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</div>
<div class="action-panel-control-wrap">
<label for="email-from" class="action-panel-label">
From:
</label>
<input type="email" aria-label="Sender Email Address" placeholder="email@example.com" id="email-from" class="email-from" pattern="^[a-zA-Z0-9_.+-]+@[a-zA-Z0-9-]+\.[a-zA-Z0-9-.]+$" maxlength="256">
</div>
<div class="action-panel-control-wrap">
<label for="email-citation-format" class="action-panel-label">
Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="vsLw6GvMVa0Zt5zPJfAYHG0i2v0Y7xg2MfvBJwY8vLNtnwFFmyd0bOwWAzIa08Om">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="vsLw6GvMVa0Zt5zPJfAYHG0i2v0Y7xg2MfvBJwY8vLNtnwFFmyd0bOwWAzIa08Om">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="vsLw6GvMVa0Zt5zPJfAYHG0i2v0Y7xg2MfvBJwY8vLNtnwFFmyd0bOwWAzIa08Om">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="vsLw6GvMVa0Zt5zPJfAYHG0i2v0Y7xg2MfvBJwY8vLNtnwFFmyd0bOwWAzIa08Om">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="vsLw6GvMVa0Zt5zPJfAYHG0i2v0Y7xg2MfvBJwY8vLNtnwFFmyd0bOwWAzIa08Om">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="27214040">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://dx.plos.org/10.1371/journal.pgen.1006080"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=4656&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=27214040&amp;db=pubmed&amp;nlmid=101239074"
title="See full text options at Public Library of Science"
data-ga-category="full_text"
data-ga-action="Public Library of Science"
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><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--journals.plos.org-resource-img-external-pgen_120x30.png" alt="Public Library of Science full text link"><span class="text">
Public Library of Science
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/27214040/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=27214040&amp;db=pubmed&amp;nlmid=101239074"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="27214040"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/27214040/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/27214040/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="27214040" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F27214040%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="vsLw6GvMVa0Zt5zPJfAYHG0i2v0Y7xg2MfvBJwY8vLNtnwFFmyd0bOwWAzIa08Om"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
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><sup class="key">1</sup> Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.</li>
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><sup class="key">2</sup> Department of Microbiology and Molecular Genetics, University of California - Davis, Davis, California, United States of America.</li>
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><sup class="key">3</sup> Molecular Microbiology Department, John Innes Centre, Norwich, United Kingdom.</li>
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><sup class="key">4</sup> Research Core Unit Metabolomics, Hannover Medical School, Hannover, Germany.</li>
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><sup class="key">5</sup> School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.</li>
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Generally, the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be-at least partially-functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus.
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<p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one">The authors have declared that no competing interests exist.</p>
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aria-describedby="figure-caption-0"
role="button"
data-image-width="674"
data-image-height="667"
data-image-alt="Fig 1"
data-pmc-id="PMC4877007"
data-figure-id="pgen.1006080.g001">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-0"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b3e9/4877007/65f7a610af9c/pgen.1006080.g001.gif"
alt="Fig 1" />
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<strong class="figure-label">
<p> Fig 1. c-di-GMP accumulates at increased levels… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 1. c-di-GMP accumulates at increased levels in developing and starving <i> M </i> . <i> xanthus </i> cells. </p>
</strong>
<div class="figure-caption-contents"><p> DK1622… </p></div>
</div>
<figcaption id="figure-caption-0" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 1. c-di-GMP accumulates at increased levels in developing and starving <i>M</i>. <i>xanthus</i> cells.
</strong>
<div class="figure-caption-contents">DK1622 WT cells were starved in MC7 buffer on a solid surface in submerged culture or in suspension. At the indicated time points the c-di-GMP levels were determined and correlated to protein concentration. The c-di-GMP level is shown as mean ± standard deviation (SD) calculated from three biological replicates. * p < 0.05, ** p < 0.001 in Students t-test in which samples from individual time points were compared to the relevant 0 hrs sample.</div>
</figcaption>
</figure>
<figure class="figure-item "
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itemprop="contentUrl"
aria-describedby="figure-caption-1"
role="button"
data-image-width="673"
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data-image-alt="Fig 2"
data-pmc-id="PMC4877007"
data-figure-id="pgen.1006080.g002">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-1"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b3e9/4877007/7c943d9da8da/pgen.1006080.g002.gif"
alt="Fig 2" />
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<strong class="figure-label">
<p> Fig 2. c-di-GMP level is important for… </p>
</strong>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 2. c-di-GMP level is important for development. </p>
</strong>
<div class="figure-caption-contents"><p> (A) c-di-GMP levels in cells expressing the… </p></div>
</div>
<figcaption id="figure-caption-1" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 2. c-di-GMP level is important for development.
