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<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="jf7fjDUD1V7rI7OH53rJpwENEsgDtcKV8N7sX3qVGXMd3bsNWqRGBu9ZbWlZAqg3">
<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="jf7fjDUD1V7rI7OH53rJpwENEsgDtcKV8N7sX3qVGXMd3bsNWqRGBu9ZbWlZAqg3">
<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="jf7fjDUD1V7rI7OH53rJpwENEsgDtcKV8N7sX3qVGXMd3bsNWqRGBu9ZbWlZAqg3">
<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="jf7fjDUD1V7rI7OH53rJpwENEsgDtcKV8N7sX3qVGXMd3bsNWqRGBu9ZbWlZAqg3">
<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="jf7fjDUD1V7rI7OH53rJpwENEsgDtcKV8N7sX3qVGXMd3bsNWqRGBu9ZbWlZAqg3">
<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>
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<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://journals.asm.org/doi/10.1128/aem.00064-22?url_ver=Z39.88-2003&amp;rfr_id=ori:rid:crossref.org&amp;rfr_dat=cr_pub 0pubmed"
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pmc
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href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/35254100/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=35254100&amp;db=pubmed&amp;nlmid=7605801"
title="Free full text at PubMed Central"
data-ga-category="full_text"
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Free PMC article
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<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="/35254100/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/35254100/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="35254100" 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%2F35254100%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="jf7fjDUD1V7rI7OH53rJpwENEsgDtcKV8N7sX3qVGXMd3bsNWqRGBu9ZbWlZAqg3"><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"
name="add-to-new-collection"
id="collections-action-dialog-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"
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<div class="abstract" id="abstract">
<h2 class="title">
Abstract
</h2>
<div class="abstract-content selected"
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<p>
The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including motile-to-sessile lifestyle transitions. Although the external stimuli that modulate cellular c-di-GMP contents are not fully characterized, there is growing evidence that certain amino acids act as environmental cues for c-di-GMP turnover. In the plant-beneficial bacterium Pseudomonas putida KT2440, both arginine biosynthesis and uptake influence second messenger contents and the associated phenotypes. To further understand this connection, we have analyzed the role of ArgR, which in different bacteria is the master transcriptional regulator of arginine metabolism but had not been characterized in P. putida. The results show that ArgR controls arginine biosynthesis and transport, and an <i>argR</i>-null mutant grows poorly with arginine as the sole carbon or nitrogen source and also displays increased biofilm formation and reduced surface motility. Modulation of c-di-GMP levels by exogenous arginine requires ArgR. The expression of certain biofilm matrix components, namely, the adhesin LapF and the exopolysaccharide Pea, as well as the diguanylate cyclase CfcR is influenced by ArgR, likely through the alternative sigma factor RpoS. Our data indicate the existence of a regulatory feedback loop between ArgR and c-di-GMP mediated by FleQ. <b>IMPORTANCE</b> Identifying the molecular mechanisms by which metabolic and environmental signals influence the turnover of the second messenger c-di-GMP is key to understanding the regulation of bacterial lifestyles. The results presented here point at the transcriptional regulator ArgR as a central node linking arginine metabolism and c-di-GMP signaling and indicate the existence of a complex balancing mechanism that connects cellular arginine contents and second messenger levels, ultimately controlling the lifestyles of Pseudomonas putida.
</p>
</div>
<p>
<strong class="sub-title">
Keywords:
</strong>
Pseudomonas; amino acid biosynthesis; amino acid transport; arginine synthesis; biofilm; c-di-GMP; cell signaling; gene regulation; metabolism; second messenger; transport.
