Search

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 celiodextrins. 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.sub.2] to [G.sub.5]), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose ([G.sub.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.sub.3] to [G.sub.5]) are assimilated faster than cellobiose.

The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CelS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 [h.sup.-1], the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 [h.sup.-1]) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate--the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.

The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability, [alpha]-Glucuronidases are family 67 glycosidases that cleave the [alpha]-1,2-glycosidic bond between 4-O-methyl-o-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of [alpha]-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the [alpha]-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial [alpha]-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in [alpha]-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35[degrees]C, compared to 65[degrees]C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9[degrees]C, was almost identical to that of the wild-type, 73.4[degrees]C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.

Sequencing of a cellulosome-integrating gene cluster in Acetivibrio cellulolyticus was completed. The cluster contains four tandem scaffoldin genes (scaA, scaB, scaC, and scaD) bounded upstream and downstream, respectively, by a presumed cellobiose phosphorylase and a nucleotide methylase. The sequences and properties of scaA, scaB, and scaC were reported previously, and those of scaD are reported here. The scaD gene encodes an 852-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. The calculated molecular weight of the mature ScaD is 88,960; a 67-residue linker segment separates cohesins 1 and 2, and two ~30-residue linkers separate cohesin 2 from 3 and cohesin 3 from the SLH module. The presence of an SLH module in ScaD indicates its role as an anchoring protein. The first two ScaD cohesins can be classified as type II, similar to the four cohesins of ScaB. Surprisingly, the third ScaD cohesin belongs to the type I cohesins, like the seven ScaA cohesins. ScaD is the first scaffoldin to be described that contains divergent types of cohesins as integral parts of the polypeptide chain. The recognition properties among selected recombinant cohesins and dockerins from the different scaffoldins of the gene cluster were investigated by affinity blotting. The results indicated that the divergent types of ScaD cohesins also differ in their preference of dockerins. ScaD thus plays a dual role, both as a primary scaffoldin, capable of direct incorporation of a single dockerin-borne enzyme, and as a secondary scaffoldin that anchors the major primary scaffoldin, ScaA and its complement of enzymes to the cell surface.

A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an 'X' domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature.

The three-dimensional crystal structure of the cohesin-dockerin heterodimer that sheds light on the critical amino acids that contribute to high affinity protein-protein interaction is reviewed. It is concluded that the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promise new insight into the assembly and consequences of the multienzyme complex.

Clostridium thermocellum produces an extracellular multienzyme complex, termed the cellulosome, that allows efficient solubilization of crystalline cellulose. The complex is organized around a large noncatalytic protein subunit, termed CipA or scaffoldin, and is found either free in the supernatant or cell bound. The binding of the complex to the cell is mediated by three cell surface anchoring proteins, OlpB, Orf2p, and SdbA, that interact with the CipA scaffoldin. The transcriptional level of the olpB, orf2, sdbA, and cipA genes was determined quantitatively by RNase protection assays in batch and continuous cultures, under carbon and nitrogen limitation. The mRNA level of olpB, orf2, and cipA varied with growth rate, reaching 40 to 60 transcripts per cell under carbon limitation at a low growth rate of 0.04 [h.sup.-1] and 2 to 10 transcripts per cell at a growth rate of 0.35 [h.sup.-1] in batch culture. The mRNA level of sdbA was about three transcripts per cell and was not influenced by growth rate. Primer extension analysis revealed two major transcriptional start sites, at -81 and -50 bp, upstream of the translational start site of the cipA gene. The potential promoters exhibited homology to the known sigma factors [[sigma].sup.A] and [[sigma].sup.L] ([[sigma].sup.54]) of Bacillus subtilis. Transcription from the [[sigma].sup.L]-like promoter was found under all growth conditions, whereas transcription from the [[sigma].sup.A]-like promoter was significant only under carbon limitation. The overall expression level obtained in the primer extension analysis was in good agreement with the results of the RNase-protection assays.

