• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Jan 2009; 191(1): 203–209.
Published online Oct 24, 2008. doi:  10.1128/JB.01190-08
PMCID: PMC2612431

Cellodextrin and Laminaribiose ABC Transporters in Clostridium thermocellum[down-pointing small open triangle]

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 β-1,3 and β-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 (G2 to G5), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G3), 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 (G3 to G5) are assimilated faster than cellobiose.

Global warming and the dwindling of fossil-based energy sources have prompted current awareness of the need for alternative energy sources to replace petroleum-based fuels (14). Lignocellulose is the most abundant natural resource available for the production of bioethanol, and its use as a renewable energy source does not contribute net CO2 to the atmosphere (24). Thus, there is great incentive to utilize cellulosic biomass via its breakdown to soluble sugars that can then be fermented to ethanol. In nature, the decomposition of lignocellulose is mediated by microorganisms, representing a key step in the carbon cycle on Earth (4, 19, 36, 37). The hydrolysis of cellulose is very challenging, since it has a highly ordered, tightly packed crystalline structure; in addition, the polymer is associated with hemicellulose and surrounded by a lignin seal (16). Whereas cellulase systems of many aerobic cellulolytic microorganisms appear to be comprised of free cellulases exclusively, many anaerobic microorganisms produce an intricate multienzyme complex called the cellulosome (5, 7, 12, 28, 29). The organization of the cellulases into the cellulosome is believed to play a crucial role in their high reactivity toward crystalline cellulose (5, 10, 29). The cellulosome was first identified and described in the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum on the basis of combined biochemical, immunochemical, ultrastructural, and genetic techniques (6, 18). Interestingly, C. thermocellum is also capable of producing ethanol and is thus considered an attractive microorganism to be used for consolidated bioprocessing, by which hydrolysis of cellulose and hemicellulose and fermentation of ethanol occur in a single step (20).

C. thermocellum can grow efficiently only on β-glucans (β-1,4 and β-1,3 glucans), although it can also utilize a limited number of other carbon sources (e.g., glucose, fructose, and sorbitol) following a long adaptation period, during which spontaneous mutants probably appear (17, 23). The exact nature of these mutations is not known, and it is not clear whether they affect the transport of the sugars into the cell or their subsequent metabolism. Sugar transport in microorganisms is usually mediated by phosphoenolpyruvate-dependent phosphotransferase systems, ATP-binding cassette (ABC) transport systems, or proton-linked transport systems. In Clostridium acetobutylicum, the transport of glucose and lactose is mediated by the phosphotransferase system (33, 39), whereas in Streptomyces reticuli, another gram-positive soil bacterium, the transport of cellobiose and cellotriose is mediated by ABC transporters (26). Strobel et al. have suggested that in C. thermocellum, cellodextrins enter the cells via ATP-dependent transport systems, i.e., the ABC transporters (31). Inside the cell, the assimilated cellodextrins undergo phosphorolytic cleavage (1-3), in which a phosphate anion is the nucleophile. The net result is that two ATP molecules are consumed for each sugar molecule taken up (regardless of its size); therefore, the utilization of long cellodextrins provides more ATP per glucose molecule than that obtained from cellobiose or glucose. Indeed, this concept was demonstrated elegantly by Zhang and Lynd by use of continuous cultures of C. thermocellum growing on cellodextrins of various lengths (40). These authors also demonstrated that when growing on crystalline cellulose, cellodextrins with degrees of oligomerization of at least 4 are preferentially utilized by the cells.

ABC transporters are multicomponent systems, consisting of two membrane-spanning domains (MSDs) that constitute the channel across the membrane and two components inside the cytoplasm (nucleotide-binding domains [NBDs]) which hydrolyze ATP and use its energy for protein conformational change that drives the transport. In many cases, these transporters also utilize a solute-binding protein (SBP) located outside the cytoplasm, which binds and presents the solute molecule to the transporter channel. In gram-negative bacteria the solute-binding protein is located in the periplasm, whereas in gram-positive bacteria it is either tethered to the cell surface via an N-terminal Cys residue covalently attached to the lipid membrane or fused directly to the transporter (9). Even though ABC transporters exhibit substrate specificity in the absence of the binding proteins, the presence of binding proteins decreases the Km for the substrate and therefore contributes to the high-affinity properties of the system (8). The identification and characterization of the sugar transport systems in C. thermocellum is of great interest, since it may allow improvement of cellulose utilization by this microorganism and provide new tools for introducing cellodextrin utilization capabilities into robust ethanol producers (e.g., Saccharomyces cerevisiae) (35).

