SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of Bacillus cereus Toxins and Cell Wall-Associated Proteins
Identifieur interne : 000353 ( Pmc/Corpus ); précédent : 000352; suivant : 000354SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of Bacillus cereus Toxins and Cell Wall-Associated Proteins
Auteurs : Aniko Vörös ; Roger Simm ; Leyla Slamti ; Matthew J. Mckay ; Ida K. Hegna ; Christina Nielsen-Leroux ; Karl A. Hassan ; Ian T. Paulsen ; Didier Lereclus ; Ole Andreas Kstad ; Mark P. Molloy ; Anne-Brit KolstSource :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
The aim of this study was to explore the role of SecDF in protein secretion in
Url:
DOI: 10.1371/journal.pone.0103326
PubMed: 25083861
PubMed Central: 4118872
Links to Exploration step
PMC:4118872Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of <italic>Bacillus cereus</italic> Toxins and Cell Wall-Associated Proteins</title><author><name sortKey="Voros, Aniko" sort="Voros, Aniko" uniqKey="Voros A" first="Aniko" last="Vörös">Aniko Vörös</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Simm, Roger" sort="Simm, Roger" uniqKey="Simm R" first="Roger" last="Simm">Roger Simm</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Slamti, Leyla" sort="Slamti, Leyla" uniqKey="Slamti L" first="Leyla" last="Slamti">Leyla Slamti</name><affiliation><nlm:aff id="aff3"><addr-line>INRA, UMR1319 Micalis, Domaine de La Minière, Guyancourt, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mckay, Matthew J" sort="Mckay, Matthew J" uniqKey="Mckay M" first="Matthew J." last="Mckay">Matthew J. Mckay</name><affiliation><nlm:aff id="aff2"><addr-line>Australian Proteome Analysis Facility (APAF), Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hegna, Ida K" sort="Hegna, Ida K" uniqKey="Hegna I" first="Ida K." last="Hegna">Ida K. Hegna</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Nielsen Leroux, Christina" sort="Nielsen Leroux, Christina" uniqKey="Nielsen Leroux C" first="Christina" last="Nielsen-Leroux">Christina Nielsen-Leroux</name><affiliation><nlm:aff id="aff3"><addr-line>INRA, UMR1319 Micalis, Domaine de La Minière, Guyancourt, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hassan, Karl A" sort="Hassan, Karl A" uniqKey="Hassan K" first="Karl A." last="Hassan">Karl A. Hassan</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Paulsen, Ian T" sort="Paulsen, Ian T" uniqKey="Paulsen I" first="Ian T." last="Paulsen">Ian T. Paulsen</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lereclus, Didier" sort="Lereclus, Didier" uniqKey="Lereclus D" first="Didier" last="Lereclus">Didier Lereclus</name><affiliation><nlm:aff id="aff3"><addr-line>INRA, UMR1319 Micalis, Domaine de La Minière, Guyancourt, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><addr-line>AgroParistech, UMR Micalis, Jouy-en-Josas, France</addr-line></nlm:aff></affiliation></author><author><name sortKey=" Kstad, Ole Andreas" sort=" Kstad, Ole Andreas" uniqKey=" Kstad O" first="Ole Andreas" last=" Kstad">Ole Andreas Kstad</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Molloy, Mark P" sort="Molloy, Mark P" uniqKey="Molloy M" first="Mark P." last="Molloy">Mark P. Molloy</name><affiliation><nlm:aff id="aff2"><addr-line>Australian Proteome Analysis Facility (APAF), Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kolst, Anne Brit" sort="Kolst, Anne Brit" uniqKey="Kolst A" first="Anne-Brit" last="Kolst">Anne-Brit Kolst</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25083861</idno><idno type="pmc">4118872</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118872</idno><idno type="RBID">PMC:4118872</idno><idno type="doi">10.1371/journal.pone.0103326</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000353</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000353</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of <italic>Bacillus cereus</italic> Toxins and Cell Wall-Associated Proteins</title><author><name sortKey="Voros, Aniko" sort="Voros, Aniko" uniqKey="Voros A" first="Aniko" last="Vörös">Aniko Vörös</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Simm, Roger" sort="Simm, Roger" uniqKey="Simm R" first="Roger" last="Simm">Roger Simm</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Slamti, Leyla" sort="Slamti, Leyla" uniqKey="Slamti L" first="Leyla" last="Slamti">Leyla Slamti</name><affiliation><nlm:aff id="aff3"><addr-line>INRA, UMR1319 Micalis, Domaine de La Minière, Guyancourt, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mckay, Matthew J" sort="Mckay, Matthew J" uniqKey="Mckay M" first="Matthew J." last="Mckay">Matthew J. Mckay</name><affiliation><nlm:aff id="aff2"><addr-line>Australian Proteome Analysis Facility (APAF), Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hegna, Ida K" sort="Hegna, Ida K" uniqKey="Hegna I" first="Ida K." last="Hegna">Ida K. Hegna</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Nielsen Leroux, Christina" sort="Nielsen Leroux, Christina" uniqKey="Nielsen Leroux C" first="Christina" last="Nielsen-Leroux">Christina Nielsen-Leroux</name><affiliation><nlm:aff id="aff3"><addr-line>INRA, UMR1319 Micalis, Domaine de La Minière, Guyancourt, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Hassan, Karl A" sort="Hassan, Karl A" uniqKey="Hassan K" first="Karl A." last="Hassan">Karl A. Hassan</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Paulsen, Ian T" sort="Paulsen, Ian T" uniqKey="Paulsen I" first="Ian T." last="Paulsen">Ian T. Paulsen</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lereclus, Didier" sort="Lereclus, Didier" uniqKey="Lereclus D" first="Didier" last="Lereclus">Didier Lereclus</name><affiliation><nlm:aff id="aff3"><addr-line>INRA, UMR1319 Micalis, Domaine de La Minière, Guyancourt, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><addr-line>AgroParistech, UMR Micalis, Jouy-en-Josas, France</addr-line></nlm:aff></affiliation></author><author><name sortKey=" Kstad, Ole Andreas" sort=" Kstad, Ole Andreas" uniqKey=" Kstad O" first="Ole Andreas" last=" Kstad">Ole Andreas Kstad</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author><author><name sortKey="Molloy, Mark P" sort="Molloy, Mark P" uniqKey="Molloy M" first="Mark P." last="Molloy">Mark P. Molloy</name><affiliation><nlm:aff id="aff2"><addr-line>Australian Proteome Analysis Facility (APAF), Macquarie University, Sydney, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kolst, Anne Brit" sort="Kolst, Anne Brit" uniqKey="Kolst A" first="Anne-Brit" last="Kolst">Anne-Brit Kolst</name><affiliation><nlm:aff id="aff1"><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The aim of this study was to explore the role of SecDF in protein secretion in <italic>Bacillus cereus</italic> ATCC 14579 by in-depth characterization of a markerless <italic>secDF</italic> knock out mutant. Deletion of <italic>secDF</italic> resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the Δ<italic>secDF</italic> mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of <italic>secDF</italic> indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen <italic>B. cereus</italic>, and further supports the notion that <italic>B. cereus</italic> enterotoxins are secreted by the Sec-system.