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<div class="conflict-of-interest" id="conflict-of-interest">
<h2 class="title">
Conflict of interest statement
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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 declare no conflict of interest.</p>
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<div class="figures" id="figures">
<h2 class="title">
Figures
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<img class="figure-thumb" itemprop="thumbnail"
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b6a/9004404/a3d83c84b381/aem.00064-22-f001.gif"
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<p> FIG 1 </p>
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<div class="figure-caption-contents"><p> Growth of KT2440 (circles) and… </p></div>
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<strong class="figure-label">
<p> FIG 1 </p>
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<div class="figure-caption-contents"><p> Growth of KT2440 (circles) and the Δ <i> argR </i> mutant (triangles) in M9 minimal… </p></div>
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FIG 1
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<div class="figure-caption-contents">Growth of KT2440 (circles) and the Δ<i>argR</i> mutant (triangles) in M9 minimal medium with 10mM glucose (A) or 10mM <sc>l</sc>-arginine (B) as the carbon source or in M8 medium with 10mM glucose as the carbon source and 5mM <sc>l</sc>-arginine as the nitrogen source (C). Growth was monitored during incubation at 30°C with continuous shaking for 24 h using a Bioscreen apparatus, measuring the absorbance in the 420- to 580-nm range every 30min. Averages and standard deviations from one representative experiment with three replicates are shown.</div>
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<p> FIG 2 </p>
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<div class="figure-caption-contents"><p> (A) Analysis of the cotranscription… </p></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> FIG 2 </p>
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<div class="figure-caption-contents"><p> (A) Analysis of the cotranscription of <i> argT </i> and <i> argR </i> with the <i> hisQMP </i> cluster.… </p></div>
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<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
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<div class="figure-caption-contents">(A) Analysis of the cotranscription of <i>argT</i> and <i>argR</i> with the <i>hisQMP</i> cluster. Electrophoresis was performed on RT-PCR products amplified with primers designed to detect transcripts containing the intergenic regions between <i>argT</i> and <i>hisQ</i> (761 bp) (left) and between <i>hisP</i> and <i>argR</i> (783 bp) (right). Lanes: R, template RNA obtained from P. putida KT2440 cultures; , negative control without the reverse transcriptase reaction; +, positive control using DNA as the template for the reaction. (B) Determination of the transcription initiation sites for <i>argT</i> and <i>argR</i>. The base corresponding to the +1 site is shaded. The conserved 10 region for the <i>argT</i> promoter and the GC-rich region in the <i>argR</i> promoter are underlined, and the predicted ARG site overlapping the 35 site of <i>argT</i> is boxed. The stop codon for <i>hisP</i> is circled. The square brackets indicate the fragments used to construct transcriptional fusions in plasmids pLBM21 and pLBM20.</div>
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data-image-width="666"
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<img class="figure-thumb" itemprop="thumbnail"
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b6a/9004404/294e7c2a9ad4/aem.00064-22-f003.gif"
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<strong class="figure-label">
<p> FIG 3 </p>
</strong>
<div class="figure-caption-contents"><p> Expression of <i> argT </i> :: <i> lacZ… </i> </p></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> FIG 3 </p>
</strong>
<div class="figure-caption-contents"><p> Expression of <i> argT </i> :: <i> lacZ </i> (pLBM21) (A) and <i> argR </i> :: <i> lacZ </i> (pLBM20) (B)… </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
</strong>
<div class="figure-caption-contents">Expression of <i>argT</i>::<i>lacZ</i> (pLBM21) (A) and <i>argR</i>::<i>lacZ</i> (pLBM20) (B) during growth in minimal medium with glucose in the absence or presence of <sc>l</sc>-arginine. β-Galactosidase activity (Miller units) was assessed in KT2440 (wild type [wt]) and the Δ<i>argR</i> mutant harboring pLBM21 or pLBM20 during growth in minimal medium with glucose (light bars) or glucose and 10mM <sc>l</sc>-arginine (dark bars). The data correspond to averages and standard deviations from two experiments with three technical replicates each (<i>n</i>=6). Differences in panel A between the wild type and the mutant and in the wild type in the presence and absence of arginine were statistically significant at all time points, while samples not showing significant differences in panel B at a given time point are indicated as n.s. (not significant) (<i>P</i>0.05 by ANOVA).</div>
