- Open Access
Deletion of a single glycosyltransferase in Caldicellulosiruptor bescii eliminates protein glycosylation and growth on crystalline cellulose
© The Author(s) 2018
- Received: 14 July 2018
- Accepted: 19 September 2018
- Published: 24 September 2018
Protein glycosylation pathways have been identified in a variety of bacteria and are best understood in pathogens and commensals in which the glycosylation targets are cell surface proteins, such as S layers, pili, and flagella. In contrast, very little is known about the glycosylation of bacterial enzymes, especially those secreted by cellulolytic bacteria. Caldicellulosiruptor bescii secretes several unique synergistic multifunctional biomass-degrading enzymes, notably cellulase A which is largely responsible for this organism’s ability to grow on lignocellulosic biomass without the conventional pretreatment. It was recently discovered that extracellular CelA is heavily glycosylated. In this work, we identified an O-glycosyltransferase in the C. bescii chromosome and targeted it for deletion. The resulting mutant was unable to grow on crystalline cellulose and showed no detectable protein glycosylation. Multifunctional biomass-degrading enzymes in this strain were rapidly degraded. With the genetic tools available in C. bescii, this system represents a unique opportunity to study the role of bacterial enzyme glycosylation as well an investigation of the pathway for protein glycosylation in a non-pathogen.
Bacteria of the thermophilic genus Caldicellulosiruptor are of industrial interest for their ability to efficiently degrade crystalline cellulose and to utilize lignocellulosic biomass without the need for the conventional pretreatment [1, 2]. As the tools for genetic manipulation have been developed in one of the most cellulolytic species, C. bescii [3, 4], this organism has been explored both as a potential candidate for consolidated bioprocessing (CBP) [5, 6] and as a source for novel, thermophilic lignocellulose-degrading enzymes [7, 8]. C. bescii secretes a suite of biomass-degrading enzymes, the most prevalent being multifunctional in nature, the most abundant of which is Cellulase A  that plays an essential role in the cellulolytic activity of the exoproteome . Most of these multifunctional enzymes consist of two glycoside hydrolases and several carbohydrate-binding modules (CBMs) connected by linker peptide regions , CelA is one of several such tethered, multifunctional enzymes expressed by C. bescii  and is so far the single most cellulolytic gene product ever isolated from a microorganism [12, 13]. In addition, we recently showed that the extracellular form of CelA is heavily glycosylated .
Though it was long thought that bacteria did not make glycoproteins, protein glycosylation pathways are now being discovered in a wide variety of bacteria including pathogens and commensals, and these bacteria utilize a wider array of pathways and sugars than eukaryotes. There are two main types of protein glycosylation: N-linked glycosylation of the amide nitrogen on Asn residues, and O-linked glycosylation of the hydroxyl oxygen on typically on Ser and Thr residues . Archaea, bacteria, and eukaryotes possess N-linked and O-linked protein glycosylation machineries; however, some bacteria also have specialized glycosylation systems (such as the adhesion-specific glycosylation systems in E. coli and Haemophilus influenzae) [15, 16]. Bacterial glycosylation has been studied primarily in the context of cell surface proteins, such as the S layers, pili, and flagella of pathogens. The bacterial N-glycan pathway is best characterized in C. jejuni where a conserved glycan is added to specific asparagine residues in the periplasm by the oligosaccharyltransferase (OTase), the N-OTase PglB. In C. jejuni, loss of N-glycosylation reduces the colonization potential in chickens and mice [17, 18], diminishes the ability to adhere to and invade intestinal epithelial cells in vitro , results in decreased DNA uptake , and increases susceptibility to gut proteases . In contrast, Neisseria species possess an O-linked glycosylation system that results in the transfer of a glycan to serine (S) residues of select periplasmic proteins by the O-OTase PglL . Another O-linked system in the rising nosocomial pathogen Acinetobacter baumannii, driven by the O-OTase PglC, is responsible for capsule biosynthesis, and its disruption weakens biofilm formation and attenuates virulence in mice . Although unusual for O-glycosylation, in these cases, both the bacterial O-linked and N-linked systems build the oligosaccharide as a lipid-linked precursor (a polyprenyl-linked intermediate or LLO) on the cytoplasmic side of the inner membrane that is then flipped into the periplasmic space and transferred en bloc to target proteins by the respective OTases [21, 23]. Whereas, in bacteria, the pathways are not essential for viability, glycosylation deficiencies and defects in protein N-glycosylation in eukaryotes result in more severe phenotypes, classified as congenital disorders of glycosylation in mammals .
