The genome of the food-borne pathogen Campylobacter jejuni contains multiple highly mutable sites, or contingency loci. It has been suggested that standing variation at these loci is a mechanism for rapid adaptation to a novel environment, but this phenomenon has not been shown experimentally. In previous work we showed that the virulence of C. jejuni NCTC11168 increased after serial passage through a C57BL/6 IL-10-/- mouse model of campylobacteriosis. Here we sought to determine the genetic basis of this adaptation during passage. Re-sequencing of the 1.64Mb genome to 200-500X coverage allowed us to define variation in 23 contingency loci to an unprecedented depth both before and after in vivo adaptation. Mutations in the mouse-adapted C. jejuni were largely restricted to the homopolymeric tracts of thirteen contingency loci. These changes cause significant alterations in open reading frames of genes in surface structure biosynthesis loci and in genes with only putative functions. Several loci with open reading frame changes also had altered transcript abundance. The increase in specific phases of contingency loci during in vivo passage of C. jejuni, coupled with the observed virulence increase and the lack of other types of genetic changes, is the first experimental evidence that these variable regions play a significant role in C. jejuni adaptation and virulence in a novel host.

BackgroundChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo “library preparation” to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing.ResultsIn this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a “gold standard” reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling.ConclusionsWe identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3135-y) contains supplementary material, which is available to authorized users.

Human Campylobacter jejuni infection can result in an asymptomatic carrier state, watery or bloody diarrhea, bacteremia, meningitis, or autoimmune neurological sequelae. Infection outcomes of C57BL/6 IL-10−/− mice orally infected with twenty-two phylogenetically diverse C. jejuni sstrains were evaluated to correlate colonization and disease phenotypes with genetic composition of the strains. Variation between strains was observed in colonization, timing of development of clinical signs, and occurrence of enteric lesions. Five pathotypes of C. jejuni in C57BL/6 IL-10−/− mice were delineated: little or no colonization, colonization without disease, colonization with enteritis, colonization with hemorrhagic enteritis, and colonization with neurological signs with or without enteritis. Virulence gene content of ten sequenced strains was compared in silico; virulence gene content of twelve additional strains was compared using a C. jejuni pan-genome microarray. Neither total nor virulence gene content predicted pathotype; nor was pathotype correlated with multilocus sequence type. Each strain was unique with regard to absences of known virulence-related loci and/or possession of point mutations and indels, including phase variation, in virulence-related genes. An experiment in C. jejuni 11168-infected germ-free mice showed that expression levels of ninety open reading frames (ORFs) were significantly up- or down-regulated in the mouse cecum at least two-fold compared to in vitro growth. Genomic content of these ninety C. jejuni 11168 ORFs was significantly correlated with the capacity to colonize and cause enteritis in C57BL/6 IL-10−/− mice. Differences in gene expression levels and patterns are thus an important determinant of pathotype in C. jejuni strains in this mouse model.

Evolution experiments in the laboratory have focused heavily on model organisms, often to the exclusion of clinically relevant pathogens. The foodborne bacterial pathogen Campylobacter jejuni belongs to a genus whose genomes are small compared to those of its closest genomic relative, the free-living genus Sulfurospirillum, suggesting genome reduction during the course of evolution to host association. In an in vitro experiment, C. jejuni serially passaged in rich medium in the laboratory exhibited loss of flagellar motility-an essential function for host colonization. At early time points the motility defect was often reversible, but after 35 days of serial culture, motility was irreversibly lost in most cells in 5 independently evolved populations. Population re-sequencing revealed disruptive mutations to genes in the flagellar transcriptional cascade, rpoN (σ54)-therefore disrupting the expression of the genes σ54 regulates-coupled with deletion of rpoN in all evolved lines. Additional mutations were detected in virulencerelated loci. In separate in vivo experiments, we demonstrate that a phase variable (reversible) motility mutant carrying an adenine deletion within a homopolymeric tract resulting in truncation of the flagellar biosynthesis gene fliR was deficient for colonization in a C57BL/6 IL-10 −/− mouse disease model. Re-insertion of an adenine residue partially restored motility and ability to colonize mice. Thus, a pathogenic C. jejuni strain was rapidly attenuated by experimental laboratory evolution and demonstrated genomic instability during this evolutionary process. The changes observed suggest C. jejuni is able to evolve in a novel environment through genome reduction as well as transition, transversion, and slip-strand mutations.