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<img class="figure-thumb" itemprop="thumbnail"
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b6a/9004404/51ff9fc8c0a1/aem.00064-22-f004.gif"
alt="FIG 4" />
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<strong class="figure-label">
<p> FIG 4 </p>
</strong>
<div class="figure-caption-contents"><p> ArgR functions as a repressor… </p></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> FIG 4 </p>
</strong>
<div class="figure-caption-contents"><p> ArgR functions as a repressor of <sc> l </sc> -arginine biosynthesis genes. β-Galactosidase activities (Miller… </p></div>
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<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
</strong>
<div class="figure-caption-contents">ArgR functions as a repressor of <sc>l</sc>-arginine biosynthesis genes. β-Galactosidase activities (Miller units) of <i>argG</i>::<i>lacZ</i> (pLBM13) (A) and <i>argH</i>::<i>lacZ</i> (pLBM14) (B) transcriptional fusions were measured in KT2440 (white bars) and the Δ<i>argR</i> mutant (gray bars) in LB at the indicated times. The data are averages and standard deviations from two independent experiments with three technical repetitions each (<i>n</i>=6). Statistically significant differences between the wild type and the Δ<i>argR</i> mutant were detected from 3 h onward (A) and at 5 h and 7 h (B), respectively (<i>P</i>0.05 by Students <i>t</i> test).</div>
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<img class="figure-thumb" itemprop="thumbnail"
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b6a/9004404/30b0fb45217d/aem.00064-22-f005.gif"
alt="FIG 5" />
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<p> FIG 5 </p>
</strong>
<div class="figure-caption-contents"><p> Influence of ArgR on the… </p></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> FIG 5 </p>
</strong>
<div class="figure-caption-contents"><p> Influence of ArgR on the changes in cellular c-di-GMP contents in response to… </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
</strong>
<div class="figure-caption-contents">Influence of ArgR on the changes in cellular c-di-GMP contents in response to exogenous <sc>l</sc>-arginine. (A) Strain KT2440 (wild type) and the Δ<i>argR</i> mutant harboring pCdrA::<i>gfp<sup>C</sup></i> were inoculated into microtiter plates containing diluted LB (1:3) supplied with different final concentrations of <sc>l</sc>-arginine (0, 5, and 15mM). Fluorescence and turbidity were recorded every 30min for 24 h using a Varioskan Lux fluorimeter. Data (GFP counts) correspond to fluorescence values normalized by growth (OD<sub>600</sub>) and are averages and standard deviations from one representative experiment using three experimental replicates under each condition. The line color intensity indicates increasing concentrations of the amino acid. (B) Values corresponding to the area under the curve derived from fluorescence measurements normalized by culture growth (OD<sub>600</sub>) were calculated for KT2440 (white bars) and the Δ<i>argR</i> mutant (gray bars), to obtain a global overview of fluorescence along the whole growth curve, where 100% corresponds to the wild type without <sc>l</sc>-arginine supplementation. Statistically significant differences between groups (<i>P</i>0.01 by ANOVA) are indicated by different lowercase letters.</div>
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<img class="figure-thumb" itemprop="thumbnail"
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b6a/9004404/4af804f511a8/aem.00064-22-f006.gif"
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<strong class="figure-label">
<p> FIG 6 </p>
</strong>
<div class="figure-caption-contents"><p> (A) Surface motility of KT2440… </p></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> FIG 6 </p>
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<div class="figure-caption-contents"><p> (A) Surface motility of KT2440 and the Δ <i> argR </i> mutant. Cultures grown overnight… </p></div>
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<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
</strong>
<div class="figure-caption-contents">(A) Surface motility of KT2440 and the Δ<i>argR</i> mutant. Cultures grown overnight were diluted to an OD<sub>660</sub> of 1, 2μL was spotted onto the center of the plate, and images were taken after 72 h of growth at 25°C. The assay was done in duplicate, with spotting on six plates in each case, and one representative image is shown for each strain. (B) Biofilm formation by KT2440 (wild type) and its Δ<i>argR</i> derivative during growth in LB in 96-well polystyrene plates under static conditions. At the indicated times, adhered biomass was quantified after crystal violet staining (<i>A</i><sub>595</sub>). Data correspond to averages and standard deviations from two independent experiments with 15 replicates per time point. Statistically significant differences between KT2440 and the Δ<i>argR</i> mutant were detected from 5 h onward (<i>P</i>0.05 by Students <i>t</i> test). (C) Effect of exogenous arginine on biofilm formation by KT2440 (white bars) and the Δ<i>argR</i> mutant (gray bars) after 10 h of growth in FAB medium with glucose as the carbon source supplied with 0, 5, or 15mM <sc>l</sc>-arginine. The setup and attached biomass quantification were done as described above for panel B. Data correspond to averages and standard deviations from three independent experiments with three technical replicates. Statistically significant differences (<i>P</i>0.05 by ANOVA) are indicated (*).</div>
