Generation of enzymatically inactive Wzk variant.

<p>(A) Expression of different <i>H</i>. <i>pylori</i> Wzk variants was induced by IPTG in <i>E</i>. <i>coli</i> DH5α for 4 h. Cell lysates from normalized cultures were separated on SDS-PAGE, followed by immunoblotting to detect the expression levels of Wzk mutants relative to wild type enzyme. Among the different Wzk variants, WzkE525A displayed expression levels comparable to that of wild type. (B) WzkE525A flippase activity is diminished <i>in vivo</i>. Both AcrA from <i>C</i>. <i>jejuni</i> and the accessory gene cluster required for its <i>N</i>-glycosylation were reconstituted in <i>E</i>. <i>coli</i>, with the exception of the native flippase. Flippase activity of Wzk and its E525A variant was tested by monitoring the glycosylation levels of <i>C</i>. <i>jejuni</i> AcrA via immunoblotting. Monoclonal anti-histidine was used to detect the expression of histidine-tagged AcrA (green), while the <i>C</i>. <i>jejuni</i> glycan was detected by the rabbit polyclonal anti-<i>C</i>. <i>jejuni</i> glycan (red). The glycosylated form of AcrA (G) is marked by the colocalization of both signals (yellow). Unglycosylated form of AcrA is marked U.</p

Wzk can substitute for MurJ in <i>E</i>. <i>coli</i>.

<p>(A) Summary of complementation experiments. The loss of <i>murJ</i> in <i>E</i>. <i>coli</i> can only be accomplished with a plasmid encoding wild-type <i>wzk</i>. The <i>wzkE525A</i> allele, which carries a mutation in the Walker B motif that is required for ATPase activity, cannot complement a Δ<i>murJ</i> allele. (B) Growth of <i>E</i>. <i>coli</i> strain NR3647 [MG1655 Δ<i>lacIZYA</i>::<i>FRT</i> Δ<i>murJ</i>::<i>kan</i> (pIH23)] in LB broth supplemented with 40 μM IPTG as determined by OD₆₀₀. The data represents the average and standard deviation of six independent cultures. (C) <i>E</i>. <i>coli</i> strain NR2920 (MG1655 Δ<i>lacIYZA</i>::<i>FRT</i> Δ<i>murJ</i>::<i>kan</i> (pRC7KanMurJ, pIH23) expressing both <i>murJ</i> and <i>wzk</i> exhibits the rod-shape cellular morphology typical of wild-type <i>E</i>. <i>coli</i> cells (MurJ⁺ Wzk⁺ panel) under a 100X phase-contrast objective. Δ<i>murJ</i> cells complemented with <i>wzk</i> (Wzk⁺ panels) exhibit morphology characteristic of cells with PG defects: larger size, aberrant morphology, and lysis (marked with white arrow heads).</p

MurJ and Wzk translocate Und-PP linked saccharides across the cytoplasmic membrane.

<p>Nucleotide-linked sugars (not shown) are used to build the PG precursor lipid II and the O-antigen precursor Und-PP-O-antigen on the membrane-bound lipid carrier Und-P. The first Und-P glycosyltransferase in each respective pathway are MraY (not shown) and WecA. After each Und-PP-linked intermediate is synthesized in the inner leaflet of the cytoplasmic membrane, flippases MurJ and Wzk translocate them across the bilayer. At the periplasmic leaflet of the cytoplasmic membrane, the disaccharide-pentapeptide component of lipid II is used to build glycan chains that are crosslinked into the preexisting PG matrix, while the O polysaccharide portion of Und-PP-O-antigen is transferred onto LPS molecules by the WaaL ligase (not shown). After these steps, the lipid carrier is recycled (green dotted arrows). The O polysaccharide is composed of <i>N</i>-acetyl glucosamine (GlcNAc, grey hexagon labeled G), galactose (green squares), and fucose (orange triangles); the disaccharide in lipid II is composed of <i>N</i>-acetyl muramic acid (MurNAc, black hexagon labeled M) and GlcNAc (grey hexagon labeled G), while the pentapeptide is composed of L-Ala (light blue circle), D-Glu (green circle), <i>meso</i>-2,6-diaminopimelic acid (<i>meso</i>-A₂pm, red circle) and two D-Ala (dark blue circle) residues.</p

Participant Demographics.

<p>^a LG = low grade HG = high grade</p><p>^b Denotes samples collected at a second time point (P2 [t = 2 years]; P7 [t = 4 months]; P9 [t = 3 years])</p><p>^c Denotes <i>H</i>. <i>pylori</i>-positive participant</p><p>Participant Demographics.</p

Upper gastrointestinal microbiome similarity with replicate sampling.