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Isobe, M" uniqKey="Isobe M">M Isobe</name></author><author><name sortKey="Ishikawa, T" uniqKey="Ishikawa T">T Ishikawa</name></author><author><name sortKey="Suwan, S" uniqKey="Suwan S">S Suwan</name></author><author><name sortKey="Agata, N" uniqKey="Agata N">N Agata</name></author><author><name sortKey="Ohta, M" uniqKey="Ohta M">M Ohta</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Beecher, Dj" uniqKey="Beecher D">DJ Beecher</name></author><author><name sortKey="Schoeni, Jl" uniqKey="Schoeni J">JL Schoeni</name></author><author><name sortKey="Wong, 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25083861</article-id><article-id pub-id-type="pmc">4118872</article-id><article-id pub-id-type="publisher-id">PONE-D-14-14114</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0103326</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Medicine and Health Sciences</subject><subj-group><subject>Pathology and Laboratory Medicine</subject><subj-group><subject>Pathogens</subject><subj-group><subject>Virulence Factors</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Proteomics</subject></subj-group></subj-group><subj-group><subject>Cell Biology</subject><subj-group><subject>Cellular Structures and Organelles</subject><subj-group><subject>Flagella</subject><subj-group><subject>Bacterial Flagella</subject></subj-group></subj-group></subj-group></subj-group><subj-group><subject>Computational Biology</subject><subj-group><subject>Genome Analysis</subject><subj-group><subject>Transcriptome Analysis</subject><subj-group><subject>Genome Expression Analysis</subject></subj-group></subj-group></subj-group></subj-group><subj-group><subject>Genetics</subject><subj-group><subject>Genomics</subject><subj-group><subject>Microbial Genomics</subject><subj-group><subject>Bacterial Genomics</subject><subj-group><subject>Bacterial Genomes</subject></subj-group></subj-group></subj-group></subj-group><subj-group><subject>Gene Expression</subject></subj-group></subj-group><subj-group><subject>Microbiology</subject><subj-group><subject>Bacteriology</subject><subj-group><subject>Bacterial Physiology</subject><subj-group><subject>Secretion Systems</subject></subj-group></subj-group><subj-group><subject>Gram Positive Bacteria</subject></subj-group></subj-group><subj-group><subject>Medical Microbiology</subject><subj-group><subject>Microbial Pathogens</subject><subj-group><subject>Bacterial Pathogens</subject><subj-group><subject>Bacillus</subject><subj-group><subject>Bacillus Cereus</subject><subject>Bacillus Subtilis</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group><subject>Microbial Control</subject><subj-group><subject>Antimicrobials</subject></subj-group></subj-group><subj-group><subject>Microbial Physiology</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of <italic>Bacillus cereus</italic> Toxins and Cell Wall-Associated Proteins</article-title><alt-title alt-title-type="running-head">SecDF Facilitates Efficient Toxin Secretion</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Vörös</surname><given-names>Aniko</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Simm</surname><given-names>Roger</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="author-notes" rid="fn1"><sup>¤</sup></xref></contrib><contrib contrib-type="author"><name><surname>Slamti</surname><given-names>Leyla</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>McKay</surname><given-names>Matthew J.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hegna</surname><given-names>Ida K.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Nielsen-LeRoux</surname><given-names>Christina</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hassan</surname><given-names>Karl A.</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Paulsen</surname><given-names>Ian T.</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lereclus</surname><given-names>Didier</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Økstad</surname><given-names>Ole Andreas</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Molloy</surname><given-names>Mark P.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kolstø</surname><given-names>Anne-Brit</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Australian Proteome Analysis Facility (APAF), Macquarie University, Sydney, Australia</addr-line></aff><aff id="aff3"><label>3</label><addr-line>INRA, UMR1319 Micalis, Domaine de La Minière, Guyancourt, France</addr-line></aff><aff id="aff4"><label>4</label><addr-line>AgroParistech, UMR Micalis, Jouy-en-Josas, France</addr-line></aff><aff id="aff5"><label>5</label><addr-line>Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Freitag</surname><given-names>Nancy E.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>University of Illinois at Chicago College of Medicine, United States of America</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>a.b.kolsto@farmasi.uio.no</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: AV RS ABK. Performed the experiments: AV RS LS IH CNL MJM KH. Analyzed the data: AV RS KH ITP DL OAØ MPM ABK. Contributed reagents/materials/analysis tools: KH MPM CNL DL ITP ABK. Contributed to the writing of the manuscript: AV RS OAØ ABK. Revising the article for important intellectual content: AV LS IKH CNL KH ITP DL MPM ABK. Final approval: AV RS LS MJM IH CNL KH ITP DL OAØ MPM ABK.