position until the DNA is again replicated. In E. coli, these pili operons have important roles in virulence in urinary tract infections. It has been proposed that inhibitors of Dam may function as antibiotics. On the other hand, DNA cytosine methylase targets CCAGG and CCTGG sites to methylate cytosine at the C5 position (C meC(A/T) GG). The other methylase enzyme, EcoKI, causes methylation of adenines in the sequences AAC(N6)GTGC and GCAC(N6)GTT. In Clostridioides difficile, DNA methylation at the target motif CAAAAA was shown to impact sporulation, a key step in disease transmission, as well as cell length, biofilm formation and host colonization. Molecular cloning Most strains used by molecular biologists are derivatives of E. coli K-12, and possess both Dam and Dcm, but there are commercially available strains that are dam-/dcm- (lack of activity of either methylase). In fact, it is possible to unmethylate the DNA extracted from dam+/dcm+ strains by transforming it into dam-/dcm- strains. This would help digest sequences that are not being recognized by methylation-sensitive restriction enzymes. The restriction enzyme DpnI can recognize 5'-GmeATC-3' sites and digest the methylated DNA. Being such a short motif, it occurs frequently in sequences by chance, and as such its primary use for researchers is to degrade template DNA following PCRs (PCR products lack methylation, as no methylases are present in the reaction). Similarly, some commercially available restriction enzymes are sensitive to methylation at their cognate restriction sites and must as mentioned previously be used on DNA passed through a dam-/dcm- strain to allow cutting. Detection DNA methylation can be detected by the following assays currently used in scientific research: Mass spectrometry is a very sensitive and reliable analytical method to detect DNA methylation. MS, in general, is however not informative about the sequence context of the methylation, thus limited in studying the function of this DNA modification. Methylation-Specific PCR (MSP), which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR. However, methylated cytosines will not be converted in this process, and primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated. Whole genome bisulfite sequencing, also known as BS-Seq, which is a high-throughput genome-wide analysis of DNA methylation. It is based on the aforementioned sodium bisulfite conversion of genomic DNA, which is