Pneumococcal chromosome conformation variation between epigenetic variants is driven by episomal mobile genetic elements

Streptococcus pneumoniae (pneumococcus) is a genetically diverse opportunistic bacterial pathogen that expresses two phase-variable loci encoding restriction-modification systems. Comparisons of two genetically-distinct pairs of epigenetically-distinct variants, each distinguished by a stabilised arrangement of one of these phase-variable loci, found the consequent changes in genome-wide DNA methylation patterns were associated with differential expression of mobile genetic elements (MGEs). This relationship was hypothesised to be mediated through changes in xenogenic silencing (XS) or nucleoid organisation. Therefore the chromosomal conformation of the both variants of each isolate were characterised using Illumina Hi-C, and Nanopore Pore-C, sequencing. Both methods concurred that the organisation of the pneumococcal chromosome was dominated by small-scale structures, with most pairwise interactions between loci <25 kb apart. Neither found substantial evidence for higher-order structure or XS in the pneumococcal genome, with more complex contact patterns only evident around the replication origin. Comparisons between the variants identified phage-related chromosomal islands (PRCIs) as the foci of differential contact densities between the variants. This was driven by copy number variation, resulting from variable excision and replication of the episomal PRCIs. However, the methods were discordant in their identification of the variant in which the PRCI was more actively replicating in both pairs. Validatory experiments demonstrated that the prevalence of circular PRCIs was not determined by DNA modification, but instead varied stochastically between colonies in both backgrounds, and was metastable during vegetative growth. PRCI excision was inducible by mitomycin C, but independent of the presence of a phage. Yet transcriptional activation of these elements was affected by both signals, indicating transcription and replication are separately regulated. Therefore pneumococcal MGEs do not appear to be subject to XS, resulting in heterogeneity being generated within these bacterial populations through the frequent local disruption of chromosome conformation resulting from the stochastic excision and reintegration of episomal elements. Author summaryThe pneumococcus is a bacterium with a circular chromosome that is organised by DNA-binding proteins and often contains mobile genetic elements (MGEs), genes able to transmit between bacteria. All pneumococci encode defences against MGEs, some of which create epigenetic modifications (typically methylation) genome-wide at particular sequence motifs. Changes in these epigenetic patterns are associated with altered MGE gene expression and replication in otherwise genetically-identical bacteria. To test whether this was the result of methylation remodelling the organisation of the chromosome, we compared the contact patterns across the chromosome using two sequencing technologies. Both methods concurred that the pneumococcal genome is generally folded into small structures, with the biggest differences between the variants caused by the replication of MGEs. However, the methods disagreed on the variant in which the MGEs replicated fastest. Further experiments showed that MGE replication was stable over the course of culturing over hours, but would randomly change level between days, explaining the inconsistent observations. MGE replication was found to rise in response to DNA damage, whereas gene expression also depended on the presence of other signals, explaining the discrepancies in these activities between variants. Hence MGEs significantly contribute to the heterogeneity that rapidly accumulates within pneumococcal populations.