</p></fn><fn id="fn1" fn-type="current-aff"><label>¤</label><p>Current address: Department of Biochemistry, Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway and Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>1</day><month>8</month><year>2014</year></pub-date><volume>9</volume><issue>8</issue><elocation-id>e103326</elocation-id><history><date date-type="received"><day>29</day><month>3</month><year>2014</year></date><date date-type="accepted"><day>26</day><month>6</month><year>2014</year></date></history><permissions><copyright-year>2014</copyright-year><copyright-holder>Vörös et al</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>The aim of this study was to explore the role of SecDF in protein secretion in <italic>Bacillus cereus</italic> ATCC 14579 by in-depth characterization of a markerless <italic>secDF</italic> knock out mutant. Deletion of <italic>secDF</italic> resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the Δ<italic>secDF</italic> mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of <italic>secDF</italic> indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen <italic>B. cereus</italic>, and further supports the notion that <italic>B. cereus</italic> enterotoxins are secreted by the Sec-system.</p></abstract><funding-group><funding-statement>The work was funded by The Norwegian Research Council (FUGE II) and EU grant IRSES-GA-2009_247634. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="17"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>The authors confirm that all data underlying the findings are fully available without restriction. All detailed procedures and raw data relating to the microarray analyses were deposited according to MIAME guidelines in the Arrayexpress database accession number E-MTAB-1759. <ext-link ext-link-type="uri" xlink:href="https://www.ebi.ac.uk/arrayexpress/arrays/browse.html?directsub=on">https://www.ebi.ac.uk/arrayexpress/arrays/browse.html?directsub=on</ext-link>.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>The authors confirm that all data underlying the findings are fully available without restriction. All detailed procedures and raw data relating to the microarray analyses were deposited according to MIAME guidelines in the Arrayexpress database accession number E-MTAB-1759. <ext-link ext-link-type="uri" xlink:href="https://www.ebi.ac.uk/arrayexpress/arrays/browse.html?directsub=on">https://www.ebi.ac.uk/arrayexpress/arrays/browse.html?directsub=on</ext-link>.</p></notes></front><body><sec id="s1"><title>Introduction</title><p><italic>Bacillus cereus sensu stricto</italic> is a Gram-positive spore-forming bacterium producing several toxins associated with food-borne disease. While cereulide has been shown to cause the emetic syndrome <xref rid="pone.0103326-Isobe1" ref-type="bibr">[1]</xref>, the pore-forming toxins cytotoxin K (CytK), haemolysin BL (Hbl) and nonhaemolytic enterotoxin (Nhe) inflict diarrhea <xref rid="pone.0103326-Beecher1" ref-type="bibr">[2]</xref>–<xref rid="pone.0103326-Lund2" ref-type="bibr">[4]</xref>. Fagerlund and co-workers have advocated that secretion of CytK and Nhe- and Hbl-components in <italic>B. cereus</italic> is directed via the Sec-translocase system <xref rid="pone.0103326-Fagerlund1" ref-type="bibr">[5]</xref>. SecDF is widely conserved across bacterial genera but is believed to be an accessory, non-essential protein component of the Sec-complex, the main protein secretion machinery in bacteria <xref rid="pone.0103326-Pogliano1" ref-type="bibr">[6]</xref>–<xref rid="pone.0103326-LycklamaaNijeholt1" ref-type="bibr">[8]</xref>. S<italic>ecDF</italic> deletion has been shown to result in low-temperature sensitivity, aberrant cell division and impaired protein secretion in <italic>Escherichia coli</italic>, <italic>Staphylococcus aureus</italic> and <italic>Bacillus subtilis</italic><xref rid="pone.0103326-Bolhuis1" ref-type="bibr">[9]</xref>–<xref rid="pone.0103326-Quiblier2" ref-type="bibr">[12]</xref>. SecDF exhibits the typical structure of RND-type (<underline>R</underline>esistance-<underline>N</underline>odulation-Cell <underline>D</underline>ivision) transporters with 12 transmembrane helices and two large extracytoplasmatic loops. However, tertiary and quarternary structures differ from the well described drug efflux-mediating RND transporters. Members of the RND transporter family are generally required for effective efflux of potentially cytotoxic compounds from the cell <xref rid="pone.0103326-Daniels1" ref-type="bibr">[13]</xref>, and their overexpression can confer multi-drug resistance in human pathogens <xref rid="pone.0103326-Webber1" ref-type="bibr">[14]</xref>. However, drug efflux is not necessarily the major function of most of the exporters, and their involvement in processes such as metal-ion homeostasis, quorum sensing, maintenance of cell homeostasis, interaction with plant or animal hosts, or efflux of toxic metabolic intermediates, fatty acids or other substances produced by the bacteria themselves, has been reported <xref rid="pone.0103326-Bazzini1" ref-type="bibr">[15]</xref>–<xref rid="pone.0103326-Sthler1" ref-type="bibr">[19]</xref>.</p><p>The exact role of SecDF during the protein translocation process has not yet been elucidated in detail. Based on SecDF crystal structures and <italic>in vitro</italic> experiments Tsukazaki and co-workers presented a model describing the proton motive force-dependent role of SecDF during later stage of protein translocation, where efficient protein translocation by SecDF is facilitated by preventing the emerging preprotein from backsliding into the SecYEG channel <xref rid="pone.0103326-Tsukazaki1" ref-type="bibr">[20]</xref>. Indeed, the charged residues shown to be important for H<sup>+</sup> translocation by other RND-type transporters are conserved in the SecDF proteins <xref rid="pone.0103326-LycklamaaNijeholt1" ref-type="bibr">[8]</xref>. Interestingly, in an early work Schiebel <italic>et al.</italic> estimated that in the absence of the PMF the costs of protein translocation increase from under 200 ATP units to several thousand ATP molecules per protein <xref rid="pone.0103326-Schiebel1" ref-type="bibr">[21]</xref>.