Abstract
Background(Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium that produces the most extensive and intricate cellulosomal system known in nature. Recently, the elaborate architecture of the B. cellulosolvens cellulosomal system was revealed from analysis of its genome sequence, and the first evidence regarding the interactions between its structural and enzymatic components were detected in vitro. Yet, the understanding of the cellulolytic potential of the bacterium in carbohydrate deconstruction is inextricably linked to its high-molecular-weight protein complexes, which are secreted from the bacterium.ResultsThe current proteome-wide work reveals patterns of protein expression of the various cellulosomal components, and explores the signature of differential expression upon growth of the bacterium on two major carbon sources—cellobiose and microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing components (mainly enzymes), in addition to free enzymatic subunits. The dockerin-bearing components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family members were represented among 102 carbohydrate-degrading enzymes, including the omnipresent, most abundant GH48 exoglucanase. Specific cellulosomal components were found in different molecular-weight fractions associated with cell growth on different carbon sources. Overall, microcrystalline cellulose-derived cellulosomes showed markedly higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different cellulosic and/or hemicellulosic carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by Clostridium thermocellum purified cellulosomes.ConclusionsThe current research demonstrates the involvement of key cellulosomal factors that participate in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies on various carbon sources was revealed. The novel reservoir of cellulolytic components of the cellulosomal degradation machineries may serve as a pool for designing new cellulolytic cocktails for biotechnological purposes.
Highlights
The rising demand for renewable alternative fuels derives from our rapidly growing, global population and concerns about climate change and environmental pollution [1]
Purification and fractionation of secreted cellulosome complexes In order to evaluate the proteomic composition of B. cellulosolvens cellulosomes, we purified the extracellular medium of B. cellulosolvens cells, after anaerobic growth of the bacterium on two types of carbon source: cellobiose (CB) and microcrystalline cellulose (MCC)
SDS-PAGE examination of isolated fractions generated a similar profile of cellulosomal components between high-molecular-weight fractions of cellobiose (CB_I) and microcrystalline cellulose (MCC_I) as well as between the lower-molecular-weight fractions
Summary
The rising demand for renewable alternative fuels derives from our rapidly growing, global population and concerns about climate change and environmental pollution [1]. Biofuels are produced from biological materials, mainly renewable plant biomass [2]. Aside from being the primary building material for plants, cellulose has many other uses. There are 19 known species of cellulosome-producing bacteria (both mesophilic and thermophilic) [6]. Some of these bacteria, e.g., Clostridium cellulolyticum, Clostridium josui, and C. papyrosolvens, produce simple cellulosomal systems with a single major scaffoldin that bears only type I cohesins for integration of the dockerin-containing enzymes. E.g., C. thermocellum, C. clariflavum, and Acetivibrio cellulolyticus, produce complex cellulosomal systems, in which primary scaffoldins bear type I cohesins whereas a second class of scaffoldin contains type II cohesins that anchor the cellulosome to the bacterial cell surface [6]
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