In contrast, relatively little is understood about the impact of glycosylation on secreted bacterial enzymes. While previous work analyzed the impact of glycosylation specifically on fungal cellulase enzymes , the results varied greatly in terms of enzyme activity, binding affinity, thermostability, susceptibility to cleavage, and protein transport. For the well-characterized Trichoderma reesei cellulase, Cel7A, O-glycosylation of the linker peptides enhances substrate affinity  and enhances resistance to proteolysis . This proteolysis protection has also been demonstrated for bacterial cellulases in Cellulomonas fimi . Sequence analysis of CelA, using the GlycoPP webserver , indicated favorable sites for N-linked glycosylation in both the GH9 and GH48 domains and sites for O-linked glycosylation spanning the linker regions and CBMs . The linkers of CelA consist primarily of alternating Thr and Pro residues with Ser residues always found near the interface with CBMs and catalytic regions; the same also holds for the other multifunctional enzymes in the exoproteome. In this work, we took an in vivo approach to begin to dissect the pathway for, and impact of, glycosylation in C. bescii. A glycosyltransferase family 39 gene, likely to be involved in protein glycosylation, was identified bioinformatically and deleted by marker replacement. A periodic acid Schiff (PAS) glycoprotein stain revealed that the extracellular enzymes secreted by the resulting GT39 mutant were devoid of any glycosylation. Western analysis of CelA revealed that it is cleaved to a far greater degree in the absence of glycosylation when compared to wild type. Growth curves on cellobiose and Avicel reveal that glycosylation is essential to the ability of C. bescii to digest crystalline cellulose, but not to growth on simple sugars. An understanding of the glycosylation pathway in C. bescii will provide new insight into related systems in other bacteria and could guide future heterologous expression system design for CelA and other thermophilic enzymes.
Bioinformatic analysis identified a putative glycosyltransferase located in close proximity to gene-encoding prominent cellulolytic enzymes, including CelA
Deletion of a glycosyltransferase family 39 gene results in a loss of protein glycosylation
To generate a deletion of Cbes_1864 in the C. bescii chromosome, vector pJRW012 (Additional file 1: Fig. S1) was constructed for the targeted deletion of 1500 bases in the 5′-prime end of the 1701 base pair open-reading frame by joining 1 kb regions upstream and downstream of this region for homologous recombination and marker replacement. The last 200 bp of the 3′-prime ORF sequence was retained to avoid the disruption of potential regulatory sequences for the adjacent gene, Cbes1863. Plasmid pJRW012 contains a wild-type allele of the pyrF gene from Clostridium thermocellum (Clo1313_1266) and does not contain an origin of replication for C. bescii. pJRW012 was transformed into C. bescii JWCB018 that contains a deletion of the pyrFA gene, rendering it a uracil auxotroph. Transformants were selected for uracil prototrophy and plasmid integration at the Cbes1864 locus is shown in (Fig. 1b). Counter selection of the pyrF wild-type allele with 5-fluoroorotic acid (5-FOA), which is converted to the toxic 5-fluorouracil in the presence of the wild-type pyrF allele, was used to select for the elimination of plasmid DNA. PCR with primers binding upstream and downstream of the open-reading frame as well as outside of the flanking regions for integration were used to screen for deletion of Cbes1864 (Fig. 1c). Deletion resulted in a 2.21-kb fragment, distinguishable from the 3.70-kb wild-type fragment followed by sequencing of the 2.21-kb PCR product. Additional PCR screening with one or both primers inside the Cbes1864 reading frame was also performed, producing the expected fragment for the wild-type strains and not from the deletion strain (Additional file 1: Figs. S2, S3).
