We examined two variants of the genome-sequenced strain, Campylobacter jejuni NCTC11168, which show marked differences in their virulence properties including colonization of poultry, invasion of Caco-2 cells, and motility. Transcript profiles obtained from whole genome DNA microarrays and proteome analyses demonstrated that these differences are reflected in late flagellar structural components and in virulence factors including those involved in flagellar glycosylation and cytolethal distending toxin production. We identified putative 28 and 54 promoters for many of the affected genes and found that greater differences in expression were observed for 28 -controlled genes. Inactivation of the gene encoding 28 , fliA, resulted in an unexpected increase in transcripts with 54 promoters, as well as decreased transcription of 28 -regulated genes. This was unlike the transcription profile observed for the attenuated C. jejuni variant, suggesting that the reduced virulence of this organism was not entirely due to impaired function of 28 . However, inactivation of flhA, an important component of the flagellar export apparatus, resulted in expression patterns similar to that of the attenuated variant. These findings indicate that the flagellar regulatory system plays an important role in campylobacter pathogenesis and that flhA is a key element involved in the coordinate regulation of late flagellar genes and of virulence factors in C. jejuni.

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. Clostridium difficile is a Gram-positive spore-forming bacillus which is recognized to be the major cause of nosocomial diarrhea associated with antibiotic therapy (35). The incidence of C. difficile infection has been rapidly increasing in both Europe and North America, and this increase in infections has been associated with a significantly high mortality rate (2, 35). The broad spectrum of diseases caused by C. difficile, which range from antibiotic-associated diarrhea to the potentially lethal, pseudomembranous colitis, has been shown to depend on the level of toxin produced (1), and this production is recognized as a critical determinant of pathogenicity. Following antibiotic therapy when the microbiota of the gastrointestinal tract is disrupted, infection by C. difficile is mediated by spores which germinate in the gut, followed by vegetative cell proliferation and the subsequent secretion of the two major virulence factors, the Rho glucosylating toxins TcdA and TcdB.The toxin-encoding genes (tcdA and tcdB) are localized to a 19.6-kb pathogenicity locus (PaLoc) which includes three other accessory genes, tcdR, tcdE and tcdC (43). TcdR is an alternative sigma factor required for transcription of the two toxin genes; TcdE has been described to be a putative holin-like protein involved in toxin secretion, although this role has recently been a source of debate (17, 32); and TcdC is an anti-sigma factor that negatively regulates tcdR-dependent transcription (28, 29). In addition, four other regulators of toxin synthesis have recently been identified: CepA (3), CodY ...

We have used comparative genomic hybridization (CGH) on a full-genome Campylobacter jejuni microarray to examine genome-wide gene conservation patterns among 51 strains isolated from food and clinical sources. These data have been integrated with data from three previous C. jejuni CGH studies to perform a metaanalysis that included 97 strains from the four separate data sets. Although many genes were found to be divergent across multiple strains (n ‫؍‬ 350), many genes (n ‫؍‬ 249) were uniquely variable in single strains. Thus, the strains in each data set comprise strains with a unique genetic diversity not found in the strains in the other data sets. Despite the large increase in the collective number of variable C. jejuni genes (n ‫؍‬ 599) found in the meta-analysis data set, nearly half of these (n ‫؍‬ 276) mapped to previously defined variable loci, and it therefore appears that large regions of the C. jejuni genome are genetically stable. A detailed analysis of the microarray data revealed that divergent genes could be differentiated on the basis of the amplitudes of their differential microarray signals. Of 599 variable genes, 122 could be classified as highly divergent on the basis of CGH data. Nearly all highly divergent genes (117 of 122) had divergent neighbors and showed high levels of intraspecies variability. The approach outlined here has enabled us to distinguish global trends of gene conservation in C. jejuni and has enabled us to define this group of genes as a robust set of variable markers that can become the cornerstone of a new generation of genotyping methods that use genome-wide C. jejuni gene variability data.Campylobacter jejuni is a human pathogen, a commensal inhabitant of many domestic animals, and globally, the most common cause of acute bacterial enteritis (for a review, see reference 31). Two well-established serotyping methods, namely, Penner typing based on heat-stable antigens and Lior typing based on heat-labile antigens, have been in use for more than two decades to study species diversity, to track epidemiological trends, and to determine important epidemiological correlations (15,23). Technical limitations on the production of high-quality typing sera have limited the availability of these reagents. Culturing conditions can affect the expression of serotyping determinants, which affects serotyping results, and several strains are nontypeable (32). Additionally, serotype relatedness is not always indicative of genetic relatedness since members of different serotypes of C. jejuni are genetically related, despite differences in heat-stable antigen expression (16).The need for alternative subtyping schemes has been recognized, leading to the development of a number of different methods based on differences at the DNA level (i.e., genotyping). The techniques used at present range from analysis of polymorphisms in groups of housekeeping genes (multilocus sequence typing [5,26]), amplified fragment length polymorphism analysis (28), restriction fragment length polymorphism analysi...

