GSE169460
Epigenomic landscape of Lyme disease spirochetes reveals novel motifs
Borrelia burgdorferi, the etiological agent of Lyme disease, persists in nature through an enzootic cycle consisting of a vertebrate host and an Ixodes tick vector. The sequence motifs modified by two well-characterized restriction/modification loci of B. burgdorferi type strain B31 were recently described, but the methylation profiles of other Lyme disease Borrelia have not been characterized. Herein, the methylomes of B. burgdorferi type strain B31 and 7 clonal derivatives, along with B. burgdorferi N40, B. burgdorferi 297, B. burgdorferi CA-11, B. afzelii PKo, B. afzelii BO23, and B. garinii PBr, were defined through PacBio SMRT sequencing. This analysis revealed 9 novel sequence motifs methylated by the plasmid-encoded restriction/modification enzymes of these Borrelia strains. Furthermore, while a previous analysis of B. burgdorferi B31 revealed an epigenetic impact of methylation on the global transcriptome, the current data contradict those findings; our analyses of wild-type B. burgdorferi B31 revealed no consistent differences in gene expression among isogenic derivatives lacking one or more restriction/modification enzyme(s). Overall design: 11 B. burgdorferi type strain B31 derivatives either containing or lacking the plasmid-encoded restriction-modification genes bbe02, bbq67, and bbh09 underwent RNA-sequencing. Total RNA was isolated from three biological replicates of B. burgdorferi strain B31-A3, B31-A3?bbe02, B31-A3?bbq67, and B31-A3?bbe02?bbq67, and from two biological replicates of B. burgdorferi B31-A3, B31-A3?bbe02, B31-A3-68, B31-A3-68?bbe02, B31-A3?lp28-3, B31-A34, and B31-A34 Tn::H09 in mid-log phase (5-9 x 107 celEls/mL). RNA was quantified and subjected to Agilent Bionalyzer 2200 Tape Station quality assessment. RNAs possessing RIN values > 7.4 were used for downstream analysis. cDNA libraries were synthesized using Ribo-Zero and ScriptSeq complete bacteria kit with indexing primers for synthesis of directional libraries per the manufacturer's instructions. The quality and quantity of the resulting cDNA libraries were assessed with an Agilent DNA 1000 assay on an Agilent 2100 Bioanalyzer and with a KAPA Library Quantification Kit prior to RNA-seq. RNA-seq libraries were sequenced on an Illumina MiSeq using v3 chemistry at 2 x 75 bp reads.RNA-seq reads were compiled and filtered to remove any reads with PHRED scores less than 10 and aligned to the B. burgdorferi B31 genome (RefSeq AE000783-AE000794 and AE001575-AE001584) using bowtie2. Reads for annotated genes were determined using featureCounts.