Anaerocellum Thermophilum Research Articles

Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from Caldicellulosiruptor bescii 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.

Углевод-связывающий модуль 28 семейства термостабильной эндо-1,4-β-глюканазы СelD из анаэробного микроорганизмаСaldicellulosiruptor besciiнеобходим для максимальной активности фермента и необратимо связывается с аморфной целлюлозой

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 one of 749 aa encoding a multimodular endo-1,4-beta-glucanase CelD (85019 Da). N-terminal region of the protein includes the signal peptide and the catalytic module of glycoside hydrolase family 5 (GH5), followed by the substrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic one and CBM28, were produced in E. coli cells and purified to homogeneity. Analysis of the catalytic properties showed CelD to be endo-1,4-beta-glucanase whose maximum activity was exhibited on beta-glucan of barley at pH 6.2 and 70 degrees C. The enzyme was stable at 50 degrees C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 decreased on cellulose substrates, and its thermostability was dropped. Binding of CBM28 to amorphous cellulose was almost irreversible as it could not be removed from this substrate in a range of pH 4-11, temperatures--of 0-75 degrees C, and NaCl concentration--of 0-5 M. Only 100% formamide or 1% SDS were able to remove the protein.

Preparation of mycobacterial antigens fused with a cellulose-binding domain with the aim of developing a subunit antituberculous vaccine

The genes of ESAT-6, CFP-10 and Ag85A secreted antigens of Mycobacterium tuberculosis were cloned in Escherichia coli. Fusion proteins Esat6-CBD, Ag85A-CBD and CFP10-CBD with thermostable cellulose binding domain (CBD) of Anaerocellum thermophilum providing high affinity to cellulose were constructed. Content of the fused proteins in overproducing strains was 20% of total protein for CFP10-CBD and 15% for both Esat6-CBD and Ag85A-CBD. Immobilization of the fusion proteins on cellulose is a one-stage process that provides simultaneous antigen purification and absorption on cellulose. The proteins retain their antigenic properties and can be used as the components for subunit tuberculosis vaccine production or in diagnostic kits.

Glycoside hydrolase inventory drives plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus

The genome of Caldicellulosiruptor saccharolyticus encodes a range of glycoside hydrolases (GHs) that mediate plant biomass deconstruction by this bacterium. Two GH-based genomic loci that appear to be central to the hydrolysis of hemicellulosic and cellulosic substrates were examined. XynB-XynF (Csac_2404-Csac_2411) encodes intracellular and extracellular GHs that are active towards xylan and xylan side-chains, as well as carboxymethyl cellulose (CMC). XynD (Csac_2409) and XynE (Csac_2410) were produced recombinantly and confirmed to be xylanases. XynF (Csac_2411) was produced in two separate polypeptides, each with one GH43 catalytic domain displaying α-L-arabinofuranosidase activity. CelA-ManB (Csac_1076-Csac_1080) encodes four multi-domain, extracellular GHs, including CelB (Csac_1078), a 118 kDa extracellular enzyme not present in the other genome-sequenced member of this genus, Caldicellulosiruptor bescii (formerly Anaerocellum thermophilum). CelB contains both GH10 and GH5 domains, separated by a family 3 carbohydrate-binding module (CBM3). CelB encoded in Csac_1078 differed from the version originally reported (Saul et al., 1990, Appl Environ Microbiol 56:3117–3124) with respect to linker regions. CelB hydrolyzed xylan and CMC, as well as barley b-glucan, glucomannan, and arabinoxylan. For all substrates tested, intact CelB was significantly more active than either the individual GH5 and GH10 domains or the two discrete domains together, indicating that the multi-domain architecture is essential for complex carbohydrate hydrolysis. Transcriptomes for C. saccharolyticus grown at 70°C on glucose, xylose, xyloglucan, switchgrass, and poplar revealed that certain GHs were particularly responsive to growth on switchgrass and poplar and that CelB was in the top decile of all transcripts during growth on the plant biomass.

Further Identification of Novel Lantibiotic Operons Using LanM-Based Genome Mining.

