Journal: iScience

Article Title: Cytosine methylation contributes to the fitness of Caulobacter cells naturally expressing a Vsr-like protein

doi: 10.1016/j.isci.2026.114749

Figure Lengend Snippet: ScmA methylates the first cytosine in YG C CGGCR motifs (A) Schematic showing the organization of genes around scmA ( CCNA_01085 ) on the C. crescentus NA1000 chromosome. Genes of unknown function are shown using their CCNA numbers. (B) Map of the pBX-1motif plasmid (left) showing the position of the unique YGCCGGCR motif and of the 40 other CCGG motifs (orange lines). These motifs are all cut by the 5mC-sensitive HpaII and MspI restriction endonucleases. The right images show the size (bp) of the expected large restriction fragments following digestion with HpaII or MspI in WT or ΔscmA C. crescentus cells if ScmA can methylate the first cytosine of the unique YG C CGGCR motif found on that plasmid. (C) Image of an agarose gel showing the size of the restriction fragments detected after a digestion of the pBX-1motif plasmid using HpaII, MspI or no enzyme (non-digested; ND). Prior to digestion, pBX-1motif was extracted from E. coli TOP10 cells (left) or from WT (JC450; +) or ΔscmA (JC2005; -) C. crescentus cells (right). Blue arrows highlight the two large restriction fragments (846 and 1,358 bp) obtained if the first C in the YGCCGGCR motif is not methylated (HpaII and MspI can then cut the CCGG motif included in that larger motif); the red arrow highlights the unique large restriction fragment obtained (2,204 bp) if the first C in the YG C CGGCR motif is methylated (HpaII and MspI can then not cut the C CGG motif included in that larger motif). L: DNA ladder (kbp).

Article Snippet: 700 ng of pBX-1motif were digested for 2h at 37°C using the 5mC-sensitive HpaII or MspI restriction endonucleases (New England Biolabs, USA).

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Methylation