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Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum.

ABSTRACT: Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only beta-1,3 and beta-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G(2) to G(5)), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G(3)), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins (G(3) to G(5)) are assimilated faster than cellobiose.

SUBMITTER: Nataf Y

PROVIDER: S-EPMC2612431 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum.

Nataf Yakir Y Yaron Sima S Stahl Frank F Lamed Raphael R Bayer Edward A EA Scheper Thomas-Helmut TH Sonenshein Abraham L AL Shoham Yuval Y

Journal of bacteriology 20081024 1

Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only beta-1,3 and beta-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consol ...[more]

PMID: 18952792

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Project description:Sugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an environmentally prominent, thermophilic, cellulolytic bacterium. The bacterium employs five putative ATP-binding cassette transporters which purportedly take up cellulose hydrolysates. Here, we first applied combined genetic manipulations and biophysical titration experiments to decipher the key glucose and cellodextrin transporters. In vivo gene inactivation of each transporter and in vitro calorimetric and nuclear magnetic resonance (NMR) titration of each putative sugar-binding protein with various saccharides supported the conclusion that only transporters A and B play the roles of glucose and cellodextrin transport, respectively. To gain insight into the structural mechanism of the transporter specificities, 11 crystal structures, both alone and in complex with appropriate saccharides, were solved for all 5 putative sugar-binding proteins, thus providing detailed specific interactions between the proteins and the corresponding saccharides. Considering the importance of transporter B as the major cellodextrin transporter, we further identified its cryptic, hitherto unknown ATPase-encoding gene as clo1313_2554, which is located outside the transporter B gene cluster. The crystal structure of the ATPase was solved, showing that it represents a typical nucleotide-binding domain of the ATP-binding cassette (ABC) transporter. Moreover, we determined that the inducing effect of cellobiose (G2) and cellulose on cellulosome production could be eliminated by deletion of transporter B genes, suggesting the coupling of sugar transport and regulation of cellulosome components. This study provides key basic information on the sugar uptake mechanism of C. thermocellum and will promote rational engineering of the bacterium for industrial application. IMPORTANCE Highly efficient sugar uptake is important to microbial cell factories, and sugar transporters are therefore of great interest in the study of industrially relevant microorganisms. Clostridium thermocellum is a lignocellulolytic bacterium known for its multienzyme complex, the cellulosome, which is of great potential value in lignocellulose biorefinery. In this study, we clarify the function and mechanism of substrate specificity of the five reported putative sugar transporters using genetic, biophysical, and structural methods. Intriguingly, the results showed that only one of them, transporter B, is the major cellodextrin transporter, whereas another, transporter A, represents the major glucose transporter. Considering the importance of transporter B, we further identified the missing ATPase gene of transporter B and revealed the correlation between transporter B and cellulosome production. Revealing the mechanism by which C. thermocellum utilizes cellodextrins will help pave the way for engineering the strain for industrial applications.

Project description:The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

Dataset Information

Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum.

Publications

Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum.

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