CelA is secreted in the absence of glycosylation, but is unstable in the cell supernatant
A western blot of the intracellular and extracellular fractions of JWCB001, JWCB018, JWCB143, and the ΔcelA strain JWCB029, using monoclonal anti-CBM3c antibodies, tracked CelA from these strains (Fig. 2d). An array of bands was present in all three extracellular fractions containing CelA, as expected, since CelA is known to exist both in an intact, full-length form as well as in several truncated forms in wild-type cell supernatants . In the glycosyltransferase deletion strain (JWCB143), the array of CelA bands is markedly shifted to molecular weights below 100 kDa when compared to the wild-type and parent strains, indicating that, in the absence of glycosylation, CelA is susceptible to increased degradation or cleavage. This is consistent with past work associating protein glycosylation with proteolytic protection of cellulases in both fungi and bacteria [26, 27]. This result explains the disappearance of the high-molecular-weight bands from the JWCB143 extracellular fraction in (Fig. 2c), a distinct phenotype that may allow a simple screen for other glycosylation-related C. bescii mutants. This result also suggests that glycosylation is not required for protein transport as CelA is present in the extracellular fraction of JWCB143 at similar apparent densities as in the parent and background strains. In the intracellular fractions, the JWCB143 CelA band migrates at a lower molecular weight, as previously observed for CelA expressed without a signal peptide . These observations for CelA seem to hold for other high MW enzymes (Fig. 2a–c), as all high MW bands disappear in both the coomassie and glycostained gels. This is not surprising given the similar sequence and structure of these other multidomain enzymes, especially in the linker regions.
Deletion of glycosyltransferase family 39 gene causes no general growth defect, but does impact the ability of C. bescii to grown on crystalline cellulose
Complementation of the glycosyltransferase deletion restores glycosylation and glycoprotein stability
The deletion of a single glycosyltransferase gene eliminated glycosylation in C. bescii, resulted in loss of the ability to grow on crystalline cellulose and destabilization of high-molecular-weight extracellular enzymes. The phenotype of the glycosyltransferase deletion was, in fact, the same as that of a CelA deletion mutant. Complementation with the wild-type allele restored glycosylation and enzyme stability, suggesting that a major role of this transferase is to glycosylate and stabilize long extracellular enzymes. While in vivo characterization in this work establishes the importance of glycosylation to stability and cellulolytic activity, detailed analysis of these enzymes may reveal additional contributions of glycosylation. The identification of Cbes_1864 as necessary for glycosylation in C. bescii is a step towards describing a novel glycosylation pathway in this hyperthermophilic Gram-positive bacterium. From an industrial perspective, this transferase may facilitate heterologous expression of these enzymes, including CelA for cost-effective production of fully functional enzymes. Past efforts to express CelA in industrial production hosts have resulted in either severe proteolytic degradation (E. coli) [39, 40] or substantially altered molecular weight and activity characteristics (Bacillus megaterium) . Beyond CelA and Caldi enzymes, this transferase may also facilitate heterologous expression of other industrially relevant enzymes. Recently, a core group of CAZymes was identified (including CelA) that account for the entire cellulolytic activity of the C. bescii exoproteome . The proposed use of this cassette to confer cellulolytic ability to other thermophiles will likely require protein glycosylation. All four of the identified enzymes are multidomain proteins with linker regions and CBMs. The presence of Cbes_1864 homologues in other cellulolytic bacteria provides an interesting foothold into studying the potential common utilization of protein glycosylation across very different native plant biomass deconstruction strategies.