In this study, we have identified and characterized five sugar ABC transport systems in C. thermocellum, based on its recently published genome sequence (http://genome.jgi-psf.org/). Four of the transport systems are specific for β-1,4-linked glucose oligomers (cellodextrins), whereas one is specific for a β-1,3-linked glucose dimer (laminaribiose).

MATERIALS AND METHODS

Bacterial strains, plasmids, and chemicals.

Clostridium thermocellum DSM 1237 (ATCC 27405) was purchased from DSMZ GmbH, Braunschweig, Germany. Escherichia coli strains used were XL1-Blue (Stratagene, La Jolla, CA) for general cloning and BL21(DE3) (Novagen, Madison, WI) for expression via the T7 RNA polymerase expression system. pET9d (Novagen) and pGEM (Promega, Madison, WI) vectors were used for cloning. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.

Growth conditions.

Cells were grown in batch culture at 60°C in Duran anaerobic bottles (Schott Corporation, Mainz, Germany) in a medium containing (per liter) 0.65 g K2HPO3·3H2O, 0.5 g KH2PO4, 1.3 g (NH4)SO4, 42 g morpholinepropanesulfonic acid (MOPS), 5 g yeast extract, 1 g cysteine, 0.5 g MgCl2, and 2 mg resazurin. The medium included 10% cellobiose (Acros Organics, Geel, Belgium) or microcrystalline cellulose (20-μm cellulose powder; Aldrich, Milwaukee, WI).

RNA extraction.

Total RNA was isolated from the mid-exponential phase of a C. thermocellum culture supplemented with cellulose as the sole carbon source. Cell pellets of about 109 cells were suspended in 1 ml of Tri reagent (Sigma), frozen in liquid nitrogen, and stored at −80°C. The samples were sonicated (sonicator model W-375; Heat System-Ultrasonics, Inc., Plainview, NY), and the RNA was extracted according to the Tri reagent protocol. The RNA was then subjected to DNase I treatment (Qiagen GmbH, Hilden, Germany) to remove any contaminating genomic DNA, following which a cleanup protocol was performed using the RNeasy kit (Qiagen).

Transcriptional analyses.

The rapid amplification of cDNA ends (RACE) technique was used to amplify portions of the putative transcripts at their 5′ ends (Clontech, Mountain View, CA). Briefly, reverse transcription was generated with reverse transcriptase that exhibits terminal transferase activity and random primers. The cDNA was amplified with universal primers (UPM) as well as specific primers antiparallel and complementary to a sequence located 100 bp downstream of the first gene (Table (Table1).1). The PCR products were cloned into pGEM vector, electrotransformed to E. coli XL1-Blue, and sequenced.

TABLE 1.
Oligonucleotides used for this study

Cloning and protein purification.

Seven open reading frames (ORFs) potentially coding for sugar-binding proteins in C. thermocellum have been identified (cbpA, gi 125972914; cbpB, gi 125973535; cbpC, gi 125974617; cbpD, gi 125974931; lbp, gi 125974085; 125973561; and 125974458) (http://www.ncbi.nlm.nih.gov/sites/entrez) and cloned in pET9d vector via PCR without their N-terminal lipoprotein coding sequence and with the addition of six-histidine codons to provide His-tagged fusion proteins. All the primers were designed to allow in-frame cloning of the genes into the T7 polymerase expression vector (pET9d) by the use of an NcoI restriction site at the 5′ terminus and a BamHI restriction site at the 3′ terminus (Table (Table1).1). DNA sequencing verified the sequences of the cloned genes. E. coli BL21(DE3) cultures harboring the appropriate pET9d vector were grown overnight in Terrific Broth medium (25) with 30 μg·ml−1 kanamycin (0.5 liter in 2-liter shake flasks) at 37°C to obtain a typical optical density at 600 nm of 20. The cultures were harvested, resuspended in 30 ml of buffer (20 mM imidazole, 500 mM NaCl, 20 mM phosphate buffer, pH 7.0), disrupted by two passages through a French press (Spectronic Instruments, Inc., Rochester, NY), and centrifuged (14,000 × g for 20 min) for the production of clear crude protein extracts. The proteins were purified with the aid of a 5-ml Histrap column mounted on an AKTA-explorer fast protein liquid chromatography system (GE Healthcare), according to the manufacturer's instructions.

