Primers used in this study.

<p>Primers used in this study.</p

Map of the study area.

<p>The top maps in panel (a) show the study area in North Jeolla province, South Korea. Water collection sites (numeric characters) for coastal streams are described in panels (b) and (c). The waste water treatment plant (●, site 9) is shown in the top map. Each water sampling site in the streams was located downstream of drainage for domestic sewage that originated from neighboring dwellings. Circles with dashed lines (- - -) represent temporary locations of dwellings in the vicinity of the streams. The asterisk (*, site 10) in panel (c) indicates the sampling site for wild-growing clams.</p

Occurrence of novel GII.17 and GII.21 norovirus variants in the coastal environment of South Korea in 2015 - Fig 2

<p><b>Neighbor-joining tree based on the partial capsid gene (region C, 303 nt) (a) and partial polymerase gene (ORF1, 745 nt) (b) of norovirus nucleotide sequences from the study area.</b> The norovirus capsid region C nucleotide sequences (a), which were harvested from the study area, are indicated as open circles (○, sequences of the region C amplicons) and closed circles (●, region C sequences of the partial ORF1-ORF2 amplicons). The norovirus partial polymerase (ORF1) nucleotide sequences (b), which were harvested from the study area, are indicated as closed circles (●, ORF1 of the partial ORF1-ORF2 amplicons). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown at the branches (bootstrap values of ≥70 are shown). The sampling site numbers are shown next to each sample label. Each sample label number on a tree indicates the sampling date (e.g., 20150321 = March 21, 2015).</p

Amino acid sequence variation of VP1 among related strains of the GII.17 in South Korea.

<p>VP1 amino acid sequences of GII.17 from the study area were aligned with former related sequences. The VP1 amino acid sequences were aligned using DNAMAN, and heterogeneous sites were exported to a table, which were ordered according to time. Each color represents an amino acid change that occurred between strains or between sub-clusters. An amino acid position coinciding with the putative histo-blood group antigen (HBGA) binding sites of GII.4 pandemic variants (horizontal stripes) are indicated at the bottom of the alignment table. Black regions at bottom of tables indicate the putative B cell epitopes of GII.17. The asterisk (*) indicates commonly changed amino acid sites in the novel strains of GII.17 (cluster III-b) compared with former related strains.</p

Phylogenetic tree of the partial VP1 (ORF2, 1,560 nt) in environmental samples determined by the maximum likelihood method.

<p>The VP1 nucleotide sequences of noroviruses harvested from the study area are indicated as closed squares (■). The sampling site numbers are shown next to each sample label. The percentages of trees in which the associated taxa clustered together are shown next to the branches (bootstrap values of ≥95 are shown). The tree is drawn to scale with branch lengths measured in the number of substitutions per site. Each sample label number on the tree indicates the sampling date (e.g., 20150321 = March 21, 2015). An outgroup reference strain (GI.1; GenBank ID: M87661) is not shown in this figure.</p

HNoV GII genotypes in water and clam samples in 2015.

<p>HNoV GII genotypes in water and clam samples in 2015.</p

Microarray analysis of VtrA and VtrB regulon in <i>V. parahaemolyticus</i>.

a<p>Fold change in gene transcripts between the wild-type and <i>ΔvtrA</i> or <i>ΔvtrB</i> mutant as determined by microarray analysis. Statistically significant changes (≥2-fold difference with <i>P</i><0.05) are highlighted in bold as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008678#s4" target="_blank"><i>Materials and Methods</i></a>.</p

VtrA and VtrB are not necessary for T3SS1-dependent cytotoxicity but necessary for T3SS2-dependent cytotoxicity.

