Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.The production of biofuels from nonfood cellulosic biomass would benefit the economy, the environment, and national energy security (17, 32). The largest technological and economical obstacle is the release of soluble fermentable sugars at prices competitive with those from sugarcane or corn kernels (17, 31). One of the approaches is discovering new cellulases from cellulolytic microorganisms, followed by cellulase engineering for enhanced performance on pretreated solid substrates. However, cellulase engineering remains challenging because enzymatic cellulose hydrolysis is complicated, involving heterogeneous substrates (33, 37), different action mode cellulase components (18), synergy and/or competition among cellulase components (36, 37), and declining substrate reactivity over the course of conversion (11, 26). Directed enzyme evolution, independent of knowledge of the protein structure and the enzyme-substrate interactions (6, 34), has been conducted to generate endoglucanase mutants, such as enhanced activities on soluble substrates (14, 16, 22), prolonged thermostability (20), changed optimum pH (24, 28), or improved expression levels (21). Here, we cloned and characterized a family 5 glycoside hydrolase (Cel5A) from a cellulolytic bacterium, Clostridium phytofermentans ISDg (ATCC 700394) (29, 30), and engineered it for enhanced thermostability.     

Processivity,Synergism, and Substrate Specificity of Thermobifida fusca Cel6B

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.Cellulose is a linear homopolymer of β-1,4-linked anhydrous glucosyl residues with a degree of polymerization (DP) of up to 15,000 (5). Adjacent glucose residues in cellulose are oriented at an angle of 180° to each other, making cellobiose the basic unit of cellulose structure (5). The β-1,4-glycosidic bonds of cellulose are enzymatically hydrolyzed by three classes of cellulases. Endocellulases (EC 3.2.1.4) cleave cellulose chains internally, generating products of variable length with new chain ends, while exocellulases, also called cellobiohydrolases (EC 3.2.1.91), act from one end of a cellulose chain and processively cleave off cellobiose as the main product. The third class is the processive endocellulases, which can be produced by bacteria (2, 20).Processivity and synergism are important properties of cellulases, particularly for hydrolysis of crystalline substrates. Processivity indicates how far a cellulase molecule proceeds and hydrolyzes a substrate chain before there is dissociation. Processivity can be measured indirectly by determining the ratio of soluble products to insoluble products in filter paper assays (14, 19, 39). Although this approach might not discriminate exocellulases from highly processive endocellulases (12), it is very helpful for comparing mutants of the same enzyme (19). The processivity of some glycoside hydrolases also can be determined from the ratio of dimers to monomers in the hydrolysate (13).Four types of synergism have been demonstrated in cellulase systems: synergism between endocellulases and exocellulases, synergism between reducing- and nonreducing-end-directed exocellulases, synergism between processive endocellulases and endo- or exocellulases, and synergism between β-glucosidases and other cellulases (3). Synergism is dependent on a number of factors, including the physicochemical properties of the substrate and the ratio of the individual enzymes (10).Great effort has been focused on improving enzymatic hydrolysis of cellulases in biomass (24). However, studying biomass is difficult due to its complexity; instead, nearly pure cellulose, amorphous cellulose, or carboxymethyl cellulose (CMC) are commonly used as substrates (22).Random mutagenesis approaches and rational protein design have been used to study cellulose hydrolysis (18), to improve the activity of catalytic domains and carbohydrate-binding modules (19), and to thermostabilize cellulases (9). Increased knowledge of cellulase structures and improvements in modeling software (1) have facilitated rational protein design. The structures of five glycoside hydrolase family 6 cellulases from four microorganisms, Trichoderma reesei (23), Thermobifida fusca (26), Humicola insolens (6, 29), and Mycobacterium tuberculosis (30), have been determined. Structural analysis showed that the active sites of the exocellulases are enclosed by two long loops forming a tunnel, while the endocellulases have an open active site groove. Movement of one of these loops is important for enzymatic activity (6, 35, 37).In nature, as well as for industrial applications, mixtures of cellulase are required; therefore, a better strategy for designing individual enzymes to improve the activity of mixtures is critical. In this study, we used Cel6B, a nonreducing-end-directed, inverting exocellulase from Thermobifida fusca, a thermophilic soil bacterium, as a model cellulase to investigate the impact of improved exocellulases in mixtures with endocellulases since T. fusca Cel6B is important for achieving the maximum activity of synergistic mixtures (35). Cel6B activity is similar to that of the fungal T. reesei exocellulase Cel6A, but Cel6B has higher thermostability and a much broader pH optimum (36). Six noncatalytic residues in the active site tunnel of T. fusca exocellulase Cel6B were mutated to obtain insight into the role of these residues in processivity and substrate specificity. Two mutant enzymes that showed higher activity with filter paper and processivity were investigated further for production of oligosaccharides and synergism to analyze the relationship between processivity and synergism.     

Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode

We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.In nature, some anaerobic cellulolytic bacteria produce cellulosomes, which are organized by the action of scaffoldin subunits that usually contain a single carbohydrate-binding module (CBM) and multiple cohesin modules (2, 7, 13, 14, 28, 36). This arrangement allows the integration of several dockerin-containing enzymes into a complex, which is then targeted to the cellulosic substrate by the common CBM. The cellulosomal enzymes then exhibit enhanced synergistic activity, presumably due to their spatial proximity and coordinated interaction. In contrast, the enzyme systems of aerobic bacteria and fungi comprise free (uncomplexed) enzymes, which differ from cellulosomal systems in that many of them contain their own CBM that delivers the individual catalytic module to the surface of the substrate (39, 41, 42).In previous work, we used the designer cellulosome concept (5) to construct unique minicellulosomes of defined content (16, 32, 33). In order to construct designer cellulosomes, chimeric scaffoldins have been prepared which contained two or more cohesins that matched the dockerins of the enzymes (native cellulosomal or dockerin-fused chimeras). Enzymes that contain dockerins that match the specificity of a scaffoldin-borne cohesin can then be selectively integrated into the designer cellulosome at a specified site. Cellulosomal enzymes containing either a native dockerin or a divergent dockerin can be inserted on different sites of a chimeric scaffoldin. Alternatively, a free, noncellulosomal enzyme can be included in designer cellulosomes by replacing its native CBM with a dockerin of choice. In some cases, designer cellulosomes displayed enhanced synergistic activity over the parallel free-enzyme system (15, 17). This increased activity was shown to be a function of both a substrate-targeting effect (contributed by the CBM on the chimeric scaffoldin) and the enzyme proximity effect, thus supporting the initial hypothesis.In recent studies, we have investigated the free-cellulase system of Thermobifida fusca for use in designer cellulosome systems. This aerobic thermophilic cellulolytic bacterium contains a limited set of six free cellulases, each composed of a catalytic module and a crystalline-cellulose binding family 2 CBM (CBM2) module on either the N or C terminus of the protein. T. fusca contains three endoglucanases (Cel5A, Cel6A, and Cel9B), two exocellulases (Cel6B and Cel48A), and one processive endoglucanase (Cel9A). Previously, we converted both family 6 cellulases and the family 48 exoglucanase from the free to the cellulosomal mode of action by replacing their native CBM2s with a dockerin module (11, 12). All three chimeric enzymes exhibited cellulose-degrading activity on both soluble and crystalline substrates. The results indicated that the family 48 exoglucanase appeared to be well adapted to the cellulosomal mode of action, whereas the family 6 exoglucanase is less appropriate for inclusion into cellulosomes. Indeed, family 48 cellulases have been found to be a major component in every native cellulosome thus far described, in contrast to the family 6 cellulases, which have been identified only in free-cellulase systems.An important feature of the free-acting fungal and bacterial cellulases is that they contain a linker segment, often rich in prolines and threonines, that connects the catalytic module to the CBM (37). The role of such flexible linkers is thought to ensure independent action of the adjacent functional modules, thus allowing progressive and efficient hydrolysis of cellulose by the catalytic modules (6, 9, 10, 20, 25-27, 34, 36, 38, 40). The present communication focuses on the effect of linker length and dockerin position (relative to the catalytic module) on enzymatic activity within a designer cellulosome. For this purpose we have employed the highly active family 5 endoglucanase Cel5A from T. fusca (21, 22, 29), which was converted to the cellulosomal mode by replacement of its CBM with a dockerin module. Chimeric dockerin derivatives were prepared on either the N or C terminus of the Cel5A catalytic module, separated by linker segments of different lengths. In most cases, binary designer cellulosomes, comprising the respective Cel5A chimera together with a Cel48A chimera, were shown to be more efficient on crystalline cellulosic substrates than the combination of the wild-type free enzymes.     

Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.Cellulose, composed of β-1,4-linked glucosyl units, is the most abundant naturally produced biopolymer on earth and can be utilized as a sustainable and renewable energy resource in place of fossil fuel. Establishing conditions for the efficient degradation of cellulose will contribute to the enhanced use of bioethanol, a biobased alternative to gasoline, which will increase biomass recycling and reduce carbon dioxide emissions (21, 30). Hence, efficient degradation of cellulose is an issue of great importance today.Bacteria and fungi produce cellulases that catalyze the hydrolysis of β-1,4-glycosidic bonds and are involved in the degradation of cellulose. Cellulases are divided into three major types according to their substrate specificities and the mode of hydrolysis: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). The most efficient hydrolysis of cellulose is thought to result from the combined synergistic actions of cellulases, whereby the enzymatic activity of an enzyme mixture is substantially higher than the sum of the activities of the individual enzymes. Several types of synergy have been described as the cooperative actions of endo- and exo-acting enzymes (20, 25, 32, 33, 45). Such cellulose-degrading enzymes are routinely used in the manufacture of beverages and industrial products, e.g., beer and wine, animal feed, paper, textiles, laundry detergents, and food ingredients (5). Hence, reducing cellulase manufacturing costs by increasing the productivity of cellulases with high specific activities through biotechnological modification is a desired research goal.Fungal cellobiohydrolases belong to glycosyl hydrolase families 6 and 7 (GH-6 and -7) and act most efficiently on highly ordered crystalline cellulose, hydrolyzing from either the reducing or the nonreducing terminus to liberate predominantly cellobiose (C2) with a minor amount of cellotriose (C3) (6, 39, 40). Also, Trichoderma reesei Cel6A can hydrolyze 1,3-1,4-β-glucan (1, 18), but it is unclear whether in vivo it is hydrolysis of 1,3-1,4-β-glucan that occurs mainly or hydrolysis of cellulose derivatives. The resulting accumulation of cellobiose inhibits the activity of cellobiohydrolase (13, 15, 28, 31, 36, 37, 44). Some microorganisms possess cellulosomes, multienzyme complexes that contribute to the efficient degradation of cellulose. Cellobiohydrolase is a documented component of cellulosomes in Clostridium thermocellum (2, 31).The three-dimensional (3D) structures of two GH-6 family members have been elucidated, including the cellobiohydrolase of T. reesei and that of Humicola insolens in complex with glucose, cellooligosaccharide, and a nonhydrolyzable substrate analogue (35, 41-43). The proposed structures have identified the significant amino acids associated with the catalytic core domain, where the catalytic site is buried inside a tunnel-shaped cavity and an enzyme-cellooligosaccharide hydrogen bond network. The structure suggests that the mode of action proceeds in a processive manner as cellobiohydrolase progresses along the cellulose chain (7, 19, 34, 40).The ascomycete fungus Magnaporthe grisea is the pathogen that causes rice blast, the most devastating fungal disease of rice. Since the complete genome sequence of M. grisea has been published (10), mining the database for candidate genes involved in pathogen-plant interactions, cell wall degradation, etc., is quite feasible. The cell wall-degrading enzymes of the genus Magnaporthe that are involved in the infection process have been of particular interest (22-24). Based on the complete genome sequence, M. grisea has three putative GH-6 family cellobiohydrolases and four GH-7 family cellobiohydrolases. Using primers designed from the database of M. grisea cellobiohydrolases, we cloned putative GH-6 and GH-7 family cellobiohydrolases, designated MoCel6A and MoCel7A, respectively, from Magnaporthe oryzae by PCR. The cloned MoCel6A and MoCel7A from M. oryzae were completely identical to those of M. grisea. In this paper, we demonstrate the properties of MoCel6A prepared by homologous overexpression in M. oryzae and examine the effects of cellobiose on the hydrolytic activity of MoCel6A. Furthermore, the effects of cellobiose on the activities of both MoCel6A and a T. reesei GH family 6 cellobiohydrolase (TrCel6A, formerly referred to as CBH II), which were overexpressed in Aspergillus oryzae, were also examined.     

Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol

By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomyces cerevisiae strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.Alternatives to fossil fuels for transportation are under extensive investigation due to the increasing concerns about energy security, sustainability, and global climate change (22, 24, 35). Lignocellulosic biofuels, such as bioethanol, have been widely regarded as a promising and the only foreseeable alternative to petroleum products currently used in transportation (11, 35, 39, 41). The central technological impediment to more widespread utilization of lignocellulose is the absence of low-cost technology to break down its major component, cellulose (19, 41). Cellulose (a linear homopolymer of glucose linked by β-1,4-glycosidic bonds) is insoluble, forms a distinct crystalline structure, and is protected by a complex plant cell wall structural matrix (10, 32). As a result, a separate processing step is required to produce large amounts of cellulases for the hydrolysis of cellulose into fermentable glucose, which makes cellulosic ethanol too expensive to compete with gasoline. Therefore, consolidated bioprocessing (CBP), which combines enzyme production, cellulose hydrolysis, and fermentation in a single step, has been proposed to significantly lower the cost of cellulosic ethanol production (23, 24). However, the great potential of CBP cannot be realized using microorganisms available today.One engineering strategy to construct CBP-enabling microbes is to endow ethanologenic microorganisms, such as Saccharomyces cerevisiae, with the ability to utilize cellulose by heterologously expressing a functional cellulase system. Nature has provided two ways of designing such systems: (i) noncomplexed cellulase systems, in which free enzymes are secreted and act discretely, and (ii) complexed cellulase systems, namely, cellulosomes, in which many enzymes are held together by a noncatalytic scaffoldin protein through high-affinity interactions between its cohesins and enzyme-borne dockerins (24). By mimicking the noncomplexed cellulase system, several groups successfully constructed cellulolytic S. cerevisiae strains that directly ferment amorphous cellulose to ethanol, although the titer and yield were relatively low (12, 16, 17). Compared to the noncomplexed cellulase systems, the cellulosome exhibits much greater degradative potential as a result of its highly ordered structural organization that enables enzyme proximity synergy and enzyme-substrate-microbe complex synergy (2, 13, 14, 21). Therefore, the second strategy could provide a “quantum leap” in development of biomass-to-biofuel technology (3).Recent studies revealed the modular nature of cellulosome assembly; by simply appending a dockerin domain, up to three enzymes (either cellulosomal or noncellulosomal) with different origins could be incorporated into a chimeric miniscaffoldin consisting of divergent cohesin domains to form a minicellulosome in vitro. The chimeric miniscaffoldin was in the form of either purified (7, 15, 26) or yeast surface-displayed protein (34). In both cases, the resulting recombinant minicellulosomes showed enhanced hydrolysis activity with cellulose. These results indicate that the high-affinity cohesin-dockerin interactions are sufficient to dictate assembly of a functional cellulosome. Therefore, in theory, the same results could also be achieved in vivo by coexpressing the cellulosomal components in a recombinant host. To date, in vivo production of recombinant cellulosomes has been limited to unifunctional complexes containing only one type of cellulolytic enzyme (1, 8, 27). Since complete enzymatic hydrolysis of cellulose requires synergistic action of three types of cellulases, endoglucanases (EGs) (EC 3.2.1.4), exoglucanases (including cellodextrinases [EC 3.2.1.74] and cellobiohydrolases [CBHs] [EC 3.2.1.91]), and β-glucosidases (BGLs) (EC 3.2.1.21) (24), none of the engineered microorganisms were shown to utilize cellulose directly.In this study, we report the first successful assembly of trifunctional minicellulosomes in S. cerevisiae. The resulting recombinant strain was able to simultaneously hydrolyze and ferment amorphous cellulose to ethanol, demonstrating the feasibility of constructing cellulolytic and fermentative yeasts by displaying recombinant minicellulosomes on the cell surface. We chose the cell surface display format over secretory proteins to potentially incorporate the cellulose-enzyme-microbe complex synergy unique to native cellulolytic microorganisms (21). Coupled with flow cytometry, yeast surface display provides a more convenient engineering platform, avoiding labor-intensive protein purification steps. Such a cell-bound format is also amenable to analysis of enzyme activity with insoluble substrates (31). Therefore, the system described here could be a useful tool for studying and engineering recombinant cellulosomes for various industrial and biotechnological applications.     

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.Cellulose hydrolysis by aerobic fungi, such as Trichoderma reesei, is usually explained in terms of the synergistic activities of endo-β-1,4-glucanases and exocellobiohydrolases. Models that describe the attack of cellulose at susceptible regions by endoglucanases, followed by cellobiohydrolase attack at the newly formed chain ends, continue to form the basis of most discussions of enzymatic cellulose hydrolysis (2, 24).Although the occurrence of endoglucanases and cellobiohydrolases in fungi is firmly established, the extent to which the cellulase systems of aerobic bacteria resemble those from fungi was unclear until recently, because there was little evidence for the presence of cellobiohydrolases in bacteria. However, it now appears that at least some cellulolytic bacteria produce enzymes similar to the fungal cellobiohydrolases. For example, Cellulomonas fimi produces at least six β-1,4-glucanases, of which four (CenA, CenB, CenC, and CenD) are endoglucanases and two (CbhA and CbhB) appear to be cellobiohydrolases that are the functional equivalents of T. reesei CBHI and CBHII (6, 15, 21, 22). Similar cellobiohydrolases have been described for the actinomycete Thermomonospora fusca (9).

C. fimi cellobiohydrolases.

The preferential attack of cellulose at the ends of glucan chains by C. fimi cellobiohydrolases CbhA and CbhB is strongly suggested by hydrolysis experiments using cellooligosaccharides or carboxymethylcellulose (CMC) (14, 15, 21, 22). However, we lack direct evidence for exohydrolytic activity on cellulose itself. Accordingly, we have examined the activities of CbhA and CbhB on cellulose by measurement of molecular size distributions during hydrolysis. Analysis of CenA was also included to allow comparison of cellobiohydrolase and endoglucanase activities.

C. fimi CenC.

Previous studies have indicated that CenA attacks susceptible linkages in soluble CMC in a relatively nonprocessive manner (7, 14); i.e., the enzyme dissociates from the substrate after each hydrolytic event. While CenB and CenD attack CMC in a similar way (14, 23), C. fimi CenC seems to act in a more processive fashion (16, 23). Therefore, CenC activity was analyzed in order to determine if the enzyme behaves in a similarly processive manner on cellulose.