Lantibiotics are gene-encoded antimicrobial peptides that are distinguished by the presence of the unusual structures, lanthionine and β-methyllanthionine, which are introduced through enzyme-catalysed post-translational modification. Lantibiotics can be subdivided on the basis of the nature of the enzyme(s) which catalyse this reaction. Lantibiotic synthetases, generically designated LanM, which catalyse the dehydration of serines (and threonines) followed by the formation lanthionine (and β-methyllanthioine), are responsible for the synthesis of the largest subdivision, type 2. One can take advantage of the conserved nature of LanM proteins to screen for and bioinformatically characterize novel lantibiotic-encoding operons in genome-sequenced microorganisms. Having employed this strategy with success previously, here we update the investigation to reveal the existence of 124 LanM homologs encoded within genome-sequenced microbes. Further analysis focussed specifically on 9 novel lantibiotic gene clusters in Anaerocellum thermophilum DSM 6725, Anaerococcus tetradius ATCC 35098, Corynebacterium matruchotti ATCC 33806, Streptococcus suis 98HAH33, Geobacillus sp. G11MC16, Nostoc punctiforme PCC 73102 (× 2; one on plasmid and one on the chromosome) and Streptococcus pneumoniae CDC 0288-04 and TIGR4. Furthermore, screening of metagenomic datasets revealed 11 additional LanM-encoding genes from a variety of environments. The alignment of these LanM proteins facilitated a detailed investigation of conserved domains and led to the design of an improved set of degenerate primers which can be employed in the laboratory to identify strains containing type 2 lantibiotic gene clusters.

Classification of ‘Anaerocellum thermophilum’ strain DSM 6725 as Caldicellulosiruptor bescii sp. nov.

The thermophilic, cellulolytic, anaerobic bacterium 'Anaerocellum thermophilum' strain Z-1320 was isolated from a hot spring almost two decades ago and deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) as DSM 6725. The organism was classified as representing a new genus, 'Anaerocellum', primarily on its growth physiology, cell-wall type and morphology. The results of recent physiological studies and of phylogenetic and genome sequence analyses of strain DSM 6725 of 'A. thermophilum' obtained from the DSMZ showed that its properties differed from those originally described for strain Z-1320. In particular, when compared with strain Z-1320, strain DSM 6725 grew at higher temperatures and had an expanded range of growth substrates. Moreover, the 16S rRNA gene sequence of strain DSM 6725 fell within the Caldicellulosiruptor clade. It is therefore suggested that 'Anaerocellum thermophilum' should be classified as a member of the genus Caldicellulosiruptor, for which the name Caldicellulosiruptor bescii sp. nov. is proposed (type strain DSM 6725(T)=ATCC BAA-1888(T)). C. bescii sp. nov. DSM 6725(T) is the most thermophilic cellulose-degrading organism known. The strain was able to grow up to 90 degrees C (pH 7.2) and degraded crystalline cellulose and xylan as well as untreated plant biomass, including potential bioenergy plants such as poplar and switchgrass.

Efficient Degradation of Lignocellulosic Plant Biomass, without Pretreatment, by the Thermophilic Anaerobe “ Anaerocellum thermophilum ” DSM 6725

Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that "Anaerocellum thermophilum" DSM 6725, an anaerobic bacterium that grows optimally at 75 degrees C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75 degrees C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70 degrees C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.

Genome Sequence of the Anaerobic, Thermophilic, and Cellulolytic Bacterium “ Anaerocellum thermophilum ” DSM 6725

"Anaerocellum thermophilum" DSM 6725 is a strictly anaerobic bacterium that grows optimally at 75 degrees C. It uses a variety of polysaccharides, including crystalline cellulose and untreated plant biomass, and has potential utility in biomass conversion. Here we report its complete genome sequence of 2.97 Mb, which is contained within one chromosome and two plasmids (of 8.3 and 3.6 kb). The genome encodes a broad set of cellulolytic enzymes, transporters, and pathways for sugar utilization and compared to those of other saccharolytic, anaerobic thermophiles is most similar to that of Caldicellulosiruptor saccharolyticus DSM 8903.