A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin. The gene immediately downstream of cipV was sequenced and designated scaB. The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin. Scab cohesins 1 and 2 are very closely linked. Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin. The scaC gene downstream of scab codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. A long, ca. 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times. The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162. The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein. The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins. Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative Scab cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the Scab dockerin. The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA. In addition, Scab is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the Scab adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture.

The mode of inhibition of mechanism-based inhibitor (2,Ki = 0.4 (mu)M) of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which is designed to mimic the combined key features of its natural substrates arabinose-5-phosphate and phoshoenolpyruvate into a single molecule, is investigated. A novel frequency-selective rotational-echo double-resonance (REDOR) experiment is developed and applied.

Crystals of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase, complexed with phosphoenolpyruvate substrate and the enzyme inhibitor, crystalize in the cubic space group with one subunit per asymmetric unit. The crystal complexes exhibit almost identical structures with an rms difference of 0.41 angstrom.

Research reveals two linked genes in Ruminococcus flavefaciens 17, which encode key structural components of a cellulosome complex. Data indicate that the gene products ScaA and ScaB represent scaffoldin-related proteins with several copies of cellulosome sequences, which exhibit dockerin-cohesin interactions.

Results demonstrate that the enzyme binds phosphoenolpyruvate and arabinose-5-phosphate via arginine and lysine residues by different mechanisms. Data reveal that the active site residues for phosphoenolpyruvate are structurally rigid residues lysine-138 and arginine-168 wheras arginine 63, which binds to arabinose-5-phosphate is flexible.

Results show that the protein CipBc is a 2,292-amino-acid residue polypeptide exhibiting 11 type II cohesin domains all of which possessing similar sequence. Data further reveal that one of the domains interacts with the cellulosomal enzyme which contains components of a cellulosomal scaffoldin.

Researchers from Israel developed a method to remove s-Triazine herbicides including atrazine from industrial waste water by using enzymes before disposing the water.

The cellulosomal scaffoldin has a molecular weight of 199,496 with cellulose-binding domains and a catalytic module belonging to family 9 glycosyl hydrolases. Comparative sequence and phylogenetic analyses of different functional modules of other scaffoldins indicate that the scaffolding gene seem to have evolved from different microbial sources.

The cellulose-binding domain (CBD) present on a noncatalytic scaffoldin subunit of the cellulosome in Clostridium thermocellum is similar in structure, substrate affinity and binding capacity to the CBD present in C. cellulovorans. However the polyclonal antibodies produced against the CBD of C. thermocellum do not act against the CBD of C. cellulovorans possibly because the exposed epitopes in the two CBD's are different. Chemical changes in the cysteine of the CBD do not affect the CBD's ability to bind cellulose.

Anion-exchange and hydrophobic interaction chromatographies help purify alpha-L-arabinofuranosidase produced by Bacillus stearothermophilus T-6. Analysis of the enzyme reveals the molecular weight as 256,000 and gel electrophoresis and gel filtration reveal the presence of four identical subunits in the enzyme. Cofactors are not necessary for the activity of the enzyme and the presence of Hg2+ inhibits the activity. The N-terminal sequence of the enzyme is compared with that of alpha-L-arabinofuranosidase from Streptomyces lividans 66.

T-6 xylanase encoding gene cloned from Bacillus stearothermophilus contains a 1, 236 bp open reading frame with ribosomal binding site and transcriptional termination site and a 379 amino acid sequence with molecular mass 43,808. The gene has highly conserved regions and xylose induction of the enzyme synthesis is regulated on a transcriptional level.

An extracellular xylanase produced by Bacillus stearothermophilus T-6 optimally bleaches pulp at pH9 and at 65 degrees Celsius. The purification and characterization of the enzyme xylanase T-6 is reported. Xylan is degraded to xylose and xylobiose by Xylanase T-6 and endoxylanase. The protein is protected by xylane from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enxyme revealed high homology with the N-terminal region of xylanase A from the alkalophililc Bacillus sp strailn C-125.