Bacterial strains, media, and culturing conditions
Strains/plasmid used in this study
DSMZ6725 wild type (ura+/5-FOAS)
ΔpyrFA ldh::ISCbe4 Δcbe1 (ura−/5-FOAR)
ΔpyrFA ldh::ISCbe4 Δcbe1 ΔcelA (ura−/5-FOAR)
ΔpyrFA ldh::ISCbe4 Δcbe1 Δcbes1864 (ura−/5-FOAR)
JWCB143-containing pJRW013 (ura+/5-FOAS)
DH5α-containing pJRW012 (ApramycinR)
DH5α-containing pJRW013 (ApramycinR)
E. coli/C. bescii shuttle vector (ApramycinR)
C. bescii integration vector (ApramycinR)
Expression vector for Cbes_1867 (ApramycinR)
Cbes1864 deletion vector (ApramycinR)
Expression vector for Cbes_1864 (ApramycinR)
Construction of the glycosyltransferase family 39 (Cbes1864) deletion and complementation vectors
Plasmids for this work were constructed using Q5 High-Fidelity DNA polymerase (New England BioLabs, Ipswich, MA, USA) for PCR, restriction enzymes (New England BioLabs, Ipswich, MA, USA) for digestion, and the fast-link DNA ligase kit (Epicentre Biotechnologies, Madison, WI, USA) for ligation, all according to the manufacturer’s instructions. The non-replicating integration plasmid pJRW012 for deletion of Cbes_1864 was constructed as follows. Using C. bescii (JWCB001) genomic DNA as template, a 5′ flanking region (1002 bp) was amplified using primers SK162 and JR022 and a 3′ flanking region (999 bp) was amplified using primers JR023 and SK161. These flanking regions were joined into one fragment (2001 bp) using overlap extension PCR (OE-PCR) with primers SK162 and SK161 adding a 5′ KpnI restriction site and a 3′ ApaLI restriction site. A fragment-containing an apramycin resistance gene cassette, a Clostridium thermocellum pyrF cassette (Clo1313_1266), and the E. coli pSC101 replication origin was amplified from pJGW003  using primers DC081 and DC262 adding the same restriction sites. The linear PCR products were digested with restriction enzymes KpnI and ApaLI and ligated together to generate pJRW012. Ligation product was transformed into E. coli DH5α to generate E. coli JW563 and resulting plasmids were screened by diagnostic restriction digestion. The sequence of the plasmid was confirmed by automatic sequencing (Genewiz, South Plainfield, NJ, USA). The shuttle vector pJRW013 for the expression of Cbes_1864 complementation was constructed by way of an intermediate shuttle vector, pJYW022. To construct pJYW022, the entire plasmid pDCW173 , a CelA expression vector, was amplified by PCR using primers DC371 and JY080 adding a Tobacco Etch Virus protease cleavage sequence and an SphI restriction site to the 5′ end of the existing 6 residue Histidine tag. The resulting linear PCR product was digested with SphI and closed by ligation to form pJYW022 that encodes CelA with a TEV cleavable His-tag. pJRW013 was constructed by replacing the CelA-coding sequence on pJYW022 with the gene Cbes_1864 for the expression of the glycosyltransferase family 39 with a TEV cleavable His-tag. First, using C. bescii (JWCB001) genomic DNA as a template, the open-reading frame for Cbes_1864 was amplified with primers JR058 and JR035 (1713 bp) adding a 5′ BamHI restriction site and a 3′ SphI restriction site. Next, a backbone fragment-containing the apramycin resistance gene cassette, a Clostridium thermocellum pyrF cassette (Clo1313_1266), the E. coli pSC101 replication origin, and C. bescii pBAS2 replication sequence was amplified from the template pJYW022 using the primers JY081 and DC464 (7990 bp) with a 5′ SphI restriction site and a 3′ BamHI site. The two linear PCR products were digested with SphI and BamHI and ligated together to generate pJRW013. Ligation product was transformed into E. coli DH5α incubated at room temperature to generate E. coli JW626 and the resulting plasmids were screened by diagnostic restriction digestion. The sequence of pJRW013 was confirmed by Automatic sequencing (Genewiz, South Plainfield, NJ, USA).
