Analysis of transcription of hp0119 in <i>H. pylori</i> 26695/arsS-H₇Q carrying substitutions of the input domain histidines H35, H44, H90, H93, H94, H118 and H126 of ArsS to glutamine.

<p>Primer extension analysis using the radiolabelled oligonucleotide HP119PE was performed on equal amounts of RNA extracted from the <i>H. pylori</i> strain 26695/arsS-H₇Q grown at pH 7.0 (lane 1) and strains 26695 (lane 3), 26695/arsS-H94Q (lane 2), and 26695/arsS-H₇Q (lane 4) exposed to pH 5.0 for one hour.</p

In vitro phosphorylation of response regulator ArsR and its orthologs HH1608, WS1817 and CJ1261 by the histidine kinases ArsS, HH1607 and CJ1262.

<p>The recombinant histidine kinases (1 µM) HH1607 (lanes 1–4), CJ1262 (lanes 5–8) and ArsS (lanes 9–12) were incubated in the presence of γ³³P-ATP and the response regulators (5 µM) ArsR (lanes 1, 5 and 9), HH1608 (lanes 2, 6 and 10), WS1817 (lanes 3, 7, and 11) and CJ1261 (lanes 4, 8, and 12), respectively, for 10 min at room temperature. The reactions were stopped by the addition of sample buffer and the reaction mixtures were separated on a 12% SDS-polyacrylamide gel. The position of the histidine kinases and response regulators is indicated by black and grey arrows on the right.</p

Analysis of the acid-responsive expression of hp0119 in <i>H. pylori</i> G27 and mutant <i>H. pylori</i> strains expressing the ArsS orthologs of <i>H. hepaticus</i>, <i>C. jejuni</i> and <i>W. succinogenes</i>.

<p>Primer extension analysis with a radiolabelled oligonucleotide specific for hp0119 was performed on equal amounts of RNA extracted from strains <i>H. pylori</i> G27 (lanes 9 and 10), G27/HP165::km (lanes 7 and 8), G27/HH1607 (lanes 1 and 2), G27/CJ1262 (lanes 3 and 4) and G27/WS1818 (lanes 5 and 6) which were grown at pH 7.0 (lanes 1, 3, 5, 7, and 9) or were exposed to pH 5.0 for one hour (lanes 2, 4, 6, 8, and 10). The hp0119-specific cDNA is indicated by a black arrow on the left. The gray arrow indicates the position of a cDNA band representing a non-specific primer elongation product whose unchanged intensity indicates the presence of equal amounts of RNA in the different samples.</p

Analysis of transcription of the acid-responsive genes hp0119 (A.) and hp1432 (B.) in <i>H. pylori</i> strains expressing ArsS proteins with individual histidine to glutamine mutations in the input domain.

<p>A. Primer extension analysis using the radiolabelled oligonucleotide HP119PE was performed on equal amounts of RNA extracted from the <i>H. pylori</i> strains 26695 (lane 1), 26695/arsS-H0 (lane 2), 26695/arsS-H35Q (lane 3), 26695/arsS-H44Q (lane 4), 26695/arsS-H90Q (lane 5), 26695/arsS-H93Q (lane 6), 26695/arsS-H94Q (lane 7), 26695/arsS-H118Q (lane 8), 26695/arsS-H126Q (lane 9) and 26695/arsS::km (lane 10) which were exposed to pH 5.0 for one hour. The hp0119-specific cDNA is indicated by a black arrow on the right. The sequencing ladder (lanes T, G, C, A) was obtained by annealing oligonucleotide HP119PE to plasmid pSL-119PE2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006930#pone.0006930-Dietz1" target="_blank">[39]</a>. The gray arrow indicates the position of a cDNA band representing a non-specific primer elongation product whose unchanged intensity indicates the presence of equal amounts of RNA in the different samples. The sequence of the -10 element of the hp0119 promoter is shown on the left. B. Primer extension analysis using the radiolabelled oligonucleotide HP1432PE was performed on equal amounts of RNA extracted from the <i>H. pylori</i> strains 26695 (lane 9), 26695/arsS-H0 (lane 1), 26695/arsS-H35Q (lane 2), 26695/arsS-H44Q (lane 3), 26695/arsS-H90Q (lane 4), 26695/arsS-H93Q (lane 5), 26695/arsS-H94Q (lane 6), 26695/arsS-H118Q (lane 7), 26695/arsS-H126Q (lane 8) and 26695/arsS::km (lane 10) which were exposed to pH 5.0 for one hour. The hp1432-specific cDNA is indicated by a black arrow on the right.</p

Analysis of transcription of the acid-responsive gene hp0119 in <i>H. pylori</i> strains expressing ArsS proteins with histidine to alanine double mutations in the input domain.