Microcalorimetry titration studies.

Titration calorimetry measurements were performed with an isothermal titration calorimeter (ITC) (VP-ITC, Microcal, Northampton, MA) as described by Wiseman et al. (38). Protein solutions for ITC were dialyzed overnight against buffer A (50 mM Tris-HCl, pH 7.0, 100 mM NaCl, and 0.02% NaN3). Ligand solutions of cellodextrins (Sigma), laminaridextrins, arabino-oligosaccharides, xylo-oligosaccharides (Megazyme, Wicklow, Ireland), glucose, fructose, lactose, maltose, and mannose were prepared by diluting with the buffer used for the protein dialysis. Aliquots (10 μl) of the ligand solution at 8.5 to 20× the binding site concentration were added by means of a 280-μl rotating stirrer-syringe to the reaction cell, containing 1.41 ml of the 0.1 mM protein solution. The heat of dilution was determined to be negligible in separate titrations of the ligand into the buffer solution. Calorimetric data analysis was carried out with Origin 7.0 software (MicroCal). Binding parameters, such as the number of binding sites (n), the binding constant (Ka [M−1]), and the binding enthalpy (ΔHa [kcal/mol]), were determined by fitting the experimental binding isotherms. Ka was primarily determined by the slope of the isotherm at the equivalence point (13).

RESULTS AND DISCUSSION

Identification of sugar ABC transport systems in C. thermocellum.

To identify potential sugar ABC transporters in C. thermocellum, we examined its recently published genome sequence (http://genome.jgi-psf.org/finished_microbes/cloth/cloth.info.html). Seven ORFs potentially encoding for sugar-binding proteins were identified based on their homology to sugar-binding proteins in other bacteria. Five of the genes (cbpA, gi 125972914; cbpB, gi 125973535; cbpC, gi 125974617; cbpD, gi 125974931; and lbp, gi 125974085) appeared to comprise parts of five complete ABC transport systems, whereas the other two genes (gi 125973561, 125974458) were not positioned adjacent to any recognizable transport system. Of the seven proteins, four (cbpB, cbpC, 125973561, 125974458) share homology to the SBP_bac_1 family, which is known to contain sugar-binding proteins. One protein, CbpD, exhibits homology to the Peripla_BP_1 family, which is responsible for the transport of many sugar-based solutes, and the Lbp protein exhibits homology to members of the Bmp family (basic membrane proteins) (http://pfam.sanger.ac.uk). CbpA did not display significant homology to any known protein family, although it shares sequence similarity with CbpD.

A schematic genetic organization of the transport systems is presented in Fig. Fig.1.1. The putative operons of transport systems A and D contain only one MSD gene, suggesting that the transmembrane channel comprises a homodimer, whereas in transport systems B, C, and E there are two MSD genes, indicating a heterodimer channel. All of the transport systems, except system B, encode their own ATPases (NBDs). In many cases, the ATP-binding protein gene in gram-positive bacteria is not part of a specific transporter operon and can function with different transporters (27, 30). For example, in S. reticuli the ATP-binding protein (MsiK) functions with two different transport systems for cellobiose and maltose (27). Thus, transport system B presumably utilizes an unidentified NBD, which is encoded elsewhere on the chromosome. Surprisingly, the system D locus also contains a gene homologous to phosphoglycerate mutase, and in system E, there are two ORFs coding for a putative radical S-adenosylmethionine and a hypothetical protein of unknown function. The function of these genes within the context of the transport systems is, at present, unclear.

FIG. 1.
Schematic representation of the genetic organization of the putative sugar ABC transporters in C. thermocellum. cbp encodes cellodextrin-binding protein, lbp encodes laminaribiose-binding protein, msd encodes MSD, nbd encodes NBD, pgm encodes phosphoglycerate ...

Transcriptional analyses of the ABC transporter operons.