</strong>
<div class="figure-caption-contents">(A) c-di-GMP levels in cells expressing the indicated proteins during starvation in suspension. The c-di-GMP levels are shown as mean ± SD from three biological replicates relative to WT at 0 hrs. * p < 0.05 in Students t-test comparing different mutants to the WT at the respective time points. Note that the data for WT are the same as in Fig 1. (B) Fruiting body formation and sporulation under two different starvation conditions. Numbers after 120 hrs of starvation in submerged culture indicate heat- and sonication resistant spores formed after 120 hrs of starvation in submerged culture in percentage of WT (100%) from one representative experiment. Scale bars, TPM agar 500 μm, submerged culture 100 μm.</div>
</figcaption>
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<img class="figure-thumb" itemprop="thumbnail"
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alt="Fig 3" />
</a>
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<strong class="figure-label">
<p> Fig 3. Complementation experiments with Δ <i> dmxB… </i> </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 3. Complementation experiments with Δ <i> dmxB </i> , Δ <i> pmxA </i> and Δ <i> tmoK </i> mutants. </p>
</strong>
<div class="figure-caption-contents"><p> (A) Domain structure of DmxB,… </p></div>
</div>
<figcaption id="figure-caption-2" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 3. Complementation experiments with Δ<i>dmxB</i>, Δ<i>pmxA</i> and Δ<i>tmoK</i> mutants.
</strong>
<div class="figure-caption-contents">(A) Domain structure of DmxB, PmxA and TmoK. The primary sequences of the indicated proteins were analyzed for domain structure using [47]. (B) Fruiting body formation and sporulation under two different starvation conditions. Cells were treated and spores enumerated as described in Fig 2B. Scale bars: TPM agar 500 μm, submerged culture 100 μm. (C) Immunoblot detection of DmxB in total cell extracts. Total cell lysates from cells of the indicated genotypes were harvested from starvation agar at 24 hrs of development, separated by SDS-PAGE and probed with rabbit, polyclonal α-DmxB serum. Protein from the same calculated number of cells was loaded per lane. DmxB has a calculated molecular mass of 35.3 kDa. Molecular mass marker is indicated on the left. The non-specific band above the band corresponding to DmxB serves as an internal loading control.</div>
</figcaption>
</figure>
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itemprop="contentUrl"
aria-describedby="figure-caption-3"
role="button"
data-image-width="768"
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data-image-alt="Fig 4"
data-pmc-id="PMC4877007"
data-figure-id="pgen.1006080.g004">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-3"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b3e9/4877007/eec3eaf0364f/pgen.1006080.g004.gif"
alt="Fig 4" />
</a>
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<strong class="figure-label">
<p> Fig 4. <i> In vitro </i> assay for enzyme… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 4. <i> In vitro </i> assay for enzyme activity and c-di-GMP binding. </p>
</strong>
<div class="figure-caption-contents"><p> (A) DGC assay of… </p></div>
</div>
<figcaption id="figure-caption-3" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 4. <i>In vitro</i> assay for enzyme activity and c-di-GMP binding.
