Abstract
A major barrier to both metabolic engineering and fundamental biological studies is the lack of genetic tools in most microorganisms. One example is Clostridium thermocellum ATCC 27405T, where genetic tools are not available to help validate decades of hypotheses. A significant barrier to DNA transformation is restriction–modification systems, which defend against foreign DNA methylated differently than the host. To determine the active restriction–modification systems in this strain, we performed complete methylome analysis via single-molecule, real-time sequencing to detect 6-methyladenine and 4-methylcytosine and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine. Multiple active systems were identified, and corresponding DNA methyltransferases were expressed from the Escherichia coli chromosome to mimic the C. thermocellum methylome. Plasmid methylation was experimentally validated and successfully electroporated into C. thermocellum ATCC 27405. This combined approach enabled genetic modification of the C. thermocellum-type strain and acts as a blueprint for transformation of other non-model microorganisms.
Highlights
Most microbial metabolic engineering for biotechnology research is performed in model organisms, because they are well studied and have a large toolbox to enable genetic modifications [7]
The resulting polymerase chain reaction (PCR)-amplified DNA can be sequenced, and remaining cytosines in the sequence were previously methylated. This approach has not been routinely used in bacteria, and we have only identified a few studies that utilized whole-genome bisulfite sequencing (WGBS) for bacterial methylome analysis for characterization of RM systems [13, 44, 51]
C. thermocellum ATCC 27405 was grown in CTFUD medium [40], which is comprised of 3 g sodium citrate tribasic dehydrate, 1.3 g ammonium sulfate, 1.43 g potassium phosphate monobasic, 1.8 g potassium phosphate dibasic trihydrate, 0.5 g cysteine HCL, 10.5 g MOPS sodium salt, 6 g glycerol-2-phosphate disodium, 5 g cellobiose, 4.5 g yeast extract, 0.13 g calcium chloride dehydrate, 2.6 g magnesium chloride hexahydrate, 0.0001 g ferrous sulfate heptahydrate, and 0.5 ml 0.2% (w/v) resazurin
Summary
Most microbial metabolic engineering for biotechnology research is performed in model organisms, because they are well studied and have a large toolbox to enable genetic modifications [7]. Type I systems are comprised of three subunits: a DNA methyltransferase, a DNA recognition subunit, and a restriction enzyme. This type of system recognizes two motifs of 3–4 bases separated by any 5–8 bases [25], such as the EcoKI system that targets AACNNNNNNGTGC (N is any base), and motifs are methylated at the N-6 position of one adenine per DNA strand to form N6-methyladenine (m6A). Type II systems are largely studied and commonly used as tools in molecular biology Their recognition systems are often palindromic, and they can methylate bases to form m6A, N4-methylcytosine (m4C), or 5-methylcytosine (m5C) [16, 31]. Type III systems recognize non-palindromic motifs and typically methylate to form m6A [38]. The last group, Type IV, is only comprised of a restriction enzyme, which recognizes motifs that are methylated differentially than the host [19, 38]
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