Genome sequence of the polysaccharide-degrading, thermophilic anaerobe Spirochaeta thermophila DSM 6192

Spirochaeta thermophila is a thermophilic, free-living anaerobe that is able to degrade various α- and β-linked sugar polymers, including cellulose. We report here the complete genome sequence of S. thermophila DSM 6192, which is the first genome sequence of a thermophilic, free-living member of the Spirochaetes phylum. The genome data reveal a high density of genes encoding enzymes from more than 30 glycoside hydrolase families, a noncellulosomal enzyme system for (hemi)cellulose degradation, and indicate the presence of a novel carbohydrate-binding module.     

Molecular and Biochemical Analyses of CbCel9A/Cel48A,a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor bescii during Growth on Crystalline Cellulose

During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases produced by this hyperthermophilic bacterium.     

Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> maximizes enzyme activity and irreversibly binds to amorphous cellulose

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo-1,4-β-glucanase CelD (85019 Da). The N-terminal region of the protein includes a signal peptide and a catalytic module of glycoside hydrolase family 5 (GH5), followed by a carbohydrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo-1,4-β-glucanase with maximum activity on barley β-glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4–11, temperatures of 0–75°C, and NaCl concentrations of 0–5 M. Only 100% formamide or 1% SDS have been able to remove the protein.     

Cloning, nucleotide sequence and module structure of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5

The nucleotide sequence of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3,429 nucleotides. The deduced amino acid sequence of CBPB contained one module highly similar to a catalytic module of glycosyl hydrolase family 9 (GHF9), one module partially similar to a family 3 carbohydrate-binding module (CBM3), two linkers, one module similar to a CBM of cellulose-binding protein A (CBPA) from E. cellulosolvens 5, and one module almost identical to a cell wall-binding module (CWBM) of CBPA. The module similar to GHF9 showed CMCase activity, and the modules similar to CBM3 and CBM of CBPA bound to cellulose. Moreover, the module highly similar to CWBM of CBPA bound to the cell walls prepared from E. cellulosolvens 5. The amino acid sequence of CBPB had a significant homology (64.15% sequence identity) with that of CBPA. These results suggest that cbpA and cbpB genes descended from the same ancestral cellulase gene.     

Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase

In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.     

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

The genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical C-terminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley β-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/β-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages.     

Structural basis for cellulose binding by the type A carbohydrate‐binding module 64 of Spirochaeta thermophila

Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate‐binding module 64 (CBM64) appended at the C‐terminus. CBM64 adsorbs to various β1‐4‐linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly‐roll‐like fold corresponding to a surface‐binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of β‐linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855–858. © 2016 Wiley Periodicals, Inc.     

Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).     

Comparison of family 9 cellulases from mesophilic and thermophilic bacteria

Cellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three homologs: Clostridium cellulolyticum Cel9G, Thermobifida fusca Cel9A, and C. thermocellum Cel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without a T. fusca fibronectin type 3 homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, but C. thermocellum Cel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation.     

Cloning and Characterization of the Gene Encoding Endo-β-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-β-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-β-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-β-1,3-glucanases, but GluA was partially similar to two fungal exo-β-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two β-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-β-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.     

Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii

A large polypeptide encoded in the genome of the thermophilic bacterium Caldicellulosiruptor bescii was determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol. 78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases from C. bescii led to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization by C. bescii.     

Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases

Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These enzymes are often bimodular, including a catalytic domain and carbohydrate-binding module (CBM) attached via a flexible linker, and exhibit an active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain data-driven insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, trained only on the number of residues in the active-site loops as features, were able to discriminate GH7 CBHs and EGs with up to 99% accuracy, demonstrating that the lengths of loops A4, B2, B3, and B4 strongly correlate with functional subtype across the GH7 family. Classification rules were derived such that specific residues at 42 different sequence positions each predicted the functional subtype with accuracies surpassing 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. Our machine learning results recapitulate, as top-performing features, a substantial number of the sequence positions determined by previous experimental studies to play vital roles in GH7 activity. We surmise that the yet-to-be-explored sequence positions among the top-performing features also contribute to GH7 functional variation and may be exploited to understand and manipulate function.     

Crystal structure of basic 7S globulin, a xyloglucan-specific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase

β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.     