Based on sequence analyses, each of the five putative sugar ABC transporters constitutes a polycistronic operon, in which a potential promoter and a rho-independent terminator were identified. The calculated free energy (ΔGa) values for the hairpin terminators of transport systems A to E were −3.5, −4.4, −3.6, −10.0, and −13.5 kcal/mole, respectively. In all of the transcripts, the intergenic spacer regions between the genes lack any obvious hairpin-like secondary structure characteristic of rho-independent terminators. To identify the 5′ end of each transcript, the RACE technique was applied, and the RACE products were cloned in the pGEM vector and introduced into E. coli XL1-Blue by transformation. From each operon, five independent clones were sequenced, and the apparent transcriptional start points obtained were consistent in all sequences (Fig. (Fig.2).2). In all cases, −35 and −10 sequences similar to the σA binding site consensus sequences defined for B. subtilis (TTGACA and TATAAT) were identified. In addition, the promoter regions of systems D and E operons contained inverted repeat sequences that might serve as binding sites for transcription factors.

FIG. 2.
Mapping the 5′ ends of the ABC transporter transcripts by the 5′ RACE technique. Arrows indicate the transcriptional start site. Bold letters are the suggested −35 and −10 sequences of the σA binding site. Inverted ...

Binding thermodynamics of the sugar-binding proteins.

To study the specificity of the sugar-binding proteins, the relevant genes were cloned fused to a His tag at the N terminus, overexpressed in E. coli BL21(DE3), and purified, and their abilities to bind different sugars were tested using ITC. By use of this approach, multiple injections of a ligand into the calorimeter cell containing a desired protein solution result in measurable heat changes from ligand binding until saturation occurs. This analysis provides direct measurement of the binding enthalpy, ΔHa, and allows simultaneous determination of the binding parameters, which include the binding constant (Ka), entropy (ΔSa), free energy (ΔGa), and the binding stoichiometry (n). The proteins were challenged with different sugars, including various cellodextrins, laminaridextrins, arabino-oligosaccharides, and xylo-oligosaccharides along with glucose, fructose, lactose, maltose, and mannose. Representative titrations for each of the sugar-binding proteins are presented in Fig. Fig.3,3, and the thermodynamic parameters together with the binding constants are summarized in Table Table2.2. All of the sugar-binding proteins exhibited high affinity for their substrates with dissociation constants (Kd) in the submicromolar range, which is typical for other known solute-binding proteins (32). In general, most of the binding interactions were both enthalpy and entropy driven, and the titration curves fit very well into a single binding site model with a calculated n of 1. In some cases, the end of the titration still showed some measurable enthalpy, which presumably arose from nonspecific binding between the ligand and the protein.

FIG. 3.
Representative isothermal calorimetric titration curves of the interactions of various oligosaccharides with the recombinant sugar-binding proteins. (A) CbpA with cellotriose at 50°C. (B) CbpB with cellotetraose at 20°C. (C) CbpC with ...
TABLE 2.
Binding of sugars to sugar-binding proteins: thermodynamic parameters and dissociation constants

Sugar-binding protein characteristics.

The characteristics of the sugar-binding proteins are as follows.

(i) CbpA.

CbpA binds only to cellotriose (G3), with a Kd of 0.20 μM. The protein is 321 amino acid residues in length with a molecular weight of 33,897, and its primary sequence shows homology to the arabinose-binding protein (AraP; gi 190333350) from Geobacillus stearothermophilus (44% identity over a 284-residue span).

(ii) CbpB.

CbpB (459 residues; molecular weight, 49,985) shares sequence similarity with both the arabino-oligosaccharide-binding protein (AbnE; gi 190333364) from G. stearothermophilus (24% identity over a 379-residue span) and the trehalose/maltose-binding protein (TMBP) from Thermococcus litoralis (23% identity over a 328-residue span). CbpB binds to cellodextrins of different lengths (G2 to G5) with the highest affinity for G4 (Kd = 0.44 μM at 30°C). Interestingly, a significant decrease in the binding enthalpy was observed when the binding temperature was increased. From the enthalpies obtained at different temperatures, the change in heat capacity, ΔCp, for the binding reactions could be determined by plotting ΔHa versus temperature (Fig. (Fig.4).4). Large negative heat capacity values are thought to be associated with hydrophobic stacking interactions, resulting from the dehydration of highly ordered water molecules that surround the hydrophobic surfaces of the aromatic side chains of the protein upon interaction with the rings of the carbohydrate ligand (34). The ΔCp binding values for G2 to G4 were quite similar (−160, −182, −159 cal/mole·K), whereas for G5, the value for ΔCp was −342 cal/mole·K (Table (Table2).2). It has been demonstrated previously that stacking interactions of tryptophan and tyrosine residues with the sugar ring provide ΔCp values of −150 and −100 cal/mole·K, respectively (21, 41). Thus, the data suggest that the binding of G2, G3, and G4 involves one Trp residue, whereas two Trp residues appear to interact with G5. Based on the three-dimensional structure of TMBP from T. litoralis (PDB ID code 1eu8) (11), there are stacking interactions between the sugar rings of the disaccharide trehalose and Trp-257, and an additional tryptophan residue, Trp-73, is also located in the vicinity of the binding site. These two Trp residues correspond to Trp296 and Trp115 in CbpB, and on the basis of our findings we propose that they play a similar role in the binding of cellodextrins.

