Genome-Wide DNA Methylation and Restriction-Modification Systems in Casimicrobium huifangae SJ-1, One of the Core Bacteria in Activated Sludge.

Casimicrobium huifangae SJ-1 is one of the 28 core microbial groups in the activated sludge of municipal wastewater treatment plants. It exhibits strong environmental adaptability and interactions with other microbes. To develop a genetic manipulation system for this strain, we tested several broad-host-range plasmids, and pBBR1MCS-2 was successfully transformed into strain SJ-1. However, only the plasmid extracted from the transformant could be retransformed into strain SJ-1, suggesting strong host defense systems. We investigated the genome-wide DNA methylation and obtained a DNA methylation map and nine methylation-targeting motifs from C. huifangae. A modified pBBR1MCS-2 lacking all targeting motifs achieved transformation efficiency comparable to that of the host-derived plasmid, implicating the restriction modification (R-M) barrier of C. huifangae. REBASE annotation revealed nine R-M systems in the genome, including two of Type I, six of Type II, and one of Type III. Quantitative PCR showed that the restriction endonuclease component of the Type I RM-C system was transcriptionally upregulated in the transformant harboring pBBR1MCS-2. The subunits responsible for the methyltransferase and endonuclease functions of RM-C were expressed in Escherichia coli, purified, and characterized. The targeting motif of RM-C was implicated as 5'-GAGNNNNNNNTGCT-3' based on in vitro cleavage assays and SMRT methylation calls. These findings reveal the DNA methylation characteristics of C. huifangae SJ-1 and outline the possible methylation-based defense architecture, providing a reference for future genetic manipulation.