Domain evolution in the GH13 pullulanase subfamily with focus on the carbohydrate-binding module family 48

Glycoside hydrolase (GH) family 13 comprises about 30 different specificities. Four of them have been proposed to form the GH13 pullulanase subfamily: pullulanase, isoamylase, maltooligosyl trehalohydrolase and branching enzyme forming the seven CAZy GH13 subfamilies: GH13 8-GH13 14. Recently, a new family of carbohydrate-binding modules (CBMs), the family CBM48 has been established containing the putative starch-binding domains from the pullulanase subfamily, the β-subunit of AMP-activated protein kinase and some other GH13 enzymes with pullulanase and/or α-amylase-pullulanase specificity. Since all of these enzymes are multidomain proteins and the structure for at least one representative of each enzyme specificity has already been determined, the main goal of the present study was to elucidate domain evolution within this GH13 pullulanase subfamily (84 real enzymes) focusing on the CBM48 module. With regard to CBM48 positioning in the amino acid sequence, the N-terminal end of a protein appears to be a predominant position. This is especially true for isoamylases and maltooligosyl trehalohydrolases. Secondary structure-based alignment of CBM modules from CBM48, CBM20 and CBM21 revealed that several residues known as consensus for CBM20 and CBM21 could also be identified in CBM48, but only branching enzymes possess the aromatic residues that correspond with the two tryptophans forming the evolutionary conserved starch-binding site 1 in CBM20. The evolutionary trees constructed for the individual domains, complete alignment, and the conserved sequence regions of the α-amylase family were found to be comparable to each other (except for the C-domain tree) with two basic parts: (i) branching enzymes and maltooligosyl trehalohydrolases; and (ii) pullulanases and isoamylases. Taxonomy was respected only within clusters with pure specificity, i.e. the evolution of CBM48 reflects the evolution of specificities rather than evolution of species. This is a feature different from the one observed for the starch-binding domain of the family CBM20 where the starch-binding domain evolution reflects the evolution of species.     

Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes of Acetivibrio cellulolyticus and Clostridium thermocellum

We have sequenced a new gene, cel9B, encoding a family-9 cellulase from a cellulosome-producing bacterium, Acetivibrio cellulolyticus. The gene includes a signal peptide, a family-9 glycoside hydrolases (GH9) catalytic module, two family-3 carbohydrate-binding modules (CBM3c-CBM3b tandem dyad) and a C-terminal dockerin module. An identical modular arrangement exists in two putative GH9 genes from the draft sequence of the Clostridium thermocellum genome. The three homologous CBM3b modules from A. cellulolyticus and C. thermocellum were overexpressed, but, surprisingly, none bound cellulosic substrates. The results raise fundamental questions concerning the possible role(s) of the newly described CBMs. Phylogenetic analysis and preliminary site-directed mutagenesis studies suggest that the catalytic module and the CBM3 dyad are distinctive in their sequences and are proposed to constitute a new GH9 architectural theme.     

X-ray crystal structure of a non-crystalline cellulose-specific carbohydrate-binding module: CBM28

Natural cellulose exists as a composite of different forms, which have historically been broadly characterized as "crystalline" or "amorphous". The recognition of both of these forms of cellulose by the carbohydrate-binding modules (CBM) of microbial glycoside hydrolases is central to natural and efficient biotechnological conversion of plant cell wall biomass. There is increasing evidence that, at least some, individual binding modules target distinct and different regions of non-crystalline "amorphous" cellulose. Competition experiments show that CBM28 modules do not compete with CBM17 modules when binding to non-crystalline cellulose. The structure of the BspCBM28 (http://afmb.cnrs-mrs.fr/CAZY/) module from the Bacillus sp. 1139 family GH5 endoglucanase, comprising a 191 amino acid protein, has therefore been determined at 1.4A resolution using single isomorphous replacement with anomalous scattering methods. The structure reveals a "beta-jelly roll" topology, with high degree of similarity to the structure of CBM17 domains. Sequence and structural conservation strongly suggests that these two families of domains have evolved through gene duplication and subsequent divergence. The ligand-binding site "topographies" of CBMs from families 28, 17 and 4 begins to shed light on the differential recognition of non-crystalline cellulose by multi-modular plant cell wall-degrading enzymes.     

Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module

Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)(8) barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.     

Cloning and characterization of the gene encoding endo-beta-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-beta-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-beta-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-beta-1,3-glucanases, but GluA was partially similar to two fungal exo-beta-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two beta-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-beta-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.     

Complete Genome Sequence of Staphylococcus aureus Bacteriophage GH15

GH15 is a polyvalent phage that shows activity against a wide range of Staphylococcus aureus strains. In this work, the complete genome sequence of GH15 was determined. With a genome size of 139,806 bp (double-stranded DNA), GH15 is the largest staphylococcal phage sequenced to date. The complete genome encodes 214 open reading frames (ORFs) and 4 tRNAs. The closest relatives are the class III staphylococcal myobacteriophages, including K, A5W, ISP, Sb-1, and G1. Interestingly, although corresponding gene sequences demonstrate very high similarity, all the introns and inteins present in the phages listed above are absent in GH15. As such, GH15 can be considered phylogenetically unique among the staphylococcal myobacteriophages, indicating the diversity of this family.     

Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

The genome of Neurospora crassa encodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome of N. crassa and preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced in Pichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochrome c, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (k(cat) and K(m) values) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the heme b cofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) from N. crassa was expressed in P. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.