</p><p>Previous reports suggested that SecDF is not an essential part of the Sec-translocase and fulfills only a noticeable function in secretion under protein hyper-expression and/or low temperature conditions. However, since the protein is ubiquitous, a more profound biological function is plausible. An important role in protein secretion has recently been acknowledged by Quiblier and co-workers (<xref rid="pone.0103326-Quiblier1" ref-type="bibr">[11]</xref>, <xref rid="pone.0103326-Quiblier2" ref-type="bibr">[12]</xref>, and indeed, a <italic>Staphylococcus aureus secDF</italic> knock out strain displays less virulence in an insect model, and less cytotoxicity to human umbilical vein endothelial cells, than its isogenic wild type strain <xref rid="pone.0103326-Quiblier2" ref-type="bibr">[12]</xref>. In this study we report that SecDF exhibits a substantial function in protein secretion in the spore-forming opportunistic pathogen <italic>B. cereus</italic>, severely affecting cellular export of major toxins and other virulence factors and resulting in reduced virulence of the Δ<italic>secDF</italic> mutant in insect larvae, thus providing additional evidence for Sec-dependent secretion of the <italic>B. cereus</italic> enterotoxins.</p></sec><sec id="s2"><title>Results</title><sec id="s2a"><title>The ΔsecDF knock out mutant is affected in growth, shape and motility</title><p>A markerless <italic>secDF</italic> deletion mutant was investigated for phenotypic alterations relative to the isogenic wild type strain <italic>B. cereus</italic> ATCC 14579. Bolhuis <italic>et al.</italic> reported a strong activation of the <italic>B. subtilis secDF</italic> promoter by the addition of glucose to the growth medium <xref rid="pone.0103326-Bolhuis1" ref-type="bibr">[9]</xref>. There was a small but consistent lag in growth during the exponential phase of Δ<italic>secDF</italic> mutant compared to the wild type in LB medium at 30°C as well as at 37°C (<xref ref-type="fig" rid="pone-0103326-g001">Fig. 1A</xref> and data not shown). In LB medium supplemented with 1% glucose (from now on referred to as LBG) growth of the Δ<italic>secDF</italic> mutant was slightly slower than the wild type, and the Δ<italic>secDF</italic> mutant did not reach the culture densities of the wild type at either 20°C, 30°C or 37°C, during the time window investigated (<xref ref-type="fig" rid="pone-0103326-g001">Fig. 1A</xref> and data not shown). After 24 h growth, microscopy showed that most Δ<italic>secDF</italic> mutant cells appeared in uncharacteristically crooked chains (<xref ref-type="fig" rid="pone-0103326-g001">Fig. 1B</xref>). These growth-related effects of the <italic>secDF</italic> deletion could be circumvented by complementation with SecDF (<xref ref-type="supplementary-material" rid="pone.0103326.s003">Fig. S3</xref>, left). The mutant displayed a smaller colony size compared to the wild type on LB and LBG agar plates, and this was more pronounced in the presence of glucose (<xref ref-type="fig" rid="pone-0103326-g001">Fig. 1C</xref>) and at lower temperatures (data not shown). Growth of the wild type and mutant strains on <italic>B. cereus</italic> agar containing bromothymol blue as pH indicator did not indicate differential production of acidic by-products as a result of glucose fermentation (data not shown).</p><fig id="pone-0103326-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.g001</object-id><label>Figure 1</label><caption><title>Growth characteristics of the Δ<italic>secDF</italic> mutant in LB with and without glucose.</title><p>Growth of <italic>B. cereus</italic> ATCC 14579 (WT) and <italic>ΔsecDF</italic> mutant in <bold>A</bold>) LB (no glucose) and <bold>B</bold>) LBG (1% glucose) in shaking cultures at 30°C. The graphs show average OD<sub>600nm</sub> values with standard deviations of two independent cultures for each strain. <bold>C</bold>) light micrographs of cultures after 24 h growth. <bold>D</bold>) growth of WT (left) and the Δ<italic>secDF</italic> mutant (right) at 30°C for 16 h on LB and LBG agar. All pictures represent results of at least two independent experiments.</p></caption><graphic xlink:href="pone.0103326.g001"></graphic></fig><p>Microscopy analyses of LBG liquid cultures had clearly showed a decreased motility of the Δ<italic>secDF</italic> mutant compared to the wild type after 4 h of growth. When analyzed on 0.3% LB agar plates, motility of the <italic>ΔsecDF</italic> mutant was approximately half of the wild type, whereas following addition of glucose, maltose or sucrose, the corresponding relative motility was below 10% (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2A</xref>). Severe reduction in motility was also observed on 0.7% LBG agar (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2B</xref>). In <italic>B. subtilis</italic> secretion of the surface-tension reducing compound surfactin enables flagellum-independent motility <xref rid="pone.0103326-Kinsinger1" ref-type="bibr">[22]</xref>. To test if differences in surface tension could explain the mutant motility phenotype, Tween 80 was added to the medium <xref rid="pone.0103326-Niu1" ref-type="bibr">[23]</xref>. This resulted in partly restored motility of the <italic>secDF</italic> mutant to almost 80% of wild type movement on medium supplemented with Tween 80. Simultaneous addition of Tween 80 and glucose resulted in 75% inhibition of motility relative to wild type under the same conditions (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2A</xref>), showing that a missing surfactant was not the only cause of reduced motility in the <italic>ΔsecDF</italic> mutant. Atomic force microscopy (AFM) amplitude images of Δ<italic>secDF</italic> and wild type cells grown for 4 h in LBG showed that the mutant displayed about five times reduced number of flagella per cell in two independent experiments (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2C</xref>), which may explain its decreased motility (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2A and B</xref>). In addition, AFM amplitude images revealed a higher number of extracellular structures in the wild type compared to the Δ<italic>secDF</italic> mutant samples (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2C</xref>), possibly representing extracellular vesicles <xref rid="pone.0103326-Thay1" ref-type="bibr">[24]</xref>.</p><fig id="pone-0103326-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.g002</object-id><label>Figure 2</label><caption><title>Diminished motility of the Δ<italic>secDF</italic> mutant.