Abstract
BackgroundIn anaerobic cellulolytic micro-organisms, cellulolysis results in the action of several cellulases gathered in extracellular multi-enzyme complexes called cellulosomes. Their action releases cellobiose and longer cellodextrins which are imported and further degraded in the cytosol to fuel the cells. In Ruminiclostridium cellulolyticum, an anaerobic and cellulolytic mesophilic bacteria, three cellodextrin phosphorylases named CdpA, CdpB, and CdpC, were identified in addition to the cellobiose phosphorylase (CbpA) previously characterized. The present study aimed at characterizing them, exploring their implication during growth on cellulose to better understand the life-style of cellulolytic bacteria on such substrate.ResultsThe three cellodextrin phosphorylases from R. cellulolyticum displayed marked different enzymatic characteristics. They are specific for cellodextrins of different lengths and present different kcat values. CdpC is the most active enzyme before CdpA, and CdpB is weakly active. Modeling studies revealed that a mutation of a conserved histidine residue in the phosphate ion-binding pocket in CdpB and CdpC might explain their activity-level differences. The genes encoding these enzymes are scattered over the chromosome of R. cellulolyticum and only the expression of the gene encoding the cellobiose phosphorylase and the gene cdpA is induced during cellulose growth. Characterization of four independent mutants constructed in R. cellulolyticum for each of the cellobiose and cellodextrin phosphorylases encoding genes indicated that only the cellobiose phosphorylase is essential for growth on cellulose.ConclusionsUnexpectedly, the cellobiose phosphorylase but not the cellodextrin phosphorylases is essential for the growth of the model bacterium on cellulose. This suggests that the bacterium adopts a “short” dextrin strategy to grow on cellulose, even though the use of long cellodextrins might be more energy-saving. Our results suggest marked differences in the cellulose catabolism developed among cellulolytic bacteria, which is a result that might impact the design of future engineered strains for biomass-to-biofuel conversion.
Highlights
In anaerobic cellulolytic micro-organisms, cellulolysis results in the action of several cellulases gathered in extracellular multi-enzyme complexes called cellulosomes
The ABC transporter called CuaABC has a solute-binding protein which binds to cellodextrins with lengths ranging from cellobiose (G2) to cellopentaose (G5), suggesting that at least these cellodextrins might be imported in the cytosol
We have formerly shown that imported cellobiose is subsequently converted into glucose and α-glucose 1-phosphate (G-1P) by the cellobiose phosphorylase A (CbpA) [10]
Summary
In anaerobic cellulolytic micro-organisms, cellulolysis results in the action of several cellulases gathered in extracellular multi-enzyme complexes called cellulosomes Their action releases cellobiose and longer cellodex‐ trins which are imported and further degraded in the cytosol to fuel the cells. We have formerly shown that imported cellobiose is subsequently converted into glucose and α-glucose 1-phosphate (G-1P) by the cellobiose phosphorylase A (CbpA) [10] The gene encoding this enzyme forms with the genes cuaABC an operon named (cuaABC-cbpA). The transformation of the strain with a vector containing the ABC transporter genes but not the cellobiose phosphorylase-encoding gene cbpA restored growth on cellulose but not on cellobiose This observation suggests that cellodextrins of degree of polymerization (DP) greater than 2 might be imported in the cytosol, ensuring growth on cellulose of this strain. The import of long cellodextrins is believed to be more cost-effective compared to the import of short ones, since for the same ATP transport cost, long cellodextrins carry more glucose units and, generate more energy than short ones [11]
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