FIG. 4.
Changes in the binding enthalpy (ΔHa) as a function of temperature for interaction of cellodextrins with CbpB. ΔCp values were derived from the slopes of the linear correlation.

(iii) CbpC.

Like CbpB, CbpC (460 residues; molecular weight, 52,208) is homologous to AbnE from G. stearothermophilus (19% identity over a 274-residue span) but interacts with G1 to G5, with G5 giving the lowest Kd (0.28 μM). However, the binding stoichiometry ratios for glucose and cellobiose were higher than 1 (1.8 and 2.2, respectively). Since CbpC exhibits a single binding site for G3 to G5, it is not clear if the binding of glucose and cellobiose is biologically productive.

(iv) CbpD.

CbpD (372 residues; molecular weight, 39,480) binds G3 to G5, with G5 giving the lowest Kd (0.22 μM). Similar to CbpA, the protein shows homology to the AraP protein from G. stearothermophilus (27% identity over a 225-residue span).

(v) Lbp.

Lbp is a 355-residue protein with a molecular weight of 37,599 that does not share significant sequence similarity with any known sugar-binding protein. This intriguing protein binds only laminaribiose, a β-1,3-linked glucose dimer, which is the end product of the action of endo-1,3(4)-β-glucanases on natural polysaccharides that contain β-1,3 glucans, such as lichenan, callose, and laminarin.

Biological significance of the C. thermocellum sugar ABC transporters.

The ability of three of the sugar-binding proteins (CbpB to -D) to bind several cellodextrins is consistent with previous studies showing that cellodextrins inhibit the uptake of cellobiose in a competitive manner (31). Our results correlate well with previous studies which indicated that C. thermocellum prefers to utilize cellodextrins rather than cellobiose. Since this bacterium employs phosphorolytic cleavage, it gains more ATP per glucose molecule by utilizing cellodextrins, as demonstrated experimentally by Zhang and Lynd (40). C. thermocellum can also grow on laminarin (β-1,3 glucan) as a sole carbon source (15), consistent with the presence of the observed laminaribiose transport system. Recently, laminaribiose was found to be the molecular inducer of the celC operon in C. thermocellum (22), which is composed of three genes, namely, celC (endo-β-1,4-glucanase), licA [endo-β-1,3(4)-glucanase], and glyR3 (celC operon repressor). Interestingly, the promoter region of system E does not contain the same inverted repeat sequence identified in the GlyR3 binding site of the celC operon (Fig. (Fig.2),2), indicating that GlyR3 is probably not a transcriptional regulator of system E. It is also worth noting that the two putative sugar-binding proteins (gi 125973561, 125974458) that were not part of a complete transport system failed to react with any of the tested sugars and presumably are not associated with sugar transport.

In summary, we have identified five ABC sugar transporters in C. thermocellum that mediate the transport of β-1,4 and β-1,3 glucans. The number of transporters and their substrate specificities are consistent with previous observations indicating that C. thermocellum prefers to utilize cellodextrins rather than cellobiose or glucose. Our results also suggest that the bacterium lacks any other sugar ABC transporters, in agreement with the fact that this strain can grow only on β-glucans. Interestingly, C. thermocellum produces many extracellular hemicellulolytic enzymes that probably serve to unmask the cellulose fibers from a lignocellulose matrix. The identification of the sugar ABC transporters in C. thermocellum paves the way to utilize the corresponding genes in order to engineer superior transporters in Clostridium spp. or for introducing them into other non-cellulose-fermenting microorganisms to obtain new bioethanol-producing industrial strains.