</title><p><bold>A</bold>) Motility of the Δ<italic>secDF</italic> mutant compared to the wild type strain on 0.3% LB only —, or LB agar plates supplemented with: gluc, 0.4% glucose; malt, 1% maltose; sucr, 1% sucrose; xyl, 1% xylose; sorb, 1% sorbitol; galac, 1% galactose; tw, 0.02% Tween80, tw+gluc, 0.02% Tween80 and 0.4% glucose. The graph shows averages of four to ten independent experiments, error bars represent standard errors and an unpaired Students t-test with two-tailed distribution was performed between wild type and Δ<italic>secDF</italic> mutant (all conditions <italic>P</italic><0.05). A nonparametric ANOVA with Dunn's multiple comparison <italic>post hoc</italic> test was performed for “LB only” and each of the conditions using additives (*<italic>P</italic><0.01; **<italic>P</italic><0.001). No movement of the Δ<italic>secDF</italic> mutant was recorded in LB+sucrose in four experiments. <bold>B</bold>) Comparison of motility on 0.7% LBG after 7 h incubation at 30°C; top: wild type; bottom: Δ<italic>secDF</italic> mutant. <bold>C</bold>) AFM amplitude images representative of two independent experiments of cells grown in LBG for 4 h show the grade of flagellation and secretion of putative membrane vesicles. Bars: 1 µm in whole cell images; 0.2 µm in the wild type detail image indicating putative vesicles (arrows).</p></caption><graphic xlink:href="pone.0103326.g002"></graphic></fig></sec><sec id="s2b"><title>SecDF deletion reduces resistance of B. cereus to xenobiotics</title><p>The 12-transmembrane secondary structure of SecDF is shared by other RND-type transporters known to mediate the efflux of a wide range of xenobiotics. In order to test if SecDF displays similar functions in addition to its role in protein translocation, the effect of SecDF expression in <italic>E. coli ΔacrB</italic> on the susceptibility towards various compounds relative to an empty vector control was tested (<xref ref-type="supplementary-material" rid="pone.0103326.s006">Tables S1</xref> and <xref ref-type="supplementary-material" rid="pone.0103326.s007">S2</xref>). Deletion of <italic>acrB</italic> in <italic>E. coli</italic>, coding for the main xenobiotic efflux transporter in this organism, leads to hypersusceptibility to various toxic compounds <xref rid="pone.0103326-Bohnert1" ref-type="bibr">[25]</xref>. Furthermore, in search for additional phenotypic traits resulting from s<italic>ecDF</italic> deletion in <italic>B. cereus</italic>, minimal inhibitory concentration (MIC) and disk diffusion assays of several xenobiotics were conducted with the <italic>B. cereus</italic> Δs<italic>ecDF</italic> mutant and wild type strains. The Δ<italic>secDF</italic> strain exhibited reduced tolerance to SDS and to the aminoglycoside antibiotic gentamicin, and the reduction in tolerance was amplified in the presence of glucose. We also observed a four-fold decrease in the resistance towards the widely used food preservative sodium benzoate, and a two-fold decreased resistance towards the antimicrobial polymyxin B in LBG medium. Strong effects on growth of the mutant were observed with alcoholic plant extracts of peppermint, calabash plant, and tea tree (<xref ref-type="supplementary-material" rid="pone.0103326.s001">Fig. S1</xref>). While expression of SecDF from the vector pHT304-pXyl in the wild type <italic>B. cereus</italic> strain did not result in modified resistance to any of the seven compounds tested, heterologous expression of SecDF in <italic>E. coli ΔacrB</italic> produced increased sodium benzoate resistance (<xref ref-type="supplementary-material" rid="pone.0103326.s007">Table S2</xref> and data not shown), in accordance with the results from the <italic>B. cereus secDF</italic> deletion mutant.</p></sec><sec id="s2c"><title>The secDF deletion mutant exhibits a reduced level of secreted proteins</title><p>To test the effect of deleting <italic>secDF</italic> on the secretome of <italic>B. cereus</italic>, we compared the amount of proteins in the growth medium of the wild type and mutant. Since the phenotypic alterations of the Δ<italic>secDF</italic> mutant seemed to be stronger when grown in glucose-containing medium, secretome analyses were carried out in the presence of 1% glucose. Silver staining following SDS-PAGE revealed a substantial overall reduction of total protein in the growth medium of the Δ<italic>secDF</italic> mutant relative to wild type at different stages of growth (<xref ref-type="fig" rid="pone-0103326-g003">Fig. 3</xref>). In addition, an increase of small proteins in the Δ<italic>secDF</italic> mutant secretome was observed. This, however did not seem to be due to an exacerbated proteolytic activity or autolysis rate of the mutant (see <xref ref-type="sec" rid="s4">method</xref> section).</p><fig id="pone-0103326-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.g003</object-id><label>Figure 3</label><caption><title>Decreased protein secretion in the Δ<italic>secDF</italic> mutant.</title><p>The Δ<italic>secDF</italic> mutant secretes less protein than the <italic>B. cereus</italic> ATCC 14579 wild type strain. Equal volumes of normalized and 40-fold concentrated supernatants collected after 3 h (exponential phase), 4 h (transition phase) and 6 h (stationary phase) of growth were applied on 4–20% gradient SDS-PAGE gels and silver stained. The gel represents results of two independent experiments.</p></caption><graphic xlink:href="pone.0103326.g003"></graphic></fig></sec><sec id="s2d"><title>Label-free mass spectrometry reveals an important role for SecDF in secretion of virulence determinants and putative cell wall modulating enzymes</title><p>To further identify which proteins are specifically dependent on SecDF for transport, a label-free proteomic analysis was conducted on three biological replicates of sterile filtered culture supernatants from the Δ<italic>secDF</italic> strain and its respective wild type, after 4 h of growth in LBG medium at 30°C. At this time point, motility of the deletion mutant was visibly reduced and the density of the culture was typically about 65% of the wild type strain (<xref ref-type="fig" rid="pone-0103326-g001">Fig. 1A</xref>). In total, 96 proteins were confidently identified in the secretome samples (<xref ref-type="supplementary-material" rid="pone.0103326.s008">Table S3</xref>). According to the PSORTb algorithm (version 3.0.2; <xref rid="pone.0103326-Yu1" ref-type="bibr">[26]</xref>) 29 of these proteins (30%) were either extracellular or cell wall-associated, six (6%) were anticipated to be located within the cytoplasmic membrane, while the majority (55) of the proteins were of cytoplasmic origin (57%). For the remaining 6 proteins (6%) no convincing localization prediction could be made based on sequence similarities with known proteins. However, two of the six contained a putative signal peptide, suggesting an extracellular localization.