Abstract
BackgroundLike a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Action of the complexes on cellulose releases cellobiose and longer cellodextrins but to date, little is known about the transport and utilization of the produced cellodextrins in the bacterium. A better understanding of the uptake systems and fermentation of sugars derived from cellulose could have a major impact in the field of biofuels production.ResultsWe characterized a putative ABC transporter devoted to cellodextrins uptake, and a cellobiose phosphorylase (CbpA) in R. cellulolyticum. The genes encoding the components of the ABC transporter (a binding protein CuaA and two integral membrane proteins) and CbpA are expressed as a polycistronic transcriptional unit induced in the presence of cellobiose. Upstream, another polycistronic transcriptional unit encodes a two-component system (sensor and regulator), and a second binding protein CuaD, and is constitutively expressed. The products might form a three-component system inducing the expression of cuaABC and cbpA since we showed that CuaR is able to recognize the region upstream of cuaA. Biochemical analysis showed that CbpA is a strict cellobiose phosphorylase inactive on longer cellodextrins; CuaA binds to all cellodextrins (G2–G5) tested, whereas CuaD is specific to cellobiose and presents a higher affinity to this sugar. This results are in agreement with their function in transport and signalization, respectively. Characterization of a cuaD mutant, and its derivatives, indicated that the ABC transporter and CbpA are essential for growth on cellobiose and cellulose.ConclusionsFor the first time in a Gram-positive strain, we identified a three-component system and a conjugated ABC transporter/cellobiose phosphorylase system which was shown to be essential for the growth of the model cellulolytic bacterium R. cellulolyticum on cellobiose and cellulose. This efficient and energy-saving system of transport and phosphorolysis appears to be the major cellobiose utilization pathway in R. cellulolyticum, and seems well adapted to cellulolytic life-style strain. It represents a new way to enable engineered strains to utilize cellodextrins for the production of biofuels or chemicals of interest from cellulose.
Highlights
Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose
Sequence analysis of cua gene cluster and its products The cua gene-cluster from loci Ccel_2109 to Ccel_2115 encompasses seven genes predicted to encode (i) a solute binding protein (CuaD), (ii) a putative two-component system composed of a sensor (CuaS) and a response regulator (CuaR), (iii) a solute binding protein CuaA and two integral membrane proteins CuaB and C, forming a putative ABC transporter, and (iv) a putative GH94 cellobiose phosphorylase named cellobiose phosphory‐ lase (CbpA) in the present study (Fig. 1)
As for solute binding protein (SBP) found in other Gram-positive bacteria, an N-terminus typical lipoprotein signal peptidase II is predicted for both proteins
Summary
Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Cellulose is the most abundant polysaccharide on earth, mostly found in plant-cell wall This polymer is made up of linear chains of glucosyl residues linked through β-1,4 glycosidic bonds, and arranged in a quasi-crystalline structure that makes it highly recalcitrant to enzymatic hydrolysis. This large reservoir of glucose represents a remarkable potential renewable source of energy to produce biofuels or chemicals as an alternative to fossilderived products. The bacterium performs efficient extracellular degradation of plant cell wall polysaccharides into fermentable sugars thanks to multienzymatic complexes called cellulosomes [4, 5]. Uptake of the released cellodextrins is crucial for growth on cellulose, and little is known about their transport and utilization into R. cellulolyticum, as well as in other cellulosomes-producing bacteria
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