Deletion and complementation of the glycosyltransferase family 39 gene (Cbes1864) using a non-replicating vector
Preparations of pJRW012 isolated from E. coli JW563 were used to transform C. bescii JWCB018 by electroporation as described previously . After electroporation with ~ 0.5 µg of plasmid DNA, cultures were recovered in low osmolarity complex (LOC) medium at 65 °C. Recovery cultures were transferred to LOD without uracil to select for uracil prototrophy . Transformants were inoculated into non-selective LOD, with 40-µM uracil, and incubated overnight at 75 °C. Serial dilutions of this overnight culture were plated to LOD containing 4-mM 5-fluoroorotic acid (5-FOA) and 40-µM uracil as described . After a 2-day incubation, colonies resistant to 5-FOA were cultured in LOD with 40 µM uracil for genomic DNA isolation and PCR screening. A PCR to screen for the deletion was performed using Jumpstart Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA) with primers SK163 and SK164 that were designed to hybridize outside the homologous flanking regions on the C. bescii chromosome. Extension time was sufficient to amplify the wild-type allele if it was still present. After the initial screening, isolates containing the expected DNA pattern were purified by two additional rounds of non-selective plating (LOD with 40-µM uracil) and PCR screening to ensure segregation of the deletion allele. The purified deletion mutant was confirmed by PCR as described above and with two additional primer pairs with one (SK163 and JR026) and then with both (JR034 and JR026) primers binding inside the targeted region of Cbes1864. The PCR product of SK163 and SK164 was sequenced to verify the site of the deletion. The verified Cbes1864 deletion strain was designated JWCB143. pJRW013 isolated from E. coli JW626 were used to transform C. bescii JWCB143 as above. Overnight cultures of E. coli JW626 were incubated shaking at room temperature for 2 days. Electroporation recovery cultures were transferred to LOD without uracil to select for uracil prototrophy . Serial dilutions of this overnight culture were plated to LOD without uracil to maintain prototrophy. After a 2-day incubation, colonies were cultured in LOD for genomic DNA isolation and PCR screening. Genomic DNA from isolates was screened by PCR for the presence of plasmid pJRW013 using primers JR026 and DC228, and for maintenance of the Cbes_1864 deletion using primers SK163 and SK164.
Preparation of extracellular and intracellular protein fractions
Extracellular protein (ECP) from C. bescii strains (JWCB001, JWCB018, JWCB029, JWCB143, and JWCB160) was collected from a 0.5–2.0-L culture grown at 65 °C in closed bottles shaking at 90 rpm to an OD680 of 0.25–0.3 in LOD media with cellobiose as sole carbon source and 40-mM MOPS. Cultures were centrifuged (6000×g at 4 °C for 15 min) and supernatants were filtered successively with 1.5- and 0.7-µm glass fiber filters to remove cells. ECP was concentrated and buffer exchanged to 20-mM MES with 2-mM β-mercaptoethanol (pH 6.0) using a 30-kDa molecular weight cut-off column (Hollow Fiber Cartridge, GE Healthcare, Chicago, IL, USA) and further concentrated using Vivaspin Turbo 15 centrifugal concentrators (Sartorius, Bohemia, NY, USA). Cell pellets were used for preparation of intracellular protein (ICP) fractions. Pellets were washed once by resuspension in 50 mL of ice-cold 50-mM Tris–Cl buffer (pH 8.0) and centrifugation (6000×g at 4 °C for 15 min), then resuspended in 1 mL of cell lytic B buffer (Sigma-Aldrich, St. Louis, MO, USA), and subjected to four cycles of freezing (with an ethanol and dry ice bath) and thawing (in 42 °C water bath), sonication four times for 15 s at 40 amps with 1-min rests in ice water. Lysates were centrifuged at maximum speed in microcentrifuge tubes to separate protein lysate from cell debris and the clear CFE was collected. Protein concentrations for both ECP and ICP fractions were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) standards.
Detection of glycosylation and CelA protein
The ECP and ICP fractions were boiled for 15 min with SDS-cracking buffer and separated by SDS-PAGE using 4–20% gradient Mini-Protean TGX gels (Bio-Rad, Hercules, CA, USA) run at 150 V until all the loading dye was run off and then for an additional 30 min to achieve the separation of high-molecular-weight proteins. Varying protein loadings (20–60 µg) were used depending on the sample and experiment. For general protein visualization, gels were stained with Coomassie Brilliant Blue. Glycoproteins were visualized by staining with a Glycoprotein Staining Kit (G-Biosciences, St. Louis, MO, USA) according to the manufacturer’s instructions. After the initial staining and imaging of glycosylated proteins, the gel was counterstained with RAPIDstain solution from the same kit to visualize all the proteins. For detection of C. bescii CelA specifically, protein fractions were run on SDS-PAGE gels as described above and then transferred to a nitrocellulose membrane using a Bio-Rad Mini-Protean 3 electrophoretic unit at 100 V for 2 h. Membranes were probed with monoclonal α-CBM3C primary antibody (1:1000 dilution) and then with α-rabbit with horse radish peroxidase (HRP) secondary antibody (1:10,000). Membranes were developed with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions and imaged by chemiluminescence.