<p>Primer extension analysis using the radiolabelled oligonucleotide HP119PE was performed on equal amounts of RNA extracted from the <i>H. pylori</i> strains 26695 (lane 2), 26695/arsS-H0 (lane 3), 26695/arsS-H94Q (lane 4), 26695/arsS-H94A (lane 5), 26695/arsS-H94,35AA (lane 6), 26695/arsS-H94,44AA (lane 7), 26695/arsS-H94,90AA (lane 8), 26695/arsS-H94,93AA (lane 9), 26695/arsS-H94,118AA (lane 10), 26695/arsS-H94,126AA (lane 11) and 26695/arsS::km (lane 12) which were exposed to pH 5.0 for one hour. As a control primer extension analysis was also performed on RNA from the wild-type strain 26695 grown at pH 7.0 (lane 1). The hp0119-specific cDNA is indicated by a black arrow on the right. The sequencing ladder (lanes T, G, C, A) was obtained by annealing oligonucleotide HP119PE to plasmid pSL-119PE2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006930#pone.0006930-Dietz1" target="_blank">[39]</a>. The sequence of the -10 element of the hp0119 promoter is shown on the left. The gray arrow indicates the position of a cDNA band representing a non-specific primer elongation product whose unchanged intensity indicates the presence of equal amounts of RNA in the different samples.</p

Location maps of the study region, Hamanaka 2, the RK12 coring site and other archaeobotanical records discussed in the text.

<p>Map compilation showing (A) the location of the study region in the northwest Pacific region; (B) the southern Primor’e Region (Russia); (C) the Hokkaido and northern Tohoku regions; (D) Rebun Island; and (E) the northern part of Rebun with Lake Kushu (white cross indicates location of the RK12 coring site) and the Hamanaka 2 archaeological site. Dots illustrate the locations of Okhotsk culture (yellow), Satsumon culture (green), Heian period (blue), and the RFE Iron Age–Eastern Xia State (red) barley records used for seed morphological comparison (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174397#pone.0174397.s003" target="_blank">S3 Table</a> for site names and further details). Red circles in (C) show the location of the 194 Okhotsk culture sites listed in the Hokkaido archaeological site database (<a href="http://www2.wagamachi-guide.com/hokkai_bunka/" target="_blank">http://www2.wagamachi-guide.com/hokkai_bunka/</a>). The dashed line marks the proposed boundary of where the two barley morphotypes dominate. Bathymetry of Lake Kushu (0.5 m isolines) is based on survey data provided by T. Haraguchi (Osaka City University). Topographic maps are based on data from the elevation Shuttle Radar Topography Mission (SRTM) V4.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174397#pone.0174397.ref024" target="_blank">24</a>]. Isolines for the terrestrial area in (E) are drawn from a topographic map [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174397#pone.0174397.ref025" target="_blank">25</a>].</p

Sample-specific total counts of selected macrobotanical remains and floated litres from the Okhotsk culture layer units of Hamanaka 2.

<p>Sample-specific total counts of selected macrobotanical remains and floated litres from the Okhotsk culture layer units of Hamanaka 2.</p

Simplified pollen diagram representing the section dating between 50 cal yr BC and 1540 cal yr AD (67 pollen spectra) of the sediment core RK12 from Lake Kushu, Rebun Island.

<p>Chronology of prehistoric cultures in northern Hokkaido comprises Epi-Jomon (EJ; ca. 100 cal yr BC–500 cal yr AD including the Susuya tradition ca. 100–500 cal yr AD), Okhotsk culture (OK; ca. 500–950 cal yr AD), and Proto-Ainu/Formative Ainu (PA; ca. 950–1600 cal yr AD) (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174397#pone.0174397.ref026" target="_blank">26</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174397#pone.0174397.ref027" target="_blank">27</a>]).</p

Box-plots showing the L/W distribution of barley seeds from the Hamanaka 2 Okhotsk culture layers (this study) and of selected records from other regions.

<p>The boxes delineate the 25–75% quartiles (regular font) with the median (italic font) shown as an inset horizontal line. The whiskers (inner fence) are defined as the array from top/bottom of the box to the largest/smallest data point less than 1.5 times the box height from the box. Data points inside and outside (outliers) the whiskers are marked by grey and blue dots, respectively. Dotted horizontal lines indicate arithmetic means of Satsumon barley populations. Sample size (if know) and site numbers used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174397#pone.0174397.g001" target="_blank">Fig 1</a> are provided. Ages are given in calendar years.</p

Hamanaka 2 cultural layers associated with the Okhotsk culture and available flotation samples.

<p>Hamanaka 2 cultural layers associated with the Okhotsk culture and available flotation samples.</p