</strong>
<div class="figure-caption-contents">(A) DGC assay of DmxB variants. The indicated His6-tagged full-length DmxB variants were incubated with [α-<sup>32</sup>P]-GTP for the indicated periods of time followed by separation of nucleotides by TLC. Full-length DgcA<sup>WT</sup> was used as a positive control. GTP and c-di-GMP are indicated. The intermediate product indicated was described as a product formed during the DGC-dependent synthesis of c-di-GMP [49]. Numbers at the bottom indicate levels of c-di-GMP in % of the total signal in each lane. (B) PDE activity assay of PmxA. The indicated PmxA variants were incubated with [α-<sup>32</sup>P]-labeled c-di-GMP for the indicated periods of time followed by separation of nucleotides by TLC. pGpG and c-di-GMP are indicated. Numbers at the bottom indicate levels of pGpG in % of the total signal in each lane. (C) DRaCALA to detect c-di-GMP binding. The indicated full-length DmxB variants were incubated with [α-<sup>32</sup>P]-c-di-GMP with or without unlabeled c-di-GMP or GTP as competitors as indicated. 10 μl of the reaction mixtures were transferred to a nitrocellulose filter, dried and imaged.</div>
</figcaption>
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itemprop="contentUrl"
aria-describedby="figure-caption-4"
role="button"
data-image-width="797"
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data-image-alt="Fig 5"
data-pmc-id="PMC4877007"
data-figure-id="pgen.1006080.g005">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-4"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b3e9/4877007/e3af97c13669/pgen.1006080.g005.gif"
alt="Fig 5" />
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<strong class="figure-label">
<p> Fig 5. c-di-GMP levels in cells of… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Fig 5. c-di-GMP levels in cells of the indicated mutants during starvation. </p>
</strong>
<div class="figure-caption-contents"><p> (A, B) c-di-GMP… </p></div>
</div>
<figcaption id="figure-caption-4" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 5. c-di-GMP levels in cells of the indicated mutants during starvation.
</strong>
<div class="figure-caption-contents">(A, B) c-di-GMP levels in cells of the indicated genotypes were determined from three biological replicates as described in Fig 1. Note that the data for the WT are the same as in Fig 1 and in (B) the data for the WT and Δ<i>dmxB</i> strains are the same as in (A) and are shown again for the indicated time points for comparison. Note the different scale in (B) and for the Δ<i>dmxB/dmxB</i><sup>R210A</sup> strain. * p &lt; 0.05, ** p &lt; 0.001 in Students t-test comparing the different mutants to the WT at the same time points.</div>
</figcaption>
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aria-describedby="figure-caption-5"
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<p> Fig 6. Determination of <i> dmxB </i> transcript and… </p>
</strong>
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<p> Fig 6. Determination of <i> dmxB </i> transcript and DmxB accumulation levels. </p>
</strong>
<div class="figure-caption-contents"><p> (A) qRT-PCR analysis of <i> dmxB… </i> </p></div>
</div>
<figcaption id="figure-caption-5" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 6. Determination of <i>dmxB</i> transcript and DmxB accumulation levels.
</strong>
<div class="figure-caption-contents">(A) qRT-PCR analysis of <i>dmxB</i> expression. Total RNA was isolated from WT developed in submerged culture at the indicated time points. <i>dmxB</i> transcript level is shown as the mean ± standard deviation from two biological replicates with each three technical replicates relative to WT at 0 hrs. (B) Immunoblot detection of DmxB in total cell extracts. Total cell lysates were prepared from cells of the indicated genotypes harvested from starvation agar plates at the indicated time points of development. Immunoblots were prepared as described in Fig 3C. Protein from the same calculated number of cells was loaded per lane. Molecular mass marker is indicated on the left. The non-specific band above the band corresponding to DmxB serves as an internal loading control. (C) Immunoblot detection of DmxB in total cell extract of WT, Δ<i>dmxB</i> and <i>difE</i> strains. Total cell lysates from cells harvested from starvation agar after 24 hrs of development were prepared. Immunoblots were prepared as described in Fig 3C. Protein from the same calculated number of cells was loaded per lane. Molecular mass marker is indicated on the left. The non-specific band above the band corresponding to DmxB serves as an internal loading control.</div>
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<p> Fig 7. T4P formation and EPS accumulation… </p>
</strong>
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<p> Fig 7. T4P formation and EPS accumulation in various mutants. </p>
</strong>
<div class="figure-caption-contents"><p> (A) Immunoblot detection of PilA… </p></div>
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<figcaption id="figure-caption-6" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 7. T4P formation and EPS accumulation in various mutants.