</p><p>Using a paired Students T-test on normalized spectral abundance factors (NSAF, <xref rid="pone.0103326-Zybailov1" ref-type="bibr">[27]</xref>) 34 of the 96 identified proteins were shown to be present at significantly different levels when comparing growth supernatants of the Δ<italic>secDF</italic> mutant and the <italic>B. cereus</italic> wild type (<xref ref-type="table" rid="pone-0103326-t001">Table 1</xref>), indicating fundamental differences in protein secretion between the strains. All the proteins present at reduced levels in the culture supernatant of the Δ<italic>secDF</italic> strain compared to the wild type, were predicted or are known to be extracellular or cell wall-associated (<xref ref-type="table" rid="pone-0103326-t001">Table 1</xref>). Phospholipase C and sphingomyelinase were major protein components in the growth medium of the wild type cells, while they were absent or nearly absent in the <italic>secDF</italic> mutant (<xref ref-type="table" rid="pone-0103326-t001">Table 1</xref>). In addition, the Hbl and Nhe enterotoxin components and cytotoxin K were highly abundant in the extracellular environment of the wild type, while being present at low levels or absent in the mutant secretome. The M9A/M9B – type collagenase C (ColC, BC0556) was 18-fold reduced in the supernatant of the mutant. Another putative collagenase, Sfp (BC3762; also annotated as S-layer protein A), belonging to the intracellular subtilisin-related peptidase S8 group, was identified only in the wild type supernatant, in moderate amounts.</p><table-wrap id="pone-0103326-t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.t001</object-id><label>Table 1</label><caption><title>Proteins found in different amounts in the culture supernatants of the wild type and Δ<italic>secDF</italic> mutant (P-value <0.005).</title></caption><alternatives><graphic id="pone-0103326-t001-1" xlink:href="pone.0103326.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td colspan="2" align="left" rowspan="1">WT</td><td colspan="2" align="left" rowspan="1">Δ<italic>secDF</italic></td></tr><tr><td align="left" rowspan="1" colspan="1">#</td><td align="left" rowspan="1" colspan="1">Identified Proteins</td><td align="left" rowspan="1" colspan="1">Localization<xref ref-type="table-fn" rid="nt101">1</xref></td><td align="left" rowspan="1" colspan="1">locus tag</td><td align="left" rowspan="1" colspan="1">Uniprot Acc.Nr.</td><td align="left" rowspan="1" colspan="1">MW (kDa)</td><td align="left" rowspan="1" colspan="1">NSAF<xref ref-type="table-fn" rid="nt102">2</xref> avg</td><td align="left" rowspan="1" colspan="1">stdev</td><td align="left" rowspan="1" colspan="1">NSAF avg</td><td align="left" rowspan="1" colspan="1">stdev</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>less abundant proteins in the mutant</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">Cytotoxin K</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_1110</td><td align="left" rowspan="1" colspan="1">Q81GS6</td><td align="left" rowspan="1" colspan="1">37</td><td align="left" rowspan="1" colspan="1"><bold>0.14</bold></td><td align="left" rowspan="1" colspan="1">0.01</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold><xref ref-type="table-fn" rid="nt103">3</xref></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Enterotoxin/cell-wall binding protein EntB</td><td align="left" rowspan="1" colspan="1">EC <xref rid="pone.0103326-Clair1" ref-type="bibr">[28]</xref></td><td align="left" rowspan="1" colspan="1">BC_2952</td><td align="left" rowspan="1" colspan="1">Q81C32</td><td align="left" rowspan="1" colspan="1">55</td><td align="left" rowspan="1" colspan="1"><bold>0.028</bold></td><td align="left" rowspan="1" colspan="1">0.004</td><td align="left" rowspan="1" colspan="1"><bold>0.001</bold></td><td align="left" rowspan="1" colspan="1">0.001</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">Perfringolysin O</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_5101</td><td align="left" rowspan="1" colspan="1">Q815P0</td><td align="left" rowspan="1" colspan="1">57</td><td align="left" rowspan="1" colspan="1"><bold>0.004</bold></td><td align="left" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">Phospholipase C</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_0670</td><td align="left" rowspan="1" colspan="1">Q81HW1</td><td align="left" rowspan="1" colspan="1">32</td><td align="left" rowspan="1" colspan="1"><bold>0.55</bold></td><td align="left" rowspan="1" colspan="1">0.12</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Non-hemolytic enterotoxin NheB</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_1810</td><td align="left" rowspan="1" colspan="1">Q81EZ7</td><td align="left" rowspan="1" colspan="1">43</td><td align="left" rowspan="1" colspan="1"><bold>0.21</bold></td><td align="left" rowspan="1" colspan="1">0.05</td><td align="left" rowspan="1" colspan="1"><bold>0.01</bold></td><td align="left" rowspan="1" colspan="1">0.01</td></tr><tr><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">Putative murein endopeptidase</td><td align="left" rowspan="1" colspan="1">U</td><td align="left" rowspan="1" colspan="1">BC_1991</td><td align="left" rowspan="1" colspan="1">Q81EI5</td><td align="left" rowspan="1" colspan="1">44</td><td align="left" rowspan="1" colspan="1"><bold>0.028</bold></td><td align="left" rowspan="1" colspan="1">0.007</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Hemolysin BL lytic component L1</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_3103</td><td align="left" rowspan="1" colspan="1">Q7BYC6</td><td align="left" rowspan="1" colspan="1">44</td><td align="left" rowspan="1" colspan="1"><bold>0.18</bold></td><td align="left" rowspan="1" colspan="1">0.05</td><td align="left" rowspan="1" colspan="1"><bold>0.01</bold></td><td align="left" rowspan="1" colspan="1">0.01</td></tr><tr><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">Hemolysin BL lytic component L2</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_3104</td><td align="left" rowspan="1" colspan="1">Q81BP7</td><td align="left" rowspan="1" colspan="1">49</td><td align="left" rowspan="1" colspan="1"><bold>0.25</bold></td><td align="left" rowspan="1" colspan="1">0.08</td><td align="left" rowspan="1" colspan="1"><bold>0.004</bold></td><td align="left" rowspan="1" colspan="1">0.003</td></tr><tr><td align="left" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1">Sphingomyelin