Abstract
The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis.We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes.DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles.Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides.These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.
Highlights
Neisseria: pathogenicity and genomic plasticityNeisseria meningitidis is a commensal Gram-negative bacterium exclusively found in the human nasopharyngeal mucosa and is readily transmitted via respiratory secretions or saliva [1]
Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection
We assayed the DNA methylation profiles of 2 N. meningitidis strains, selecting NM1264 and its closest reference strain Z2491 for SMRT sequencing at a coverage for each strand approximating 50x on Tet1 converted genomic DNA samples
Summary
Neisseria: pathogenicity and genomic plasticityNeisseria meningitidis is a commensal Gram-negative bacterium exclusively found in the human nasopharyngeal mucosa and is readily transmitted via respiratory secretions or saliva [1]. A small proportion of individuals colonized by a virulent strain may develop invasive disease including sepsis or meningitis [2], especially devastating as epidemics in the African ‘meningitis belt’ [3]. Genome sequencing of steadily increasing numbers of N. meningitidis strains revealed substantial homologous recombination with DNA cleavage mechanism suggested to be associated with phylogenetic clades [6]. Frequent horizontal transfer of DNA elements as well as a range of genetic variation mechanisms in N. meningitidis including phase variation [7] require extra caution in the interpretation of nucleotide changes. A number of genotypes were suggested to be associated with virulence including genes involved in the synthesis of the polysaccharide capsule. To present no strict pathogenic genotype is defined which would allow to distinguish disease-causing strains from avirulent carrier strains [8]
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