</strong>
<div class="figure-caption-contents">(A) Immunoblot detection of PilA in total cell extracts and in the sheared T4P fraction. In the upper and lower blots, total cell extracts were isolated from the indicated strains developed in submerged culture at the indicated time points. In the middle blot, T4P were sheared off from the same number of cells and concentrated by MgCl2 precipitation. In all three blots, protein from the same calculated number of cells was loaded per lane. The upper and middle blots were probed with α-PilA antibodies. The lower blot was probed against PilC, which is important for T4P assembly and served as a loading control. PilA and PilC have a calculated molecular mass 23.4 kDa and 45.2 kDa, respectively. Molecular mass marker is indicated on the left. (B) Quantification of EPS accumulation in <i>dmxB</i> mutants. 20 μl aliquots of exponentially growing cells of the indicated genotypes were spotted at a density of 7 × 10<sup>9</sup> cells/ml on 1.5% agar supplemented with 0.5% CTT and 20 μg/ml trypan blue or on TPM starvation agar supplemented with 20 μg/ml trypan blue and incubated at 32°C for 24 hrs. (C) Extracellular complementation assay of Δ<i>dmxB</i> mutant. Cells of the indicated genotypes were either developed alone as described in Fig 2B in submerged culture or mixed at a 1:1 ratio and co-developed in submerged culture. Sporulation levels are enumerated after 120 hrs of starvation as the number of germinating heat- and sonication resistant spores relative to WT (100%).</div>
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data-image-width="800"
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data-figure-id="pgen.1006080.g008">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-7"
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<p> Fig 8. c-di-GMP regulates <i> epsABD </i> transcription. </p>
</strong>
<div class="figure-caption-contents"><p> Total… </p></div>
</div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
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<p> Fig 8. c-di-GMP regulates <i> epsABD </i> transcription. </p>
</strong>
<div class="figure-caption-contents"><p> Total RNA was isolated at the indicated time points… </p></div>
</div>
<figcaption id="figure-caption-7" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Fig 8. c-di-GMP regulates <i>epsABD</i> transcription.
</strong>
<div class="figure-caption-contents">Total RNA was isolated at the indicated time points from cells of WT (closed circles) and the Δ<i>dmxB</i> mutant (open squares) developed in submerged culture. Transcript levels are shown as mean ± standard deviation from two biological replicates with each three technical replicates relative to WT at 0 hrs.</div>
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data-figure-id="pgen.1006080.g009">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-8"
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<p> Fig 9. Transcriptional regulator EpsI/Nla24 binds c-di-GMP. </p>
</strong>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
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<p> Fig 9. Transcriptional regulator EpsI/Nla24 binds c-di-GMP. </p>
</strong>
<div class="figure-caption-contents"><p> (A) Pull-down experiment with the soluble fraction of… </p></div>
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<figcaption id="figure-caption-8" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
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Fig 9. Transcriptional regulator EpsI/Nla24 binds c-di-GMP.
</strong>
<div class="figure-caption-contents">(A) Pull-down experiment with the soluble fraction of <i>E</i>. <i>coli</i> cell lysates before and after induction of EpsI/Nla24 synthesis using biotinylated c-di-GMP immobilized on streptavidin magnetic beads. Cell extract from the uninduced sample was used as a negative control. Pulled-down protein is indicated with an arrow. EpsI/Nla24-His<sub>6</sub> has a calculated molecular mass of 50 kDa. (B) SPR sensorgrams and resulting affinity fit data for EpsI/Nla24 binding to biotinylated c-di-GMP. Upper panel, sensorgrams of EpsI/Nla24 binding to biotinylated c-di-GMP immobilized on sensor chip. The concentration of EpsI/Nla24 ranged from 62.5 nM (lowest curve) to 4 μM (highest curve) and concentration replicates were included as appropriate. The protein binding and dissociation phases for all sensorgrams are shown. Lower panel, affinity fit of EpsI/Nla24 binding to biotinylated c-di-GMP. For this fit, binding responses were measured 4 sec before the end of the injection.</div>
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<span class="docsum-journal-citation full-journal-citation">J Bacteriol. 2022 Apr 19;204(4):e0056121. doi: 10.1128/jb.00561-21. Epub 2021 Dec 20.</span>
<span class="docsum-journal-citation short-journal-citation">J Bacteriol. 2022.</span>
<span class="citation-part">PMID: <span class="docsum-pmid">34928179</span></span>
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<h2 class="title">
Related information
</h2>
<ul class="related-links-list"><li><a title="Link to Gene for the GeneRIF subcategory" href="https://www.ncbi.nlm.nih.gov/gene?linkname=pubmed_gene_rif&amp;from_uid=27214040"
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Gene (GeneRIF)
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