Acknowledgments

This work was supported by the United States-Israel Binational Science Foundation (BSF) (grant no. 2005-186 to A.L.S. and Y.S.) and by the Technion-Niedersachsen Research Cooperation Program (to S.Y., F.S., T.-H.S., and Y.S.). Y.S. holds the Erwin and Rosl Pollak Chair in Biotechnology at the Technion. E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry at the Weizmann Institute of Science.

Footnotes

[down-pointing small open triangle]Published ahead of print on 24 October 2008.

REFERENCES

1. Alexander, J. K. 1961. Characteristics of cellobiose phosphorylase. J. Bacteriol. 81903-910. [PMC free article] [PubMed]
2. Alexander, J. K. 1968. Purification and specificity of cellobiose phosphorylase from Clostridium thermocellum. J. Biol. Chem. 2432899-2904. [PubMed]
3. Alexander, J. K., and G. Victor. 1972. Cellodextrin phosphorylase from Clostridium thermocellum. Methods Enzymol. 28948-953.
4. Anderson, K. L. 2000. Degradation of cellulose and starch by anaerobic bacteria, p. 359-386. In R. J. Doyle (ed.), Glycomicrobiology. Kluwer Academic/Plenum Publishers, New York, NY.
5. Bayer, E. A., J. P. Belaich, Y. Shoham, and R. Lamed. 2004. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu. Rev. Microbiol. 58521-554. [PubMed]
6. Bayer, E. A., R. Kenig, and R. Lamed. 1983. Adherence of Clostridium thermocellum to cellulose. J. Bacteriol. 156818-827. [PMC free article] [PubMed]
7. Bhat, K. M., and T. M. Wood. 1992. The cellulase of the anaerobic bacterium Clostridium thermocellum: isolation, dissociation, and reassociation of the cellulosome. Carbohydr. Res. 227293-300.
8. Davidson, A. L., and J. Chen. 2004. ATP-binding cassette transporters in bacteria. Annu. Rev. Biochem. 73241-268. [PubMed]
9. Davidson, A. L., E. Dassa, C. Orelle, and J. Chen. 2008. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol. Mol. Biol. Rev. 72317-364. [PMC free article] [PubMed]
10. Demain, A. L., M. Newcomb, and J. H. Wu. 2005. Cellulase, clostridia, and ethanol. Microbiol. Mol. Biol. Rev. 69124-154. [PMC free article] [PubMed]
11. Diez, J., K. Diederichs, G. Greller, R. Horlacher, W. Boos, and W. Welte. 2001. The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A. J. Mol. Biol. 305905-915. [PubMed]
12. Doi, R. H., and A. Kosugi. 2004. Cellulosomes: plant-cell-wall-degrading enzyme complexes. Nat. Rev. Microbiol. 2541-551. [PubMed]
13. Faergeman, N. J., B. W. Sigurskjold, B. B. Kragelund, K. V. Andersen, and J. Knudsen. 1996. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry. Biochemistry 3514118-14126. [PubMed]
14. Farrell, A. E., R. J. Plevin, B. T. Turner, A. D. Jones, M. O'Hare, and D. M. Kammen. 2006. Ethanol can contribute to energy and environmental goals. Science 311506-508. [PubMed]
15. Fuchs, K. P., V. V. Zverlov, G. A. Velikodvorskaya, F. Lottspeich, and W. H. Schwarz. 2003. Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-beta-1,3-glucanase bound to the outer cell surface. Microbiology 1491021-1031. [PubMed]
16. Himmel, M. E., S. Y. Ding, D. K. Johnson, W. S. Adney, M. R. Nimlos, J. W. Brady, and T. D. Foust. 2007. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315804-807. [PubMed]
17. Johnson, E. A., F. Bouchot, and A. L. Demain. 1985. Regulation of cellulase formation in Clostridium thermocellum. J. Gen. Microbiol. 1312303-2308.
18. Lamed, R., E. Setter, R. Kenig, and E. A. Bayer. 1983. The cellulosome—a discrete cell surface organelle of Clostridium thermocellum which exhibits separate antigenic, cellulose binding and various catalytic activities. Biotechnol. Bioeng. Symp. 13163-181.
19. Leschine, S. B. 1995. Cellulose degradation in anaerobic environments. Annu. Rev. Microbiol. 49399-426. [PubMed]
20. Lynd, L. R., W. H. van Zyl, J. E. McBride, and M. Laser. 2005. Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16577-583. [PubMed]
