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

Abstract

Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence was determined to be 5′-GG/CC-3′, indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase (Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and classified CbeI as a member of a novel subfamily of HaeIII-like enzymes.

Recombinogenic Properties of Pyrococcus furiosus Strain COM1 Enable Rapid Selection of Targeted Mutants

“…Cell-free extracts (CFE) were prepared from 1-liter cultures as described previously (7). Endonuclease assays were performed in 10-l reaction mixture volumes using 0.5 to 1 g of pJFW051 DNA.…”

Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction

“…In developing methods for DNA transformation of C. bescii, we observed that restriction by a HaeIII isoschizomer, CbeI [21], was an absolute barrier to transformation of DNA from E. coli. We identified, cloned, expressed, and purified its cognate methyltransferase, M. CbeI, from C. bescii and showed that DNA methylated in vitro readily transformed C. bescii [17].…”

“…JWCH009 was grown to the late exponential phase (OD 680 approximately 0.15) in 50 mL LOD. Direct extraction of plasmid DNA from JWCH009 was performed as previously described [21,25]. The plasmid DNA was digested with enzymes HaeIII, EcoRI, HhaI, and MboI (NEB).…”

“…Both the C. bescii and the heterologous Clostridium thermocellum wild-type pyrF allele were shown to complement this deletion, restoring the mutant to uracil prototrophy. Deletion of the ChyI restriction enzyme in C. hydrothermalis, a homolog of a HaeIII-like restriction enzyme known to be a barrier to transformation in C. bescii [20,21], increased the transformation efficiency by about an order of magnitude. The new strain C. hydrothermalis JWCH008 should facilitate the assessment of plant biomass deconstruction by the Caldicellulosiruptor genus and the molecular engineering of deconstruction enzymes.…”

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

“…Such organisms require a tractable genetic system for metabolic engineering applications, and such a system has been established for C. bescii in recent years (5,6). A major step in developing a genetic system for C. bescii was the discovery that it has a restriction-modification system that severely limits its transformability (7). This barrier to transformation was overcome through the use of a methylated donor plasmid with a methylation pattern that protected the transformed DNA from restriction digestion (5).…”