Growth of glycosyltransferase deletion mutant on soluble and insoluble substrates
Cells were sub-cultured three times in LOD medium with 5-g/L maltose as the sole carbon source. The third sub-culture was used to inoculate 50 mL of LOD medium supplemented with 40-mM MOPS and 40-µM uracil and with 5 g/L of either cellobiose or Avicel as the sole carbon source. A 0.2% v/v inoculum was used and cultures were incubated at 65 °C with shaking at 150 rpm. Growth on cellobiose was measured by optical density (OD) at 680 nm using a Jenway Genova spectrophotometer. Growth on crystalline cellulose, Avicel PH-101, was measured by colony-forming units (CFU) by plating on LOD medium (maltose) supplemented with 40-µM uracil.
JFR performed experiments, analyzed data, and drafted the manuscript; SKK performed experiments and analyzed data; JD performed experiments, performed bioinformatics analysis, and analyzed data; HN performed bioinformatics analysis; MEH and YJB analyzed data and contributed to the writing of the manuscript; CMS performed bioinformatics analysis and contributed to the writing of the manuscript; JW directed the work and contributed to the writing of the manuscript. All authors read and approved the final manuscript.
We thank Shreena Patel for expert technical assistance. Funding was provided by The BioEnergy Science Center (BESC) and The Center for Bioenergy Innovation (CBI), U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. CMS is an Alberta Innovates Strategic Chair in Bacterial Glycomics.
The authors declare that they have no competing interests.
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- Blumer-Schuette SE, Kataeva I, Westpheling J, Adams MW, Kelly RM. Extremely thermophilic microorganisms for biomass conversion: status and prospects. Curr Opin Biotechnol. 2008;19(3):210–7.View ArticleGoogle Scholar
- Yang SJ, Kataeva I, Hamilton-Brehm SD, Engle NL, Tschaplinski TJ, Doeppke C, Davis M, Westpheling J, Adams MW. Efficient degradation of lignocellulosic plant biomass, without pretreatment, by the thermophilic anaerobe “Anaerocellum thermophilum” DSM 6725. Appl Environ Microbiol. 2009;75(14):4762–9.View ArticleGoogle Scholar
- Chung D, Cha M, Farkas J, Westpheling J. Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus. PLoS ONE. 2013;8(5):e62881.View ArticleGoogle Scholar
- Chung DH, Huddleston JR, Farkas J, Westpheling J. Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes. J Ind Microbiol Biotechnol. 2011;38(11):1867–77.View ArticleGoogle Scholar
- Cha M, Chung D, Elkins JG, Guss AM, Westpheling J. Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass. Biotechnol Biofuels. 2013;6(1):85.View ArticleGoogle Scholar
- Chung D, Cha M, Guss AM, Westpheling J. Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii. Proc Natl Acad Sci USA. 2014;111(24):8931–6.View ArticleGoogle Scholar
- Blumer-Schuette SE, Giannone RJ, Zurawski JV, Ozdemir I, Ma Q, Yin Y, Xu Y, Kataeva I, Poole FL 2nd, Adams MW, et al. Caldicellulosiruptor core and pangenomes reveal determinants for noncellulosomal thermophilic deconstruction of plant biomass. J Bacteriol. 2012;194(15):4015–28.View ArticleGoogle Scholar
- Dam P, Kataeva I, Yang SJ, Zhou F, Yin Y, Chou W, Poole FL 2nd, Westpheling J, Hettich R, Giannone R, et al. Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725. Nucleic Acids Res. 2011;39(8):3240–54.View ArticleGoogle Scholar
- Lochner A, Giannone RJ, Rodriguez M Jr, Shah MB, Mielenz JR, Keller M, Antranikian G, Graham DE, Hettich RL. Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis. Appl Environ Microbiol. 2011;77(12):4042–54.View ArticleGoogle Scholar
