A novel SfaNI-like restriction-modification system in Caldicellulosiruptor extents the genetic engineering toolbox for this genus

Abstract

Caldicellulosiruptor is a genus of thermophilic to hyper-thermophilic microorganisms that express and secrete an arsenal of enzymes degrading lignocellulosic biomasses into fermentable sugars. Because of this distinguished feature, strains of Caldicellulosiruptor have been considered as promising candidates for consolidated bioprocessing. Although a few Caldicellulosiruptor strains with industrially relevant characteristics have been isolated to date, it is apparent that further improvement of the strains is essential for industrial application. The earlier identification of the HaeIII-like restriction-modification system in C. bescii strain DSM 6725 has formed the basis for genetic methods with the aim to improve the strain's lignocellulolytic activity and ethanol production. In this study, a novel SfaNI-like restriction-modification system was identified in Caldicellulosiruptor sp. strain BluCon085, consisting of an endonuclease and two methyltransferases that recognize the reverse-complement sequences 5'-GATGC-3' and 5'-GCATC-3'. Methylation of the adenine in both sequences leads to an asymmetric methylation pattern in the genomic DNA of strain BluCon085. Proteins with high percentage of identity to the endonuclease and two methyltransferases were identified in the genomes of C. saccharolyticus strain DSM 8903, C. naganoensis strain DSM 8991, C. changbaiensis strain DSM 26941 and Caldicellulosiruptor sp. strain F32, suggesting that a similar restriction-modification system may be active also in these strains and respective species. We show that methylation of plasmid and linear DNA by the identified methyltransferases, obtained by heterologous expression in Escherichia coli, is sufficient for successful transformation of Caldicellulosiruptor sp. strain DIB 104C. The genetic engineering toolbox developed in this study forms the basis for rational strain improvement of strain BluCon085, a derivative from strain DIB 104C with exceptionally high L-lactic acid production. The toolbox may also work for other species of the genus Caldicellulosiruptor that have so far not been genetically tractable.

Fig 1. Prediction of the four most prominent DNA methylation patterns detected in the Nanopore sequencing dataset from strain BluCon085 using the methylation-calling tool Tombo version 1.5.

For each identified methylation pattern, raw Nanopore signals (red lines) for three related sequence motifs in the genome and the corresponding violin plots are shown. The violin plots visualize the predicted methylation frequencies of single nucleotides (y-axis/-log10^p-value) for related sequence motifs with a given proportion of methylated reads (x-axis/DNA sequence). Modified bases are highlighted in the violin plot by methylation frequencies greater than 0.8. The predicted methylated sites within the identified motifs are “A” and “T” in the related asymmetric sequences 5’-GATGC-3’ and 5’-GCATC-3’, “A” and “T” in the palindromic sequence 5’-GATC-3’, and “C” and “A” in the asymmetric sequence 5’-RCWGCAG-3’. As for the latter one, the methylated reverse-complement counterpart could not be detected, and therefore hemi-methylation is assumed.

Fig 2. Digestion of genomic DNA of strain BluCon085 with methylation-sensitive restriction endonucleases.

Restriction digests were performed by incubating genomic DNA in the presence of the restriction endonuclease SfaNI, AlwI, DpnI or PleI. A restriction digest without restriction endonuclease was included as control.

Fig 3. SDS-PAGE analysis of protein extracts from E. coli NEB 10-beta strains expressing methyltransferases M1.Cal02329 and M2.Cal02329.

Protein extracts were prepared from E. coli strain NEB 10-beta pTrc-Ec_cal02329M1 (NEB pTrc-M1), E. coli strain NEB 10-beta pTrc-Ec_cal02329M2 (NEB pTrc-M2), E. coli strain NEB 10-beta pTrcHis2 B (NEB pTrc), and Caldicellulosiruptor strain BluCon085 (BLU). Cells were first lysed by sonification, and thermotolerant proteins present in the cell lysates were purified by heat treatment at 70°C. Protein extracts before and after purification are shown.

Fig 4. Degradation of a methylated and unmethylated DNA fragment by endogenous nucleases present in a BluCon085 cell lysate.

The methylated and unmethylated DNA fragments were incubated in the absence of cell lysate (w/o), and in the presence of 14%, 29% and 43% of cell lysate. The incubation was performed at 70°C for 30 minutes.

Fig 5. Confirmation of the deletion in the pyrE gene in strain DIB 104C DpyrE.

(A) The pyrE open reading frame together with 100 bp immediately downstream of the stop codon was sequenced in strains DIB 104C and DIB 104C DpyrE. The pyrE open reading frame in strain DIB 104C is indicated in grey. (B) The pyrE gene was PCR amplified from genomic DNA of strains DIB 104C and DIB 104C DpyrE using primers BLU13/14 (S1 Table), which bind upstream and downstream of the pyrE open reading frame. (C) The uracil auxotrophy of strain DIB 104C DpyrE was checked by incubating cells in MOPS-buffered medium with filter paper and CSM-Ura. Incubation was performed at 70°C for 4 days.

Fig 6. Digestion of methylated and unmethylated pUC19-P_slp-pyrE plasmid DNA with the restriction endonuclease SfaNI.

Restriction digests were performed by incubating unmethylated plasmid DNA (-) as well as plasmid DNA methylated with either M1.Cal02329 (M1), or M2.Cal02329 (M2), or both M1.Cal02329 and M2.Cal02329 (M1/2) in the presence of the restriction endonuclease SfaNI. A restriction digest without SfaNI was included as control.

Fig 7. Confirmation of genomic integration of the pyrE gene by PCR.

(A) Confirmation of the integration of a fragment in the CIS1 region in five single cell colonies (Scc 1 to Scc 5) obtained after transformation of strain DIB 104C DpyrE with plasmid pUC19-P_slp-pyrE. Strain DIB 104C DpyrE was included as control (DpyrE). (B) Confirmation of the mutant version of the pyrE gene in the single cell colonies Scc 1 to Scc 5. (C) Confirmation of the wild type pyrE gene in five single cell colonies (Scc 6 to Scc 10) obtained after transformation of strain DIB 104C DpyrE with a linear DNA fragment containing the wild type pyrE gene.