phosphodiesterase</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_0671</td><td align="left" rowspan="1" colspan="1">Q81HW0</td><td align="left" rowspan="1" colspan="1">37</td><td align="left" rowspan="1" colspan="1"><bold>0.28</bold></td><td align="left" rowspan="1" colspan="1">0.11</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">putative murein endopeptidase</td><td align="left" rowspan="1" colspan="1">CW</td><td align="left" rowspan="1" colspan="1">BC_0991</td><td align="left" rowspan="1" colspan="1">Q81H34</td><td align="left" rowspan="1" colspan="1">65</td><td align="left" rowspan="1" colspan="1"><bold>0.004</bold></td><td align="left" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1">Cell wall endopeptidase, family M23/M37</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_0740</td><td align="left" rowspan="1" colspan="1">Q81HR4</td><td align="left" rowspan="1" colspan="1">42</td><td align="left" rowspan="1" colspan="1"><bold>0.016</bold></td><td align="left" rowspan="1" colspan="1">0.006</td><td align="left" rowspan="1" colspan="1"><bold>0.001</bold></td><td align="left" rowspan="1" colspan="1">0.002</td></tr><tr><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">Hemolysin BL binding component</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_3102</td><td align="left" rowspan="1" colspan="1">Q81BP9</td><td align="left" rowspan="1" colspan="1">42</td><td align="left" rowspan="1" colspan="1"><bold>0.10</bold></td><td align="left" rowspan="1" colspan="1">0.05</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1">Microbial collagenase</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_0556</td><td align="left" rowspan="1" colspan="1">Q81I63</td><td align="left" rowspan="1" colspan="1">109</td><td align="left" rowspan="1" colspan="1"><bold>0.088</bold></td><td align="left" rowspan="1" colspan="1">0.039</td><td align="left" rowspan="1" colspan="1"><bold>0.005</bold></td><td align="left" rowspan="1" colspan="1">0.005</td></tr><tr><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1">Bacillolysin</td><td align="left" rowspan="1" colspan="1">EC</td><td align="left" rowspan="1" colspan="1">BC_5351</td><td align="left" rowspan="1" colspan="1">Q814S1</td><td align="left" rowspan="1" colspan="1">65</td><td align="left" rowspan="1" colspan="1"><bold>0.034</bold></td><td align="left" rowspan="1" colspan="1">0.013</td><td align="left" rowspan="1" colspan="1"><bold>0.005</bold></td><td align="left" rowspan="1" colspan="1">0.006</td></tr><tr><td align="left" rowspan="1" colspan="1">15</td><td align="left" rowspan="1" colspan="1">Non-hemolytic enterotoxin NheA</td><td align="left" rowspan="1" colspan="1">EC <xref rid="pone.0103326-Clair1" ref-type="bibr">[28]</xref></td><td align="left" rowspan="1" colspan="1">BC_1809</td><td align="left" rowspan="1" colspan="1">Q81EZ8</td><td align="left" rowspan="1" colspan="1">44</td><td align="left" rowspan="1" colspan="1"><bold>0.12</bold></td><td align="left" rowspan="1" colspan="1">0.05</td><td align="left" rowspan="1" colspan="1"><bold>0.01</bold></td><td align="left" rowspan="1" colspan="1">0.01</td></tr><tr><td align="left" rowspan="1" colspan="1">16</td><td align="left" rowspan="1" colspan="1">Flagellin<xref ref-type="table-fn" rid="nt104">*</xref></td><td align="left" rowspan="1" colspan="1">EC <xref rid="pone.0103326-LaVallie1" ref-type="bibr">[101]</xref></td><td align="left" rowspan="1" colspan="1">BC_1657-9</td><td align="left" rowspan="1" colspan="1">Q81FD3-5</td><td align="left" rowspan="1" colspan="1">29</td><td align="left" rowspan="1" colspan="1"><bold>0.51</bold></td><td align="left" rowspan="1" colspan="1">0.08</td><td align="left" rowspan="1" colspan="1"><bold>0.31</bold></td><td align="left" rowspan="1" colspan="1">0.10</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>more abundant proteins in the mutant</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">DNA-binding protein HU</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_3728</td><td align="left" rowspan="1" colspan="1">Q81A62</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1"><bold>0.15</bold></td><td align="left" rowspan="1" colspan="1">0.02</td><td align="left" rowspan="1" colspan="1"><bold>0.30</bold></td><td align="left" rowspan="1" colspan="1">0.02</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Foldase protein PrsA 1</td><td align="left" rowspan="1" colspan="1">M</td><td align="left" rowspan="1" colspan="1">BC_1043</td><td align="left" rowspan="1" colspan="1">PRSA1_BACCR</td><td align="left" rowspan="1" colspan="1">32</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>0.010</bold></td><td align="left" rowspan="1" colspan="1">0.002</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">3-oxoacyl-[acyl-carrier-protein] synthase 2</td><td align="left" rowspan="1" colspan="1">M</td><td align="left" rowspan="1" colspan="1">BC_1174</td><td align="left" rowspan="1" colspan="1">Q81GL9</td><td align="left" rowspan="1" colspan="1">44</td><td align="left" rowspan="1" colspan="1"><bold>ND</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><bold>0.007</bold></td><td align="left" rowspan="1" colspan="1">0.001</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">50S ribosomal protein L10</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0119</td><td align="left" rowspan="1" colspan="1">RL10_BACCR</td><td align="left" rowspan="1" colspan="1">18</td><td align="left" rowspan="1" colspan="1"><bold>0.009</bold></td><td align="left" rowspan="1" colspan="1">0.006</td><td align="left" rowspan="1" colspan="1"><bold>0.065</bold></td><td align="left" rowspan="1" colspan="1">0.011</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">30S ribosomal protein S10</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0130</td><td align="left" rowspan="1" colspan="1">RS10_BACCR</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1"><bold>0.066</bold></td><td align="left" rowspan="1" colspan="1">0.016</td><td align="left" rowspan="1" colspan="1"><bold>0.183</bold></td><td align="left" rowspan="1" colspan="1">0.029</td></tr><tr><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">DNA-binding protein HU</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_1510</td><td align="left" rowspan="1" colspan="1">Q81FQ9</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1"><bold>0.44</bold></td><td align="left" rowspan="1" colspan="1">0.09</td><td align="left" rowspan="1" colspan="1"><bold>0.75</bold></td><td