Cellulose substrates.

Previous determinations of molecular size distribution during hydrolysis have shown that the choice of substrate is an important consideration (10). In this study we used two forms of cellulose: bacterial microcrystalline cellulose (BMCC) and phosphoric acid-swollen cellulose (PASC). These celluloses were chosen in order to simplify the interpretation of data by avoiding complications due to low surface/volume ratios and substrate heterogeneity, which are associated with the use of substrates like cotton or pulp fibers (24). Both BMCC and PASC have a high surface/volume ratio (17). BMCC is a highly crystalline form of cellulose I prepared from cellulose produced by Acetobacter xylinum. PASC is produced by swelling microcrystalline cellulose in concentrated phosphoric acid; although often described as amorphous, it is probably a low-crystallinity form of cellulose II (1). Recent data suggest that cellulose I and cellulose II contain glucan chains arranged in parallel orientation (12).     

Diversity of Bacteria and Glycosyl Hydrolase Family 48 Genes in Cellulolytic Consortia Enriched from Thermophilic Biocompost

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.The exploration and understanding of cellulose fermentation capabilities in nature could inform and enable industrial processes converting cellulosic biomass to fuels and other products. Enrichment of microbial communities that can utilize cellulose is useful in this context for the identification of novel organisms, novel metabolisms, and novel functions. Of particular interest are communities that can utilize cellulose at high temperatures and under anaerobic conditions, featuring high rates of solubilization under conditions where the energy and the reducing power of substrates are conserved in potentially useful fermentation products.Some evidence indicates that cocultures may be able to utilize cellulose more fully and produce higher concentrations of ethanol than pure cultures of model cellulolytic organisms such as Clostridium thermocellum and Clostridium straminisolvens (16, 20, 34). An initial step toward understanding the functional roles of community members in cooperative cellulose degradation is answering the question of what organisms are present in cellulolytic consortia obtained from nature. Currently, diversity estimation methods applied to cellulolytic communities range from traditional methods targeting the 16S rRNA gene (4, 12) to complex metagenomic analyses targeting the breadth of functional genes present in genomes of mixed cultures and the environment (3).From a functional gene standpoint, cellulase systems are complex assemblages of multifunctional glycosyl hydrolases. Even particularly relevant families, such as family 5 and family 9, tend to include hydrolases with multiple substrate specificities, deep evolutionary roots, and extensive sequence diversity within the same organism (19). However, family 48 glycosyl hydrolases include a select group of cellulosomal and unbound cellulases thought to play an essential role in cellulose solubilization by model cellulolytic clostridia (5, 7, 15), actinobacteria (6, 13), and anaerobic fungi (31). One key feature of this family of glycosyl hydrolases (mostly exoglucanases) is their ability to enhance cellulose solubilization in synergistic interactions with family 9 glycosyl hydrolases (2, 13). But unlike the latter, and with the notable exception of CelS and CelY in Clostridium thermocellum, family 48 hydrolases are present mostly in single copies in the genomes of cellulolytic microbes, making family 48 hydrolase genes a desirable target for primer design and molecular characterization.In this paper we describe the enrichment of microbial communities from a thermophilic compost pile and provide an assessment of diversity in stable cellulolytic enrichments by addressing total bacterial diversity using the 16S rRNA gene as well as introducing a novel method to assess functional diversity in cellulolytic consortia by targeting glycosyl hydrolase family 48 (GHF48) genes.     

Cellulosilyticum ruminicola,a Newly Described Rumen Bacterium That Possesses Redundant Fibrolytic-Protein-Encoding Genes and Degrades Lignocellulose with Multiple Carbohydrate- Borne Fibrolytic Enzymes

