The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants’ needs.

Nitrogen fixation by rhizobia in symbiosis with legumes is economically and ecologically important. The symbiosis can involve a complex bacterial transformation—terminal differentiation—that includes major shifts in the transcriptome and cell cycle.

Methylation of specific genomic DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Here, we explored DNA methylation in bacteria of the genus Ensifer, including its potential role in regulating the process of terminal differentiation occurring during nitrogen-fixing symbiosis with legumes. Using single-molecule real-time sequencing, six unique genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. These five motifs were nearly fully methylated across the genomes in all tested conditions, and they were not enriched in the promoter regions of symbiosis, carbon source, or cell cycle-regulated genes, suggesting that most DNA methylation is not a major regulatory mechanism in the genus Ensifer. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cells, methylation of GANTC motifs increased progressively from the ori to ter region in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. The GANTC methylation profile transited into a genome-wide pattern of near full methylation in the early stage of symbiotic differentiation, followed by a progressive decrease in methylation from the ori to ter regions of fully differentiated symbiotic bacteria. This is evidence of a dysregulated and constitutive CcrM activity during terminal differentiation, which we suggest is a driving factor for endoreduplication of terminally differentiated bacteroids.