- Young J, Chung D, Bomble YJ, Himmel ME, Westpheling J. Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass. Biotechnol Biofuels. 2014;7(1):142.View ArticleGoogle Scholar
- Brunecky R, Chung D, Sarai NS, Hengge N, Russell JF, Young J, Mittal A, Pason P, Vander Wall T, Michener W, et al. High activity CAZyme cassette for improving biomass degradation in thermophiles. Biotechnol Biofuels. 2018;11:22.View ArticleGoogle Scholar
- Brunecky R, Alahuhta M, Xu Q, Donohoe BS, Crowley MF, Kataeva IA, Yang SJ, Resch MG, Adams MW, Lunin VV, et al. Revealing nature’s cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA. Science. 2013;342(6165):1513–6.View ArticleGoogle Scholar
- Brunecky R, Donohoe BS, Yarbrough JM, Mittal A, Scott BR, Ding H, Taylor Ii LE, Russell JF, Chung D, Westpheling J, et al. The multi domain Caldicellulosiruptor bescii CelA cellulase excels at the hydrolysis of crystalline cellulose. Sci Rep. 2017;7(1):9622.View ArticleGoogle Scholar
- Chung D, Young J, Bomble YJ, Vander Wall TA, Groom J, Himmel ME, Westpheling J. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated. PLoS ONE. 2015;10(3):e0119508.View ArticleGoogle Scholar
- Nothaft H, Szymanski CM. Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol. 2010;8(11):765–78.View ArticleGoogle Scholar
- Nothaft H, Szymanski CM. Bacterial protein N-glycosylation: new perspectives and applications. J Biol Chem. 2013;288(10):6912–20.View ArticleGoogle Scholar
- Hendrixson DR, DiRita VJ. Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract. Mol Microbiol. 2004;52(2):471–84.View ArticleGoogle Scholar
- Szymanski CM, Burr DH, Guerry P. Campylobacter protein glycosylation affects host cell interactions. Infect Immun. 2002;70(4):2242–4.View ArticleGoogle Scholar
- Larsen JC, Szymanski C, Guerry P. N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81–176. J Bacteriol. 2004;186(19):6508–14.View ArticleGoogle Scholar
- Alemka A, Nothaft H, Zheng J, Szymanski CM. N-glycosylation of Campylobacter jejuni surface proteins promotes bacterial fitness. Infect Immun. 2013;81(5):1674–82.View ArticleGoogle Scholar
- Hartley MD, Morrison MJ, Aas FE, Borud B, Koomey M, Imperiali B. Biochemical characterization of the O-linked glycosylation pathway in Neisseria gonorrhoeae responsible for biosynthesis of protein glycans containing N,N’-diacetylbacillosamine. Biochemistry. 2011;50(22):4936–48.View ArticleGoogle Scholar
- Lees-Miller RG, Iwashkiw JA, Scott NE, Seper A, Vinogradov E, Schild S, Feldman MF. A common pathway for O-linked protein-glycosylation and synthesis of capsule in Acinetobacter baumannii. Mol Microbiol. 2013;89(5):816–30.View ArticleGoogle Scholar
- Kelly J, Jarrell H, Millar L, Tessier L, Fiori LM, Lau PC, Allan B, Szymanski CM. Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer. J Bacteriol. 2006;188(7):2427–34.View ArticleGoogle Scholar
- Freeze HH, Schachter H, Kinoshita T: Genetic disorders of glycosylation. In: Essentials of glycobiology. Varki A, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, Darvill AG, Kinoshita T, Packer NH, Prestegard JH et al, eds. 3rd edn. Cold Spring Harbor (NY); 2015.Google Scholar
- Beckham GT, Dai Z, Matthews JF, Momany M, Payne CM, Adney WS, Baker SE, Himmel ME. Harnessing glycosylation to improve cellulase activity. Curr Opin Biotechnol. 2012;23(3):338–45.View ArticleGoogle Scholar
- Payne CM, Resch MG, Chen L, Crowley MF, Himmel ME, Taylor LE 2nd, Sandgren M, Stahlberg J, Stals I, Tan Z, et al. Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci USA. 2013;110(36):14646–51.View ArticleGoogle Scholar