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.The ruminant rumens are long believed to be the anaerobic environments efficiently degrading the plant-derived polysaccharides, which is attributed to the inhabited abundant rumen microorganisms. They implement the fibrolytic degradation by the combination of the enzymes comprising of cellulases, hemicellulases, and to a lesser extent pectinases and ligninases (12). The rumen bacteria are outnumbered of the other rumen microbes; however, only a few of cellulolytic bacteria have been isolated from rumens. Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes are considered to be the most important cellulose-degrading bacteria in the rumen (18), and they produce a set of cellulolytic enzymes, including endoglucanases, exoglucanases (generally cellobiohydrolase), and β-glucosidases, as well as hemicellulases. In addition, the predominant ruminal hemicellulose-digesting bacteria such as Butyrivibrio fibrisolvens and Prevotella ruminicola lack the ability to digest cellulose but degrade xylan and pectin and utilize the degraded soluble sugars as substrates (10, 14). Although the robust cellulolytic species F. succinogenes degrades xylan, it cannot use the pentose product as a carbon source (24). Culture-independent approaches indicate that the three cellulolytic bacterial species represent only ∼2% of the ruminal bacterial 16S rRNA (43). Therefore, many varieties of rumen microbes remain uncultured (2). In recent years, rumen metagenomics studies have revealed the vast diversity of fibrolytic enzymes, multiple domain proteins, and the complexity of microbial composition in the ecosystem (9, 17). Hence, it is likely that the entire microbial community is necessary for the implementation of an efficient fibrolytic process in the rumen, including the uncultured species.In the rumen and other fibrolytic ecosystems, cellulolytic bacteria have to cope with the structural complexity of lignocelluloses and the interspecies competition; thus, not only a variety of plant polymer-degrading enzymes but also a noncatalytic assistant strategy, such as including adhesion of cells to substrates by a variety of anchoring domains, is required (8, 33, 38, 39). The (hemi)cellulolytic enzyme systems have been intensively studied for nonrumen anaerobic bacteria, including Clostridium thermocellum (19, 40), Clostridium cellulolyticum (6), Clostridium cellulovorans (13), and Clostridium stercorarium (47), as well as the rumen species, Rumicoccocus albus (35), Ruminococcus flavefaciens (32), and Fibrobacter succinogenes (4). The results indicate that most of them, except for Fibrobacter succinogenes, produce multiple cellulolytic enzymes integrated in a complex, cellulosome, and free individual proteins.The yak (Bos grunniens) is a large ruminant (∼1,000 kg) in the bovine family that lives mainly on the Qinghai-Tibetan Plateau in China at an altitude of 3,000 m above sea level. It is a local species that lives mainly on the world''s highest plateau. Yaks live in a full-grazing style with grasses, straws, and lichens as their exclusive feed, so the yak rumen can harbor a microbial flora distinct from those of other ruminants due to their fiber-component diet, since diet can be a powerful factor in regulating mammalian gut microbiome (27). A very different prokaryote community structure was revealed for yak rumen in our previous work based on the 16S rRNA diversity, which showed fewer phyla than for cattle but that a higher ratio of sequences was related to uncultured bacteria (2).We previously isolated a novel anaerobic fibrolytic bacterium, Cellulosilyticum ruminicola H1, from the rumen of a domesticated yak (11). Strain H1 grew robustly on natural plant fibers such as corn cob, alfalfa, and ryegrass as the sole carbon and energy sources, as well as on a variety of polysaccharides, including cellulose, xylan, mannan, and pectin, but not monosaccharides such as glucose, which is preferred by most ruminal bacteria. In the present study, using a draft of its genome and enzymatic characterization, we analyzed the enzymatic activities and the structures of the polymer hydrolases of strain H1 that were involved in the hydrolysis of complex polysaccharides.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Simultaneous Cellulose Degradation and Electricity Production by Enterobacter cloacae in a Microbial Fuel Cell

