitoba, Winnipeg R3T 2N2, MB, Canada.
authors.forename: Umesh
authors.initials: U
authors.lastname: Ramachandran
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg R3T 2N2, MB, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Sparling
authors.affiliation: Department of Biosystems Engineering, University of Manitoba, Winnipeg R3T 2N2, MB, Canada. Electronic address: david.levin@umanitoba.ca.
authors.forename: David B
authors.initials: DB
authors.lastname: Levin
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis*; D002475:Cellobiose / Q000737:chemistry / Q000494:pharmacology*; D002482:Cellulose / Q000737:chemistry / Q000494:pharmacology*; D003013:Clostridium / Q000254:growth & development*; D005786:Gene Expression Regulation / Q000187:drug effects*; D040901:Proteomics
chemical_list: D001426:Bacterial Proteins; D002475:Cellobiose; D002482:Cellulose
keywords: 2D HPLC-MS/MS; CAZYmes; Cellulosome; Glycoside hydrolases; Secretome; iTRAQ