In vivo study of the immunogenic properties of the recombinant cellulose-binding domain of Anaerocellum thermophilum

Development of novel technologies enabling the one-stage production of preparations of a required degree of purity is one of the most important goals of contemporary medical biotechnology. The incorporation of affinity domains capable of binding to relevant sorbents into recombinant proteins is one of the most promising approaches. The method of one-stage production of protein-cellulose injectants without using chemical conjugates is based on the construction of two-component proteins containing a target antigen and a cellulose-binding domain (CBD) capable of spontaneous binding to cellulose-containing sorbents. To study the immunogenic properties of the CBD, as well as the effect of cellulose sorbents on the immune response, a recombinant CBD was obtained and purified using the one-stage method based on the CBD immobilization on amorphous cellulose; the free CBD protein was isolated. A comparative study of the immunogenic properties of the CBD, the CBD-cellulose complex and the cellulose sorbent was performed using a rat model. The titers of specific antibodies, as well as the spectrum and concentrations of cytokines in the blood serum of rats induced by the obtained preparations, were determined using the BioPlex suspension array system. The effect of the CBD on the induction of cytokine synthesis observed on injection of the free protein and its immobilized form was studied. It has been demonstrated that the immobilization of CBD on the cellulose sorbent enhances the synthesis of specific antibodies. It was found that cellulose immunosorbent is not immunotolerant and induces the synthesis of the cytokines involved in the regulation of the humoral immune response.

Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum

The nucleotide sequence of two novel plasmids isolated from the extreme thermophilic anaerobic bacterium Anaerocellum thermophilum DSM6725 ( A. thermophilum), growing optimally at 70 °C, has been determined. pBAS2 was found to be a 3653 bp plasmid with a GC content of 43%, and the sequence revealed 10 open reading frames (ORFs). The two largest of these, namely Orf21 and Orf41, showed similarity to a Bacillus plasmid recombinase and a Pseudoalteromonas plasmid replication protein, respectively. A sequence with homology to double stranded replication origins from rolling circle plasmids was found, but no single stranded intermediates, characteristic of rolling circle replication, were found on Southern blots. The larger plasmid, pBAL, was found to be a 8294 bp plasmid with a GC content of 39%. It revealed 17 ORFs, of which three showed similarity at the amino acid (aa) level to known proteins. Orf22 showed the strongest similarity (33% aa) to replication proteins from large multiresistance Staphylococcal and Lactococcal plasmids, all of which are believed to replicate via a θ-like replication mechanism. Orf32 showed similarity to both DNA repair proteins and DNA polymerases with highest similarity to DNA repair protein from Campylobacter jejuni (25% aa). Orf34 showed similarity to sigma factors with highest similarity (28% aa) to the sporulation specific Sigma factor, Sigma 28(K) from Bacillus thuringiensis.

Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains

A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum'. Full-length CelA and a truncated enzyme species designated CelA' were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and beta-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 degrees C (CM-cellulase) and 85 degrees C (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelA-encoding gene was isolated from genomic DNA of 'A. thermophilum' by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the N-terminus of CelA purified from 'A. thermophilum'. Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family III cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor saccharolyticus (sequence identities of 96 and 97% were found for the N- and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound to microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

Cloning and Expression of Genes Coding for Carbohydrate Degrading Enzymes of Anaerocellum thermophilum in Escherichia coli

A gene library of Anaerocellum thermophilum Z-1320 was constructed in Escherichia coli using plasmid pUC18. Clones were selected by screening for hydrolysis of CMC, MU-Cel, lichenan and xylan. Recombinant clones expressing two β-1,4-glucanases and two different xylanases were obtained. Both β-1,4-glucanases were capable of degrading Avicel. Clones with xylanase activity also cleft PNPXyl, MU-Glu and MU-Cel. The genes of one β-1,4-glucanase and one xylanase are probably located in the same region of A.thermophilum chromosome.