Comparison of CcrM-dependent methylation in Caulobacter crescentus and Brucella abortus by nanopore sequencing
Bacteria rely on DNA methylation for restriction modification systems and for epigenetic control of gene expression. In Alphaproteobacteria, the CcrM orphan methyltransferase is particularly noteworthy in a range of transcriptional regulation. The wider adoption of nanopore sequencing and updated processing pipelines has made epigenome measurements in bacteria more convenient than before. Here, we validate this approach in Alphaproteobacteria by measuring CcrM-dependent DNA methylation in Caulobacter crescentus and show excellent correlation with other approaches. Continuing in Caulobacter, we directly measure the impact of Lon-mediated CcrM degradation on the epigenome and show that the AlkB demethylase has no global impact on DNA methylation during normal growth. We report on the global DNA methylation in Brucella abortus for the first time and find that CcrM-dependent methylation is reliant on Lon in an unexpected species- and chromosome-specific manner. Finally, we measure the impact of the MucR transcription factor on the Brucella methylome. Given the ease and reduced costs, our work demonstrates the utility of nanopore-based sequencing for epigenome monitoring in Alphaproteobacteria. Overall design: Caulobacter crescentus NA1000, along with its derivative strains, were initially cultured on Peptone Yeast Extract (PYE) agar plates. These plates contain a specific nutrient mix: 2 g/L of peptone, 1 g/L of yeast extract, 0.5 mM of MgSO4, 0.5 mM of CaCl2, and 1.5% agar (produced by Difco), and were incubated overnight at 30°C. Following incubation, isolated colonies were picked and further cultured in PYE liquid media overnight at 30°C within shaken flasks. The cultures were then diluted to achieve a starting optical density (OD600) of 0.1, and allowed to grow to a mid-exponential phase (OD600 of ~0.6) prior to the isolation of chromosomal DNA. Brucella abortus 2308 and derivative strains were grown on Schaedler agar (Becton, Dickinson, and Co., Sparks, MD, USA) supplemented with 5% defibrinated bovine blood (SBA) incubated at 37°C under 5% CO2 or in brucella broth (Becton, Dickinson, and Co., Sparks, MD, USA) incubated at 37°C with shaking. Growth media were supplemented with kanamycin (45 ug/mL) or chloramphenicol (10 ug/mL) when appropriate. For isolation of chromosomal DNA, Brucella abortus 2308 and derivative strains were grown overnight in brucella broth, subcultured to an OD 600 of 0.1 and then grown to  mid-exponential phase (OD 600 of ~0.6) before isolation of chromosomal DNA by guanidine thiocyanate. Caulobacter strains were sequenced using nanopore R.9.5 pores, and Brucella strains were sequenced using nanopore R.10.4.1 pores. Raw nanopore sequencing outputs (Fast5 format) for Caulobacter strains were quality-controlled and trimmed using Guppy. High-quality reads were aligned to the reference genome NC_011916.1 with Minimap2, using default parameters. Fast5 files were converted to slow5 files and aligned to the mapped reads using the 'eventalign' function within Nanopolish. Methylated bases were quantified using mCaller, employing the precompiled 'r95_twobase_model_NN_6_m6A.pkl' model for assessing modified adenines. The methylation results were then annotated using Bedtools. Raw nanopore sequencing outputs (Fast5 format) from nanopore sequencing for Brucella strains, were converted into pod5 files for optimization. Dorado's standard basecalling command was employed, utilizing the model 'dna_r10.4.1_e8.2_400bps_sup@ v4.2.0'. In addition, modified base calling was executed using the specialized model 'dna_r10.4.1_e8.2_400bps_sup @v4.2.0_6mA@v2'. The produced bed files were then aligned to NCBI reference genomes NC_007618.1 and NC_007624.1 with the mapped reads using the align and sort functions of samtools . The resulting modified bam files were run with modbam2bed with default settings.