Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5′CCCGA and 5′CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits

Universal Statistical Behavior of Neural Spike Trains

We construct a model that predicts the statistical properties of spike trains generated by a sensory neuron. The model describes the combined effects of the neuron's intrinsic properties, the noise in the surrounding, and the external driving stimulus. We show that the spike trains exhibit universal statistical behavior over short times, modulated by a strongly stimulus-dependent behavior over long times. These predictions are confirmed in experiments on H1, a motion-sensitive neuron in the fly visual system.Comment: 7 pages, 4 figure

High throughput method for analysis of repeat number for 28 phase variable loci of C. jejuni strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method for rapidly determining ON/OFF states of these 28 phase-variable loci in a large number of individual colonies. The method combines a series of multiplex PCR assays with a GeneScan assay and automated extraction of tract length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. An important output of this assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species

Nonselective Bottlenecks Control the Divergence and Diversification of Phase-Variable Bacterial Populations

Phase variation occurs in many pathogenic and commensal bacteria and is a major generator of genetic variability. A putative advantage of phase variation is to counter reductions in variability imposed by nonselective bottlenecks during transmission. Genomes of Campylobacter jejuni, a widespread food-borne pathogen, contain multiple phase-variable loci whose rapid, stochastic variation is generated by hypermutable simple sequence repeat tracts. These loci can occupy a vast number of combinatorial expression states (phasotypes) enabling populations to rapidly access phenotypic diversity. The imposition of nonselective bottlenecks can perturb the relative frequencies of phasotypes, changing both within-population diversity and divergence from the initial population. Using both in vitro testing of C. jejuni populations and a simple stochastic simulation of phasotype change, we observed that single-cell bottlenecks produce output populations of low diversity but with bimodal patterns of either high or low divergence. Conversely, large bottlenecks allow divergence only by accumulation of diversity, while interpolation between these extremes is observed in intermediary bottlenecks. These patterns are sensitive to the genetic diversity of initial populations but stable over a range of mutation rates and number of loci. The qualitative similarities of experimental and in silico modeling indicate that the observed patterns are robust and applicable to other systems where localized hypermutation is a defining feature. We conclude that while phase variation will maintain bacterial population diversity in the face of intermediate bottlenecks, narrow transmission-associated bottlenecks could produce host-to-host variation in bacterial phenotypes and hence stochastic variation in colonization and disease outcomes

Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage

Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, “phasotypes,” was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence

Genomics and population dynamics of phase variable genes in Campylobacter

Phase variation is a feature of many pathogenic bacteria, including Campylobacter jejuni – a leading cause of food-borne gastroenteritis. C. jejuni has many phase variable (PV) genes which exhibit high frequency, stochastic, reversible switching between ON and OFF expression states. This results from instability in simple sequence repeats (SSRs). This study is the first to conduct a widescale survey of the Campylobacter ‘phasome’ (the set of PV genes present in a genome). A new program, PhasomeIt, was developed to identify and compare all SSR-mediated phase variable genes in 77 complete Campylobacter genomes. Surprisingly, there are a large number of rare PV genes with few, or no, homologues in other genomes. These exist alongside a “core phasome” of PV genes associated with particular species. This suggests a significant role for phasome diversity in Campylobacter population and disease dynamics. SSR tract length influences rates of PV, however it is not known whether differences in PV rate are biologically significant or whether a threshold effect exists. A cyclical assaywas developed which allows alternation of experimental conditions favouring the ON and OFF states of cj1421c. Bacteriophage F336 selects for the OFF state whilst human serum selects for the ON state, and both produce complete selective sweeps. These agents were combined for a complete ON→OFF→ON cycle. Using an in silico model of this assay it was demonstrated that a broad range of conditions favour PV over non-PV genes whilst variation rate is primarily dependent on duration of selection. Extending this modelling approach to the interaction of non-selective bottlenecks and PV loci indicated that smaller bottlenecks have a disruptive effect on population dynamics whilst larger bottlenecks preserve increased diversity. These differences are capable of producing stochastic differences in output populations that may drive host-to-host diversity and result in rare occurrence of disease sequelae

LangoScoley_Control_Sequence_Files

This folder contains the control sequences for the specific variants used for the analyse