<p>A. <i>vtrA</i> and <i>vtrB</i> are not necessary for T3SS1-dependent cytotoxicity. Caco-2 cells were infected for 6 h with isogenic strains of POR-3 (<i>ΔtdhASΔvcrD2</i>). Bar 1: POR-3 (<i>ΔtdhASΔvcrD2</i>); bar 2: POR-3<i>ΔvtrA</i>; bar 3: POR-3<i>ΔvtrB</i>; bar 4: <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>). Cytotoxicity was evaluated by the amount of LDH released. Error bars represent standard deviations for results from triplicate experiments. B. <i>vtrA</i> and <i>vtrB</i> are essential for T3SS2-dependent cytotoxicity. Caco-2 cells were infected for 6 h with isogenic mutant strains of POR-2 (<i>ΔtdhASΔvcrD1</i>). Bar 1: POR-2 (<i>ΔtdhASΔvcrD1</i>); bar 2: POR-2<i>ΔvtrA</i> (<i>ΔtdhASΔvcrD1ΔvtrA</i>); bar 3: POR-2<i>ΔvtrA</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrA</i>+p<i>vtrA</i>); bar 4: POR-2<i>ΔvtrA</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrA</i>+p<i>vtrB</i>); bar 5: POR-2<i>ΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrB</i>); bar 6; POR-2<i>ΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrB</i>+p<i>vtrA</i>); bar 7: POR-2<i>ΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrB</i>+p<i>vtrB</i>); bar 8: POR-2<i>ΔvtrAΔvtrB</i> (<i>ΔtdhASΔvcrD1ΔvtrAΔvtrB</i>); bar 9: POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrA</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrA</i>); bar 10: POR-2<i>ΔvtrAΔvtrB</i> expressing <i>vtrB</i> (POR-2<i>ΔvtrAΔvtrB</i>+p<i>vtrB</i>); bar 11: <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>). Cytotoxicity was evaluated by the amount of LDH released. Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parent strain (*<i>P</i><0.05). C. Overexpressing of <i>vtrA</i> and <i>vtrB</i> promoted T3SS2-dependent cytotoxicity. Caco-2 cells were infected for 1.5–6 h with <i>V. parahaemolyticus</i>. Cytotoxicity was evaluated by the amount of LDH released. POR-2 (<i>ΔtdhASΔvcrD1</i>) with control vector (pSA19CP-MCS) (filled squares, solid line), POR-2 expressing <i>vtrA</i> (filled circles, solid line), POR-2 expressing <i>vtrB</i> (filled triangles, solid line), <i>ΔvcrD1ΔvcrD2</i> (<i>ΔtdhASΔvcrD1ΔvcrD2</i>) with control vector (pSA19CP-MCS) (open squares, dashed line), <i>ΔvcrD1ΔvcrD2</i> expressing <i>vtrA</i> (open circles, dashed line), and <i>ΔvcrD1ΔvcrD2</i> expressing <i>vtrB</i> (open triangles, dashed line). Error bars represent standard deviations for results from triplicate experiments. Asterisks indicate significant differences from the results obtained with the parent strain (*<i>P</i><0.05).</p

Whole-genome transcriptional profiling of <i>vtrA</i> and <i>vtrB</i> deletion strain.

<p>Genome-wide transcript analysis of the VtrA and VtrB regulons is shown. Gene expression was determined by comparing cDNA generated from WT<i>ΔvtrA</i> (A) or WT<i>ΔvtrB</i> (B) in exponential phase grown in LB medium with 0.5% NaCl with that from the WT strain. The Vp-PAI region is indicated by a bold line. Effect of the <i>vtrA</i> (C) or <i>vtrB</i> (D) deletion on expression of genes located within Vp-PAI (<i>vpa1309</i>-<i>vpa1396</i>). Representative gene functions are indicated at the top.</p

VtrA and VtrB regulate the expression levels of T3SS2-related proteins and TDH.

<p>A. Loss of <i>vtrA</i> and <i>vtrB</i> diminished the expression of T3SS2-related proteins and TDH. Western blot analysis of bacterial pellets (ppt.) and secreted proteins (sup.) from isogenic mutants of wild-type (WT) <i>V. parahaemolyticus</i>. Lane 1, wild-type <i>V. parahaemolyticus</i> (WT); lane 2, <i>vtrA</i> deletion strain (WT<i>ΔvtrA</i>); lane 3, <i>vtrB</i> deletion strain (WT<i>ΔvtrB</i>); lane 4, <i>vtrA</i> and <i>vtrB</i> double deletion strain (WT<i>ΔvtrAΔvtrB</i>). Samples from indicated strains were loaded in lane 5 to confirm the specificity of each antibody. Blots were probed with anti-VscC1, anti-VopD1, anti-VepA, anti-VscC2, anti-VopD2, anti-VopC, and anti-TDH polyclonal antibodies. B. Vector-induced <i>vtrB</i> could restore the secretory capacity of T3SS2 independent of <i>vtrA</i>. Western blot analyses of bacterial pellets (ppt.) and secreted proteins (sup.) from indicated strains are shown. Blots were probed with anti-VscC2, anti-VopD2, anti-VopC, and anti-TDH polyclonal antibodies. C. Genetic organization of the DNA region containing <i>vscC2</i> and <i>vpa1343</i> of <i>V. parahaemolyticus</i> RIMD2210633. D. VPA1343 protein expression was strictly regulated by VtrB. Western blot analysis of bacterial pellets (ppt.) from isogenic mutants of wild-type (WT) <i>V. parahaemolyticus</i> (upper panel) and their complemented strains (lower panel). Blots were probed with anti-VPA1343 polyclonal antibodies.</p