<p>(A–C) Species/genera-level profiles of microbiota detected by 454 sequencing in squamous esophagus, Barrett’s esophagus, stomach corpus and stomach antrum of individuals P7 at the time of first [t = 0] and second sample collection [t = 4 months] (A), P2 at t = 0 and t = 2 years (B) and P9 at t = 0 and t = 3 years (C). Individual species/genera are presented according to coloring scheme described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129055#pone.0129055.g002" target="_blank">Fig 2</a> (D) Phylogenetic KR distance between (inter) samples from participants P2, P7 and P9 at both time points and within those individuals comparing the 1^st and 2^nd time points from the indicated anatomic site. The central line within each box represents the median and the whiskers represent the minimum and maximum values.</p

Members of the <i>Firmicutes</i> or <i>Bacteroidetes</i> phyla dominate the upper gastrointestinal tract microbiome.

<p>(A) Phylogenetic relationship of the top 45 OTUs recovered from each of the four sites sampled in individual participants. Respective phyla are noted above main branches of the phylogenetic tree. Numbers in parentheses represent total number of pyrosequencing reads recovered for a given species or genera across all samples followed by the fraction of participants in whom a relative abundance of ≥1.3% of a given species or genera were detected. (B) Species/genera-level profiles of top 45 OTUs detected by 454 sequencing in squamous esophagus, Barrett’s esophagus, stomach corpus and stomach antrum of indicated participants. Data arranged in order of increasing <i>Firmicutes</i> dominance. Individual species/genera are color-coded according to scheme presented in (A). Sequencing reads from brush samples were used when available, otherwise, data from biopsy samples are shown. Species reads were normalized to the total number of reads per corresponding site in a given individual. ^†Denotes samples collected at a second time point (P2 [t = 4 months]; P7 [t = 2 years]; P9 [t = 3 years]); <i>Hp+</i> indicates <i>H</i>. <i>pylori</i>-positive individual. Italicized participant IDs denote data from biopsy samples in cases where brush samples were not available for analysis.</p

<i>Streptococcus</i> to <i>Prevotella</i> species ratio corresponds to phylogenetic distance sample clustering and correlates with Barrett’s esophagus risk factors.

<p>(A) Cluster analysis of KR distances between microbial communities of individual study samples. Pyroseq. <i>Strep</i>:<i>Prev</i> ratio was calculated using relative abundance of mapped reads for all <i>Streptococcus</i> and <i>Prevotella</i> species as determined by pyrosequencing. ddPCR <i>Strep</i>:<i>Prev</i> ratio was calculated using copies/μl of a <i>Streptococcus</i> or <i>Prevotella</i>-specific 16s rRNA gene segment as determined by droplet digital PCR. Samples color-coded based on the majority of calculated Pyroseq. <i>Strep</i>:<i>Prev</i> ratios in a group being <0.5 (blue), 0.5–1.5 (green), 1.5–4.0 (magenta) or >4.0 (red). (B) Boxplots comparing <i>Streptococcus</i> to <i>Prevotella</i> ratio as determined by pyrosequencing and ddPCR. The central line within each box represents the median of the data, the whiskers represent the 5^th and 95^th percentiles and data outside that range are plotted as individual points. (C) Relationship of <i>Streptococcus</i> to <i>Prevotella</i> ratio (measured by ddPCR) and waist-hip ratio of all male participants segregated by anatomic site. Strength of association between these two variables was determined by Pearson’s correlation test with correlation coefficient squared (r²) values as indicated. (D) Relationship of <i>Streptococcus</i> to <i>Prevotella</i> ratios (measured by ddPCR) and hiatal hernia length in all participants segregated by anatomic site. Strength of association tween these two variables was determined by Pearson’s correlation test with correlation coefficient squared (r²) values as indicated.</p

Brush sampling of the upper gastrointestinal tract enriches for bacterial abundance and diversity.

<p>(A) Diagram of the human upper gastrointestinal tract indicating regions sampled via biopsy or brush collection method. Anatomic sites were abbreviated with the first and second letter indicating the sampled organ and intra-organ tissue, respectively (ES—squamous esophagus; EB—Barrett’s esophagus; SC—stomach corpus; SA—stomach antrum). (B) Total bacterial versus human DNA recovered from biopsy or brush samples segregated by anatomical site as measured by qPCR and plotted as copy number of bacterial 16S rRNA gene and human 18S rRNA gene. Error bars indicate standard deviation from the mean. (C) Ratio of human 18S rRNA to bacterial 16S rRNA copy number in all biopsy (n = 26) or brush (n = 35) samples. Error bars indicate standard deviation from the mean. (D) Species diversity in biopsy and brush samples as measured by quadratic entropy analysis. The central line within each box represents the median of the data, the whiskers represent the 5^th and 95^th percentiles and data outside that range are plotted as individual points. Statistical difference between biopsy and brush samples was measured by Wilcoxon rank sum test with continuity correction (p = 0.000594).</p