BackgroundThe aim of this study was to characterize the genomes of 30 Listeria monocytogenes isolates collected at a pig slaughterhouse to determine the molecular basis for their persistence.ResultsComparison of the 30 L. monocytogenes genomes showed that successive isolates (i.e., persistent types) recovered from thew sampling site could be linked on the basis of single nucleotide variants confined to prophage regions. In addition, our study revealed the presence among these strains of the bcrABC cassette which is known to produce efflux pump-mediated benzalkonium chloride resistance, and which may account for the persistence of these isolates in the slaughterhouse environment. The presence of the bcrABC cassette was confirmed by WGS and PCR and the resistance phenotype was determined by measuring minimum inhibitory concentrations. Furthermore, the BC-resistant strains were found to produce lower amounts of biofilm in the presence of sublethal concentrations of BC.ConclusionsHigh resolution SNP-based typing and determination of the bcrABC cassette may provide a means of distinguishing between resident and sporadic L. monocytogenes isolates, and this in turn will support better management of this pathogen in the food industry.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1363-9) contains supplementary material, which is available to authorized users.

Whole-genome sequencing (WGS) of bacterial pathogens is currently widely used to support public-health investigations. The ability to assess WGS data quality is critical to underpin the reliability of downstream analyses. Sequence contamination is a quality issue that could potentially impact WGS-based findings; however, existing tools do not readily identify contamination from closely-related organisms. To address this gap, we have developed a computational pipeline, ConFindr, for detection of intraspecies contamination. ConFindr determines the presence of contaminating sequences based on the identification of multiple alleles of core, single-copy, ribosomal-protein genes in raw sequencing reads. The performance of this tool was assessed using simulated and lab-generated Illumina short-read WGS data with varying levels of contamination (0–20% of reads) and varying genetic distance between the designated target and contaminant strains. Intraspecies and cross-species contamination was reliably detected in datasets containing 5% or more reads from a second, unrelated strain. ConFindr detected intraspecies contamination with higher sensitivity than existing tools, while also being able to automatically detect cross-species contamination with similar sensitivity. The implementation of ConFindr in quality-control pipelines will help to improve the reliability of WGS databases as well as the accuracy of downstream analyses. ConFindr is written in Python, and is freely available under the MIT License at github.com/OLC-Bioinformatics/ConFindr.

Although Campylobacter jejuni is a leading cause of food-borne illness, little is known about the mechanisms by which this pathogen mediates prolonged environmental survival or host cell virulence. Although these behaviours represent distinct phenotypes, they share a common requirement of an immobilized state. In order to understand the cellular mechanisms that facilitate a surface-associated lifestyle, transcriptional and translational expression profiles were determined for sessile and planktonic C. jejuni. These investigations indicate that the immobilized bacteria undergo a shift in cellular priorities away from metabolic, motility and protein synthesis capabilities towards emphasis on iron uptake, oxidative stress defence and membrane transport. This pattern of expression partially overlaps those reported for Campylobacter during host colonization, as well as for other species of bacteria involved in biofilms, highlighting common adaptive responses to the conserved challenges within each of these phenotypes. The adaptation of Campylobacter to immobilized growth may represent a quasi-differentiated state that functions as a foundation for further specialization towards phenotypes such as biofilm formation or host cell virulence.

No abstract

The 3' regions of several group II introns within the mitochondrial genes nad1 and nad7 show unexpected sequence divergence among flowering plants, and the core domains 5 and 6 are predicted to have weaker helical structure than those in self-splicing group II introns. To assess whether RNA editing improves helical stability by the conversion of A-C mispairs to A-U pairs, we sequenced RT-PCR amplification products derived from excised intron RNAs or partially spliced precursors. Only in some cases was editing observed to strengthen the predicted helices. Moreover, the editing status within nad1 intron 1 and nad7 intron 4 was seen to differ among plant species, so that homologous intron sequences shared lower similarity at the RNA level than at the DNA level. Plant-specific variation was also seen in the length of the linker joining domains 5 and 6 of nad7 intron 3; it ranged from 4 nt in wheat to 11 nt in soybean, in contrast to the 2-4 nt length typical of classical group II introns. However, this intron is excised as a lariat structure with a domain 6 branchpoint adenosine. Our observations suggest that the core structures and sequences of these plant mitochondrial introns are subject to less stringent evolutionary constraints than conventional group II introns.