21. Makhatadze, G. I., and P. L. Privalov. 1990. Heat capacity of proteins. I. Partial molar heat capacity of individual amino acid residues in aqueous solution: hydration effect. J. Mol. Biol. 213375-384. [PubMed]
22. Newcomb, M., C. Y. Chen, and J. H. Wu. 2007. Induction of the celC operon of Clostridium thermocellum by laminaribiose. Proc. Natl. Acad. Sci. USA 1043747-3752. [PMC free article] [PubMed]
23. Nochur, S. V., A. L. Demain, and M. F. Roberts. 1990. Mutation of Clostridium thermocellum in the presence of certain carbon sources. FEMS Microbiol. Lett. 71199-204.
24. Ragauskas, A. J., C. K. Williams, B. H. Davison, G. Britovsek, J. Cairney, C. A. Eckert, W. J. Frederick, Jr., J. P. Hallett, D. J. Leak, C. L. Liotta, J. R. Mielenz, R. Murphy, R. Templer, and T. Tschaplinski. 2006. The path forward for biofuels and biomaterials. Science 311484-489. [PubMed]
25. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
26. Schlosser, A., J. Jantos, K. Hackmann, and H. Schrempf. 1999. Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader Streptomyces reticuli. Appl. Environ. Microbiol. 652636-2643. [PMC free article] [PubMed]
27. Schlosser, A., T. Kampers, and H. Schrempf. 1997. The Streptomyces ATP-binding component MsiK assists in cellobiose and maltose transport. J. Bacteriol. 1792092-2095. [PMC free article] [PubMed]
28. Schwarz, W. H. 2001. The cellulosome and cellulose degradation by anaerobic bacteria. Appl. Microbiol. Biotechnol. 56634-649. [PubMed]
29. Shoham, Y., R. Lamed, and E. A. Bayer. 1999. The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides. Trends Microbiol. 7275-281. [PubMed]
30. Shulami, S., O. Gat, A. L. Sonenshein, and Y. Shoham. 1999. The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6. J. Bacteriol. 1813695-3704. [PMC free article] [PubMed]
31. Strobel, H. J., F. C. Caldwell, and K. A. Dawson. 1995. Carbohydrate transport by the anaerobic thermophile Clostridium thermocellum LQR1. Appl. Environ. Microbiol. 614012-4015. [PMC free article] [PubMed]
32. Tam, R., and M. H. Saier, Jr. 1993. Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol. Rev. 57320-346. [PMC free article] [PubMed]
33. Tangney, M., and W. J. Mitchell. 2007. Characterisation of a glucose phosphotransferase system in Clostridium acetobutylicum ATCC 824. Appl. Microbiol. Biotechnol. 74398-405. [PubMed]
34. Tomme, P., A. L. Creagh, D. G. Kilburn, and C. A. Haynes. 1996. Interaction of polysaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC. 1. Binding specificity and calorimetric analysis. Biochemistry 3513885-13894. [PubMed]
35. van Zyl, W. H., L. R. Lynd, R. den Haan, and J. E. McBride. 2007. Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae. Adv. Biochem. Eng. Biotechnol. 108205-235. [PubMed]
36. Wilson, D. B. 2008. Aerobic microbial cellulase system, p. 374-392. In M. E. Himmel (ed.), Biomass recalcitrance. Blackwell Publishing, Oxford, United Kingdom.
37. Wilson, D. B. 2008. Three microbial strategies for plant cell wall degradation. Ann. N. Y. Acad. Sci. 1125289-297. [PubMed]
38. Wiseman, T., S. Williston, J. F. Brandts, and L. N. Lin. 1989. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179131-137. [PubMed]
39. Yu, Y., M. Tangney, H. C. Aass, and W. J. Mitchell. 2007. Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 731842-1850. [PMC free article] [PubMed]
40. Zhang, Y. H., and L. R. Lynd. 2005. Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation. Proc. Natl. Acad. Sci. USA 1027321-7325. [PMC free article] [PubMed]
41. Zolotnitsky, G., U. Cogan, N. Adir, V. Solomon, G. Shoham, and Y. Shoham. 2004. Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry. Proc. Natl. Acad. Sci. USA 10111275-11280. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...