- Amore A, Knott BC, Supekar NT, Shajahan A, Azadi P, Zhao P, Wells L, Linger JG, Hobdey SE, Vander Wall TA, et al. Distinct roles of N- and O-glycans in cellulase activity and stability. Proc Natl Acad Sci USA. 2017;114(52):13667–72.View ArticleGoogle Scholar
- Langsford ML, Gilkes NR, Singh B, Moser B, Miller RC Jr, Warren RA, Kilburn DG. Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains. FEBS Lett. 1987;225(1–2):163–7.View ArticleGoogle Scholar
- Chauhan JS, Bhat AH, Raghava GP, Rao A. GlycoPP: a webserver for prediction of N- and O-glycosites in prokaryotic protein sequences. PLoS ONE. 2012;7(7):e40155.View ArticleGoogle Scholar
- Coutinho PM, Deleury E, Davies GJ, Henrissat B. An evolving hierarchical family classification for glycosyltransferases. J Mol Biol. 2003;328(2):307–17.View ArticleGoogle Scholar
- VanderVen BC, Harder JD, Crick DC, Belisle JT. Export-mediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways. Science. 2005;309(5736):941–3.PubMedGoogle Scholar
- Wehmeier S, Varghese AS, Gurcha SS, Tissot B, Panico M, Hitchen P, Morris HR, Besra GS, Dell A, Smith MC. Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotes. Mol Microbiol. 2009;71(2):421–33.View ArticleGoogle Scholar
- Naegeli A, Michaud G, Schubert M, Lin CW, Lizak C, Darbre T, Reymond JL, Aebi M. Substrate specificity of cytoplasmic N-glycosyltransferase. J Biol Chem. 2014;289(35):24521–32.View ArticleGoogle Scholar
- Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014;42(Database issue):490–5.View ArticleGoogle Scholar
- Loibl M, Strahl S. Protein O-mannosylation: what we have learned from baker’s yeast. Biochim Biophys Acta. 2013;1833(11):2438–46.View ArticleGoogle Scholar
- Conway JM, McKinley BS, Seals NL, Hernandez D, Khatibi PA, Poudel S, Giannone RJ, Hettich RL, Williams-Rhaesa AM, Lipscomb GL, et al. Functional analysis of the glucan degradation locus (GDL) in Caldicellulosiruptor bescii reveals essential roles of component glycoside hydrolases in plant biomass deconstruction. Appl Environ Microbiol. 2017. https://doi.org/10.1128/AEM.01828-17.View ArticlePubMedPubMed CentralGoogle Scholar
- Schwartz R, Ting CS, King J. Whole proteome pI values correlate with subcellular localizations of proteins for organisms within the three domains of life. Genome Res. 2001;11(5):703–9.View ArticleGoogle Scholar
- Kall L, Krogh A, Sonnhammer EL. A combined transmembrane topology and signal peptide prediction method. J Mol Biol. 2004;338(5):1027–36.View ArticleGoogle Scholar
- Yi Z, Su X, Revindran V, Mackie RI, Cann I. Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose. PLoS ONE. 2013;8(12):e84172.View ArticleGoogle Scholar
- Zverlov V, Mahr S, Riedel K, Bronnenmeier K. 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. Microbiology. 1998;144(Pt 2):457–65.View ArticleGoogle Scholar
- Farkas J, Chung D, Cha M, Copeland J, Grayeski P, Westpheling J. Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol. 2013;40(1):41–9.View ArticleGoogle Scholar
- Groom J, Chung D, Young J, Westpheling J. Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction. Biotechnol Biofuels. 2014;7(1):132.View ArticleGoogle Scholar
- Chung D, Farkas J, Huddleston JR, Olivar E, Westpheling J. Methylation by a unique alpha-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725. PLoS ONE. 2012;7(8):e43844.View ArticleGoogle Scholar
- Cha M, Wang H, Chung D, Bennetzen JL, Westpheling J. Isolation and bioinformatic analysis of a novel transposable element, ISCbe4, from the hyperthermophilic bacterium, Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol. 2013;40(12):1443–8.View ArticleGoogle Scholar