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b6a/9004404/14b3d0d7ccce/aem.00064-22-f007.gif"
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<strong class="figure-label">
<p> FIG 7 </p>
</strong>
<div class="figure-caption-contents"><p> Influence of <i> argR </i> mutation on… </p></div>
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<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> FIG 7 </p>
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<div class="figure-caption-contents"><p> Influence of <i> argR </i> mutation on the expression of adhesin- and EPS-encoding genes, <i> rpoS… </i> </p></div>
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<strong class="figure-label">
FIG 7
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<div class="figure-caption-contents">Influence of <i>argR</i> mutation on the expression of adhesin- and EPS-encoding genes, <i>rpoS</i> and <i>cfcR</i>. Plasmids pMMGA, pMMG1, pMP220-bcs, pMP220-pea, pMP220-peb, and pMIR200, harboring transcriptional fusions of <i>lapA</i>, <i>lapF</i>, <i>pea</i>, <i>peb</i>, <i>bcs</i>, and <i>cfcR</i> to <i>lacZ</i>, and pMAMV21, harboring an <i>rpoS-lacZ</i> protein fusion, were introduced into KT2440 (white bars) and the Δ<i>argR</i> mutant (gray bars), and β-galactosidase activity was measured after 10 h of growth in LB. The data are averages and standard deviations from two biological replicates with three technical repetitions each. Statistically significant differences (<i>P</i>0.05 by Students <i>t</i> test) are indicated (*).</div>
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<img class="figure-thumb" itemprop="thumbnail"
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9b6a/9004404/828c48e0d484/aem.00064-22-f008.gif"
alt="FIG 8" />
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<p> FIG 8 </p>
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<div class="figure-caption-contents"><p> Expression of <i> argR </i> and <i> argT… </i> </p></div>
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</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> FIG 8 </p>
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<div class="figure-caption-contents"><p> Expression of <i> argR </i> and <i> argT </i> is modulated by c-di-GMP via <i> fleQ </i> . (A… </p></div>
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<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
</strong>
<div class="figure-caption-contents">Expression of <i>argR</i> and <i>argT</i> is modulated by c-di-GMP via <i>fleQ</i>. (A and B) Plasmids harboring <i>argR</i>::<i>lacZ</i> (pLBM20) (A) and <i>argT</i>::<i>lacZ</i> (pLBM21) (B) were introduced into KT2440 (circles) and its <i>fleQ</i> mutant derivative cfcK-77 (triangles). Growth (OD<sub>600</sub>) (open symbols) and β-galactosidase activity (Miller units) (closed symbols) were measured over time in LB. The data are averages and standard deviations from two biological replicates with three technical repetitions each. (C) Activity of the <i>argR</i>::<i>lacZ</i> fusion in KT2440 and cfcK-77 harboring pMAMV1 (<i>cfcR</i> in multicopy) or pBBR1-MCS5 (empty vector) after growth overnight in LB. Cultures were treated with glass beads (diameter of 425 to 600μm) for 1min to disrupt cell aggregates formed as a consequence of high c-di-GMP levels due to the presence of pMAMV1. The results correspond to averages and standard deviations from four biological replicates with three technical repetitions each. Statistically significant differences (<i>P</i>0.05 by ANOVA) are indicated by different lowercase letters.</div>
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<p> FIG 9 </p>
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<div class="figure-caption-contents"><p> Current model of the regulatory… </p></div>
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<div class="figure-caption-contents"><p> Current model of the regulatory network connecting <sc> l </sc> -arginine metabolism, c-di-GMP signaling, and… </p></div>
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FIG 9
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<div class="figure-caption-contents">Current model of the regulatory network connecting <sc>l</sc>-arginine metabolism, c-di-GMP signaling, and biofilm formation in P. putida KT2440. ArgR influences c-di-GMP levels (and, therefore, biofilm formation) indirectly through its positive regulatory role in arginine transport and negative effect on arginine synthesis. Arginine and c-di-GMP signaling are connected through the RpoS-dependent transcription of <i>cfcR</i> and potentially via other DGCs. In turn, c-di-GMP modulates the expression of <i>argR</i> via FleQ, thus establishing a feedback loop between arginine pools and c-di-GMP. See the text for further details.</div>
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