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from many different biodegradable substrates. When cellulose is used as the substrate, electricity generation requires a microbial community with both cellulolytic and exoelectrogenic activities. Cellulose degradation with electricity production by a pure culture has not been previously demonstrated without addition of an exogenous mediator. Using a specially designed U-tube MFC, we enriched a consortium of exoelectrogenic bacteria capable of using cellulose as the sole electron donor. After 19 dilution-to-extinction serial transfers of the consortium, 16S rRNA gene-based community analysis using denaturing gradient gel electrophoresis and band sequencing revealed that the dominant bacterium was Enterobacter cloacae. An isolate designated E. cloacae FR from the enrichment was found to be 100% identical to E. cloacae ATCC 13047^T based on a partial 16S rRNA sequence. In polarization tests using the U-tube MFC and cellulose as a substrate, strain FR produced 4.9 ± 0.01 mW/m², compared to 5.4 ± 0.3 mW/m² for strain ATCC 13047^T. These results demonstrate for the first time that it is possible to generate electricity from cellulose using a single bacterial strain without exogenous mediators.Exoelectrogenic microorganisms can release electrons to electron acceptors outside the cell, such as iron oxides or carbon anodes in microbial fuel cells (MFCs). Members of many genera, including Rhodoferax (6), Shewanella (13, 14), Pseudomonas (29), Aeromonas (28), Geobacter (2), Geopsychrobacter (10), Desulfuromonas (1), Desulfobulbus (9), Clostridium (27), Geothrix (3), Ochrobactrum (40), and Rhodopseudomonas (38), have been shown to produce electricity in an MFC. These bacteria have been grown on simple soluble substrates, such as glucose or acetate, that can be directly taken into the cell and used for energy production.Cellulose is the most abundant biopolymer in the world, and there is great interest in using this material as a substrate in an MFC. However, use of a particulate substrate in an MFC has not been well investigated. Cellulose must first be hydrolyzed to a soluble substrate that can be taken up by the cell. In previous MFC tests this has required the use of enzymes to hydrolyze the cellulose into sugars or the use of cocultures or mixed cultures (32, 33, 35). For example, Ren et al. (32) used a coculture of the cellulose fermentor Clostridium cellulolyticum and the exoelectrogen Geobacter sulfurreducens to generate electricity in an MFC fed with cellulose. Analysis of the anode microbial communities in other studies of cellulose-fed MFCs showed that Clostridium spp. (in a biofilm) and Comamonadaceae (in suspension) were predominant when rumen contents were used as an inoculum (35), while a rice paddy soil inoculum (12) converged to a Rhizobiales-dominated anode community (more than 30% of the population). To date, it has not been demonstrated that a single microbe can both degrade cellulose and generate current.Conventional methods of isolating exoelectrogenic microorganisms are based primarily on identifying microorganisms that can respire using soluble or insoluble metal oxides in agar plates (20-22). However, not all dissimilatory metal oxide-reducing bacteria are capable of producing electricity in an MFC, and not all bacteria that produce current in an MFC can grow using metal oxides (5, 34). Therefore, these methods may miss important electrochemically active strains of microorganisms. A new method to isolate exoelectrogenic microorganisms was recently developed (40); this method is based on dilution to extinction and a specially designed U-tube MFC that enriches exoelectrogenic bacteria on the anode. Using this method, a bacterium that could produce electricity in an MFC but not respire using iron was isolated (40).The main objective of this study was to isolate a bacterium capable of producing current from particulate cellulose. A cellulose-degrading consortium was diluted and serially transferred into U-tube MFCs using cellulose as the sole electron donor. Community analysis demonstrated the predominance of a single bacterium, which was isolated and compared to a culture collection strain for generation of current in an MFC.     

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ∼1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.Thermobifida fusca is an aerobic thermophilic soil bacterium with strong cellulolytic activity (52). The T. fusca enzyme system is an extensively studied free cellulase system in which nearly all of the cellulolytic enzymes have been fully characterized, from the individual enzyme sequences to the three-dimensional structures, as well as the biochemical activities of the native and recombinant proteins. The genome sequence has been published (36), and the number and types of carbohydrate-active enzymes produced by the organism are known. This actinomycete produces six different cellulases that have been well studied (29, 31, 32, 50, 52). T. fusca also has the ability to grow on xylan and produces several enzymes involved in xylan degradation, such as xylanases, β-xylosidase, α-l-arabinofuranosidase, and acetylesterases (1, 21).Previous research has suggested that the multienzyme cellulosome complex from Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides (33). The cellulosome system is characterized by the strong bimodular interaction between the cohesin and dockerin modules that integrates the various enzymes into the complex (5, 35, 55). Scaffoldin subunits (nonenzymatic protein components) contain the cohesin modules that incorporate the enzymes into the complex via their resident dockerins. The primary scaffoldin subunit also includes a carbohydrate (cellulose)-binding module (CBM) through which the complex recognizes and binds to the cellulosic substrate (42, 46).In order to evaluate the reasons for the apparent advantage of cellulosomes over free enzymes, it is interesting to compare the properties of the best-characterized free-enzyme systems for degradation of polysaccharides with those of the best-studied cellulosome system. We have initiated a program to convert the free-enzyme system of T. fusca into an artificial designer cellulosome (11-13). The designer cellulosome concept is based on the very high affinity (20, 44) and specific interaction (37, 43, 55) between a cohesin and a dockerin module from the same species. Since the various scaffoldin-borne cohesins of a given species essentially show the same specificity of binding for the enzyme-borne dockerins, designer cellulosomes are constructed from recombinant chimeric scaffoldins containing divergent cohesins from different species, for which matching dockerin-containing enzyme hybrids are prepared, as a platform for promoting synergistic action among enzyme components (5). Free cellulases from the T. fusca system were converted to the cellulosomal mode by replacing their native CBM with a desired dockerin module, and in some cases, the resultant “designer cellulosomes” exhibited enhanced synergistic activity on crystalline cellulosic substrates compared to that of the mixture of wild-type enzymes (11).In this study, we incorporated xylanolytic enzymes into designer cellulosomes and investigated their hydrolytic effects on purified xylans and on a native, complex cellulosic substrate (hatched wheat straw). We focused on T. fusca xylanases 11A and 10B (Xyn11A and Xyn10B), which are the most abundant xylanases produced during growth on xylan (34). Xyn11A and Xyn10B function as endoxylanases (28, 34); Xyn11A contains a C-terminal family 2 CBM that binds both cellulose and xylan, whereas Xyn10B lacks a CBM. In some experiments, one of the previously converted (dockerin-containing) T. fusca endoglucanases, f-5A (11), was also introduced into the designer cellulosomes in order to evaluate cooperation between xylanases and cellulases in hydrolysis of a natural substrate. This study contributes primary information concerning a major feature of cellulosomes that had not been suitably addressed in earlier research: although xylanases are integral components of cellulosomes, their synergistic action in the cellulosome mode has yet to be examined experimentally. The xylan-binding CBM (termed XBM for the purposes of this report) was found to contribute to the activity of the parent Xyn11A enzyme.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring

Cutinase from Thermobifida fusca is thermally stable and has potential application in the bioscouring of cotton in the textile industry. In the present study, the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBM_Cel6A) and Cellulomonas fimi cellulase CenA (CBM_CenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. Both fusion enzymes, cutinase-CBM_Cel6A and cutinase-CBM_CenA, were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that both displayed similar catalytic properties and pH stabilities in response to T. fusca cutinase. In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal temperature of 50°C. Compared to T. fusca cutinase, in the absence of pectinase, the binding activity on cotton fiber was enhanced by 2% for cutinase-CBM_Cel6A and by 28% for cutinase-CBM_CenA, whereas in the presence of pectinase, the binding activity was enhanced by 40% for the former and 45% for the latter. Notably, a dramatic increase of up to 3-fold was observed in the amount of released fatty acids from cotton fiber by both cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM. The improvement in activity and the strong synergistic effect between the fusion proteins and pectinase suggest that they may have better applications in textile bioscouring than the native cutinase.Cotton fiber has a multilayered structure, with its outermost surface being the cuticle that is cross-linked to the primary cell wall of cotton fiber by esterified pectin substances. The major component of the cuticle is cutin, an insoluble polyester composed mainly of saturated C₁₆ and C₁₈ hydroxy and epoxy fatty acids (14, 16, 27, 38). During the process of scouring in the textile industry, the cuticle layer has to be removed in order to improve the wettability of cotton fiber, which then facilitates uniform dyeing and finishing. Traditionally, this process is performed by hot hydrolysis in alkaline medium, which not only consumes large quantities of water and energy but also causes severe pollution and fiber damage (20, 21, 33). Therefore, environment-friendly scouring methods based on biocatalysts have been actively sought (2, 30, 36).Cutinase is a multifunctional esterase capable of degrading the cutin component of the cuticle. Earlier reports showed that the fungal cutinase from Fusarium solani pisi has potential use for cotton cuticle degradation and exhibits a good synergistic effect with pectinase, an enzyme utilized to degrade pectin, in the scouring of cotton fiber (1, 7, 8, 14). Moreover, site-directed mutagenesis has been performed to replace the specific amino acid residues near the active site of cutinase (3) to improve its hydrolytic activity toward polyesters. More recently, a cutinase from the thermophilic bacterium Thermobifida fusca has been identified and overexpressed in Escherichia coli in our laboratory (10). The good thermal stability and alkali resistance of this recombinant T. fusca cutinase make it potentially more amenable to textile bioscouring (10).To further improve the applicability and/or catalytic efficiency of T. fusca cutinase, the present study attempts to engineer a novel cutin-degrading enzyme, based on analysis of the surface structure of cotton fiber. It has been observed that, in addition to cutin, pectin, proteins and other components, there is also a large amount of cellulose on the surface layer of cotton fiber (23). Thus, it is tempting to hypothesize that if the enzyme can be engineered to specifically bind to cellulose through a “gain of function” modification, its concentration on the surface of cotton fiber could increase significantly. Subsequently, its catalytic efficiency for cutin breakdown could be improved due to a proximity effect. In order to design such an enzyme, a fusion protein strategy in which a cellulose-binding protein/module will be attached to cutinase is considered.It is well known that cellulase is capable of binding specifically to cellulose (25, 31). This enzyme has two separate modules: a catalytic module and a carbohydrate-binding module (CBM) (11). The two modules are discrete structural and functional units usually connected by a flexible linker (5, 17, 28). CBM has high specific capacities for cellulose binding. Previously, it has been reported that CBM is able to be fused to a chosen target protein by genetic manipulation (36), resulting in enhanced binding of this fusion protein to cellulose (6, 29). For example, fusion proteins were constructed by fusing CBM to β-glucose nucleotide enzyme (GUS) (13) or β-glycosidase (BglA) (19), which facilitates biochemical analysis of scouring efficiency for cotton fabrics.In the present study, the CBM from T. fusca cellulase Cel6A (CBM_Cel6A) and the CBM from Cellulomonas fimi cellulase CenA (CBM_CenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. The resulting fusion enzymes were compared to the native cutinase in terms of their biochemical properties, as well as the catalytic efficiency in cutin breakdown on cotton fiber. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.