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An investigation of Burkholderia cepacia complex methylomes via SMRT sequencing and mutant analysis

ABSTRACT: While nucleotide sequences determine an organism’s proteins, methylation of the nucleotides themselves can confer additional properties. In bacteria, differences in methylation can be used to identify incoming DNA, for example from bacteriophages. Restriction enzymes detect the methylation status at specific sequence motifs, and cleave improperly methylated, foreign DNA. Furthermore, methylation of key genetic regions can influence gene expression and hence phenotype. In this study, we determined the methylated motifs of three strains from the closely-related Burkholderia cepacia complex of opportunistic pathogens. Three of these motifs were novel, while two are methylated throughout this group of bacteria and beyond and had already been assigned putative causative genes. To investigate the contribution of the enzymes that methylate and cut DNA, we deleted the genes encoding them from a representative strain. We found that these genes are important for traits including motility, biofilm formation, cell shape and production of iron acquisition elements. The mutant strain showed marked defects in replicon partitioning and was more efficient at acquiring foreign DNA, illustrating the importance of methylation in maintaining genome integrity. Interestingly, we found that one methylase, part of an integrated bacteriophage genome, was essential for maintaining the phage in a dormant state.

ORGANISM(S): Burkholderia cenocepacia H111

PROVIDER: GSE147038 | GEO | 2021/06/01

REPOSITORIES: GEO

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Project description:Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: methylcytosine has been detected at very low levels in early embryos, but the genomic location and function of methylation has not been established. We have mapped cytosine methylation genomewide in Stage 5 Drosophila embryo DNA by combining immuno-enrichment for 5-methylcytosine, bisulfite conversion, and deep sequencing. Unlike methylation patterns observed in other eukaryotic species, methylation in Drosophila is punctate and highly strand-asymmetrical; we confirmed this by direct PCR amplification and sequencing of bisulfite-converted DNA. Despite the locally asymmetric nature of methylation, large-scale patterns of methylation are symmetric. Methylated regions make up ~1% of the genome, and within these regions methylation of individual cytosines averages 2-10%. Methylation is concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. It is depleted from promoters, coding sequences, and most retrotransposons, and enriched in introns and in certain simple sequence repeats containing the commonly methylated motifs. Comparison with available gene expression data indicates that methylation in a gene is associated with lower expression; the X chromosome, which is subject to gene dosage compensation, is more densely methylated than the autosomes. This study firmly establishes the presence of cytosine methylation in Drosophila; the temporal overlap of methylation with the maternal-zygotic transition raises the possibility that methylation participates in the transition to zygotic gene expression. To enrich for rare cytosine methylation in Drosophila at embryonic Stage 5 (2-3 hours post-fertilization), we enriched sonicated Stage 5 genomic DNA for methylcytosine by immunoprecipitation with antibody to 5-methylcytosine. The immunoprecipitated DNA was then bisulfite converted and Illumina sequenced to obtain direct evidence for the presence of methylation. The presence and extent of DNA methylation was confirmed by Illumina sequencing of bisulfite-converted PCR amplicons.

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An investigation of Burkholderia cepacia complex methylomes via SMRT sequencing and mutant analysis

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