List of putative proteins encoded on SMG 6 fosmid insert.

<p><b>Abbreviations:</b> aa = amino acid; % ID = % identity at amino acid level over the entire length of the protein; % G+C = Percentage guanine and cytosine content;</p><p>DUF = Domain of Unknown Function.</p

Growth of <i>E. coli</i> MKH13::pCI372 and <i>E. coli</i> MKH13 carrying a plasmid encoded copy of either <i>brpA_L</i>, <i>brpA_S</i> or <i>brpAatfA</i> in (A) LB broth or (B) LB+3% NaCl.

<p>All of the genes confer a significant salt tolerance phenotype to MKH13 relative to cells with an empty plasmid vector. All values are the average of triplicate experiments and error bars are representative of the standard error of the mean (SEM).</p

Overview of SMG 6 fosmid insert and features of specific genes.

<p>(<b>A</b>) Gene map of SMG 6 insert, displaying gene orientation and individual %G+C content indicated with a gradient colour bar. Gene numbers correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103318#pone-0103318-t002" target="_blank">Table 2</a> and are drawn approximately to scale. (<b>B</b>) Focus on genes 25 (<i>atfA</i>) and 26 (<i>brpA</i>), showing the regions cloned for each construct. (<b>C</b>) Detailed view of putative ATG and TTG start codons of <i>brpA</i>, including upstream regions, as well as predicted promoter regions (highlighted in bold) and transcription factor binding site sequences (blue and orange boxes). (<b>D</b>) TMHMM prediction of seven transmembrane regions in BrpA.</p

EZTn<i>5</i> transposon mutagenesis of SMG 6 was performed to identify mutants lacking pigmentation when grown in the presence of β-carotene.

<p>(<b>A</b>) Clones positive for a transposon in this region of SMG 6 fosmid insert were identified by PCR, with amplicons of ∼3.3 kb indicative of an insertion event. (<b>B</b>) Approximate locations of transposon insertions in relation to <i>brpA</i> and neighbouring genes. <b>(C)</b> Appearance of cell pellets of SMG 6 and transposon insertion mutants (EZTn #24, #26, #34 and #38) following growth in the presence of β-carotene.</p

Putative virulence associated genes in <i>C. ureolyticus</i> DSM 20703.

<p>Putative virulence associated genes in <i>C. ureolyticus</i> DSM 20703.</p

BrpA homologues identified when BLAST searched against Human Microbiome Project (HMP) datasets from 17 body sites at maximum e-value cut-off of (A) 1e⁻⁵⁰ and (B) 1e⁻⁰⁵.

<p>BrpA homologues identified when BLAST searched against Human Microbiome Project (HMP) datasets from 17 body sites at maximum e-value cut-off of (A) 1e⁻⁵⁰ and (B) 1e⁻⁰⁵.</p

Growth of metagenomic clones SMG 1, 6 and 52 compared to EPI300 carrying an empty fosmid vector (pCC1FOS) in (A) LB broth and (B) LB broth supplemented with 7% NaCl.

<p>Growth of metagenomic clones SMG 1, 6 and 52 compared to EPI300 carrying an empty fosmid vector (pCC1FOS) in (A) LB broth and (B) LB broth supplemented with 7% NaCl.</p

Possible mechanism(s) of action of BrpA (A) Representation of the known reaction for the formation of retinal.

<p>B-carotene is cleaved at its central 15,15′ bond by <i>brp</i> 15,15′- β-carotene monooxygenase to form two molecules of <i>­all-trans</i> retinal (Vitamin A aldehyde). We propose that <i>brpA</i> may be regulated from two promoters, with translation being initiated from one of two potential start codons (ATG and TTG), depending on environmental conditions. While speculative, we illustrate some possibilities discussed in the text. (<b>B</b>) Pigmentation phenotype: regulation of <i>brpA</i> from promoter 1 (upstream of ATG start codon) under “normal” cellular conditions, or possibly by β-carotene, could result in (<b>B1</b>) BrpA adding an acyl group to β-carotene, allowing it to interact with phosphate head groups of lipids and anchoring it in the hydrophobic core of the lipid membrane or (<b>B2</b>) BrpA may cleave β-carotene to retinal and subsequently bind the derived retinal anchoring it in the cell membrane. (<b>C</b>) Stress response: regulation of <i>brpA</i> from promoter 2 (upstream of TTG start codon), may be initiated by environmental signals such as changes in external osmolarity, resulting in increased tolerance or resistance to environmental stress, such as increased NaCl concentrations by an as yet unknown mechanism. Alternative start codons, such as TTG, have been found in a number of stress response genes.</p

Genomic Investigation into Strain Heterogeneity and Pathogenic Potential of the Emerging Gastrointestinal Pathogen <i>Campylobacter ureolyticus</i>

<div><p>The recent detection and isolation of <i>C. ureolyticus</i> from patients with diarrhoeal illness and inflammatory bowel diseases warrants further investigation into its role as an emerging pathogen of the human gastrointestinal tract. Regarding the pathogenic mechanisms employed by this species we provide the first whole genome analysis of two <i>C. ureolyticus</i> isolates including the type strain. Comparative analysis, subtractive hybridisation and gene ontology searches against other <i>Campylobacter</i> species identifies the high degree of heterogenicity between <i>C. ureolyticus</i> isolates, in addition to the identification of 106 putative virulence associated factors, 52 of which are predicted to be secreted. Such factors encompass each of the known virulence tactics of pathogenic <i>Campylobacter</i> spp. including adhesion and colonisation (CadF, PEB1, IcmF and FlpA), invasion (<i>ciaB</i> and 16 <i>virB-virD4</i> genes) and toxin production (S-layer RTX and ZOT). Herein, we provide the first virulence catalogue for <i>C. ureolyticus</i>, the components of which theoretically provide this emerging species with sufficient arsenal to establish pathology.</p></div

Evolutionary relationships of taxa Zona Occluden Toxin (ZOT).

<p>The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. DSMZ 1 and DSMZ 2 correspond to the ZOT paralogs in <i>C. ureolyticus</i> DSM20703. ACS-301-V-Sch-3b:<i>C. ureolyticus</i> ACS-301-V-Sch-3b.</p