Deciphering bacterial epigenomes using modern sequencing technologies

Abstract

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.

Figure 1:. DNA methylation in bacteria as a mechanism of phenotypic plasticity.

Chemical structures of the most common forms of DNA methylation in bacteria, including (a) 5-methylcytosine, (b) N6-methyladenine, and (c) N4-methylcytosine. (d) Methylome characterization is increasingly becoming a standard component of bacterial genomic research. The detection of methylated positions can lead to the identification of precise methylated sequence motifs. A methylated motif can then be assigned to the responsible MTase based on either querying a database of MTases with known target motifs or through experimental means, involving comparisons with strains where the MTase is inactivated (Box 2). Multiple lines of functional investigation can lead from this basic characterization of the primary features of a bacterial methylome.

Figure 2:. Technologies for detection of DNA methylation through direct sequencing of native DNA molecules.

(a-c) SMRT sequencing. (d-f) ONT sequencing. (a) Sequencing libraries for single-molecule, real-time (SMRT) sequencing from Pacific Biosciences libraries consist of double-stranded DNA fragments flanked by the hairpin SMRTbell adapters that permit the polymerase to process through both strands of the template. The libraries can take on various configurations depending upon the requirements of the application. Short insert libraries generate multiple subreads from both strands of the template molecule (useful for generating higher accuracy consensus subreads), while long insert libraries are used to generate the longest subread lengths (critical for de novo assembly and detection of structural variants). (b) SMRT sequencing relies on a sequencing-by-synthesis approach. A DNA polymerase is bound within a zeptoliter-scale observation chamber (called a zero-mode waveguide, or ZMW) and uses a strand from the native sequencing library as a template for the read, incorporating fluorescently labeled deoxyribonucleoside triphosphates (dNTPs) as they diffuse into the ZMW. Each incorporated dNTP is briefly immobilized at the polymerase active site, emitting a fluorescent pulse in the corresponding color channel. (c) When observing the fluorescent traces produced by each ZMW, which are highly multiplexed on a chip, the order of pulses provides the read sequence, while pauses between pulses indicate the presence of a covalent modification in the template DNA. (d) The 1D library preparation from Oxford Nanopore Technologies (ONT) use a lead adapter (loaded with a motor protein) and a tethering adapter, which helps co-locate the molecule near the nanopore, to enable the sequencing of a single DNA strand from the molecule. (e) ONT sequencing instruments that rely on engineered biological nanopores embedded in a lipid membrane to sequence ssDNA. While a voltage potential is applied across the membrane, ssDNA is ratcheted through the nanopore by a motor protein bound to the DNA library molecule (f). The ionic current flowing through the nanopore depends on the precise set of nucleotides (k = 4 or 5) occupying the constriction point. Methylated nucleotides in the ssDNA introduce distinct current patterns, making it possible to distinguish modified bases relative to amplified (methylation-free) DNA or precomputed models.

Figure 3:. Epigenetic mechanisms of gene regulation and its consequences.

(a) Transcription of the agn43 gene and pap gene cluster in E. coli serve as canonical examples of gene expression being regulated according to the methylation status at motif sites within its upstream regulatory sequence. The presence of methylated bases in this region can interfere with the binding of regulatory proteins, leading to either up- or down-regulation of the gene. For instance, methylation can prevent a transcription factor (TF) from binding to its transcription factor binding site (TFBS), thereby preventing transcription of the downstream gene. (b) If the gene affected by methylation status encodes a transcription factor, or another protein with promiscuous DNA-binding specificity, the local methylation status can potentially trigger a cascade of downstream changes on gene expression. (c) Some bacteria are capable of inducing genome-wide changes in methylation status and gene expression through phase- variable MTases. Spontaneous and reversible frameshift mutations in the MTase gene lead to a clonally expanded bacterial population with divergent methylation activity and distinct gene expression regimes. (d) DNA methylation is likely to be involved in alternative mechanisms of gene regulation. For example, methylation is known to affect the curvature of DNA molecules, which could potentially control which regions of a chromosome are exposed to the transcriptional machinery of the cell. (e) The presence of phase-variable methyltransferases can introduce heterogeneous methylation patterns in a clonally expanded bacterial population, leading to subpopulations with distinct gene expression regimes and phenotypes.