align="left" rowspan="1" colspan="1">0.05</td></tr><tr><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Elongation factor G</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0128</td><td align="left" rowspan="1" colspan="1">EFG_BACCR</td><td align="left" rowspan="1" colspan="1">76</td><td align="left" rowspan="1" colspan="1"><bold>0.041</bold></td><td align="left" rowspan="1" colspan="1">0.010</td><td align="left" rowspan="1" colspan="1"><bold>0.074</bold></td><td align="left" rowspan="1" colspan="1">0.005</td></tr><tr><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">30S ribosomal protein S11</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0157</td><td align="left" rowspan="1" colspan="1">RS11_BACCR</td><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1"><bold>0.005</bold></td><td align="left" rowspan="1" colspan="1">0.009</td><td align="left" rowspan="1" colspan="1"><bold>0.077</bold></td><td align="left" rowspan="1" colspan="1">0.023</td></tr><tr><td align="left" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1">Putative triosephosphate isomerase</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_5137</td><td align="left" rowspan="1" colspan="1">TPIS_BACCR</td><td align="left" rowspan="1" colspan="1">26</td><td align="left" rowspan="1" colspan="1"><bold>0.005</bold></td><td align="left" rowspan="1" colspan="1">0.009</td><td align="left" rowspan="1" colspan="1"><bold>0.063</bold></td><td align="left" rowspan="1" colspan="1">0.019</td></tr><tr><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">50S ribosomal protein L6</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0146</td><td align="left" rowspan="1" colspan="1">RL6_BACCR</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1"><bold>0.002</bold></td><td align="left" rowspan="1" colspan="1">0.004</td><td align="left" rowspan="1" colspan="1"><bold>0.081</bold></td><td align="left" rowspan="1" colspan="1">0.028</td></tr><tr><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1">Putative uncharacterized protein</td><td align="left" rowspan="1" colspan="1">U</td><td align="left" rowspan="1" colspan="1">BC_p0002</td><td align="left" rowspan="1" colspan="1">Q814F0</td><td align="left" rowspan="1" colspan="1">18</td><td align="left" rowspan="1" colspan="1"><bold>0.10</bold></td><td align="left" rowspan="1" colspan="1">0.02</td><td align="left" rowspan="1" colspan="1"><bold>0.43</bold></td><td align="left" rowspan="1" colspan="1">0.12</td></tr><tr><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">50S ribosomal protein L1</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0118</td><td align="left" rowspan="1" colspan="1">RL1_BACCR</td><td align="left" rowspan="1" colspan="1">25</td><td align="left" rowspan="1" colspan="1"><bold>0.014</bold></td><td align="left" rowspan="1" colspan="1">0.009</td><td align="left" rowspan="1" colspan="1"><bold>0.065</bold></td><td align="left" rowspan="1" colspan="1">0.019</td></tr><tr><td align="left" rowspan="1" colspan="1">13</td><td align="left" rowspan="1" colspan="1">50S ribosomal protein L3</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0131</td><td align="left" rowspan="1" colspan="1">RL3_BACCR</td><td align="left" rowspan="1" colspan="1">23</td><td align="left" rowspan="1" colspan="1"><bold>0.001</bold></td><td align="left" rowspan="1" colspan="1">0.002</td><td align="left" rowspan="1" colspan="1"><bold>0.021</bold></td><td align="left" rowspan="1" colspan="1">0.009</td></tr><tr><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1">50S ribosomal protein L15</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0150</td><td align="left" rowspan="1" colspan="1">RL15_BACCR</td><td align="left" rowspan="1" colspan="1">15</td><td align="left" rowspan="1" colspan="1"><bold>0.003</bold></td><td align="left" rowspan="1" colspan="1">0.005</td><td align="left" rowspan="1" colspan="1"><bold>0.030</bold></td><td align="left" rowspan="1" colspan="1">0.011</td></tr><tr><td align="left" rowspan="1" colspan="1">15</td><td align="left" rowspan="1" colspan="1">Fructose-bisphosphate aldolase</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_5335</td><td align="left" rowspan="1" colspan="1">Q814T5</td><td align="left" rowspan="1" colspan="1">31</td><td align="left" rowspan="1" colspan="1"><bold>0.027</bold></td><td align="left" rowspan="1" colspan="1">0.008</td><td align="left" rowspan="1" colspan="1"><bold>0.054</bold></td><td align="left" rowspan="1" colspan="1">0.010</td></tr><tr><td align="left" rowspan="1" colspan="1">16</td><td align="left" rowspan="1" colspan="1">50S ribosomal protein L21</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_4438</td><td align="left" rowspan="1" colspan="1">RL21_BACCR</td><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1"><bold>0.085</bold></td><td align="left" rowspan="1" colspan="1">0.070</td><td align="left" rowspan="1" colspan="1"><bold>0.225</bold></td><td align="left" rowspan="1" colspan="1">0.032</td></tr><tr><td align="left" rowspan="1" colspan="1">17</td><td align="left" rowspan="1" colspan="1">50S ribosomal protein L4</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_0132</td><td align="left" rowspan="1" colspan="1">RL4_BACCR</td><td align="left" rowspan="1" colspan="1">23</td><td align="left" rowspan="1" colspan="1"><bold>0.018</bold></td><td align="left" rowspan="1" colspan="1">0.007</td><td align="left" rowspan="1" colspan="1"><bold>0.035</bold></td><td align="left" rowspan="1" colspan="1">0.006</td></tr><tr><td align="left" rowspan="1" colspan="1">18</td><td align="left" rowspan="1" colspan="1">30S ribosomal protein S15</td><td align="left" rowspan="1" colspan="1">C</td><td align="left" rowspan="1" colspan="1">BC_3806</td><td align="left" rowspan="1" colspan="1">RS15_BACCR</td><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1"><bold>0.022</bold></td><td align="left" rowspan="1" colspan="1">0.014</td><td align="left" rowspan="1" colspan="1"><bold>0.067</bold></td><td align="left" rowspan="1" colspan="1">0.022</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt101"><label>1</label><p>according to prediction of PSORTb algorithm (version 3.0.2; <xref rid="pone.0103326-Yu1" ref-type="bibr">[26]</xref>): EC extracellular, C cytoplasmic, U unknown, M membrane; references for experimentally defined locations are given for proteins with predicted unknown localization.</p></fn><fn id="nt102"><label>2</label><p><bold>N</bold>ormalized <bold>S</bold>pectral <bold>A</bold>bundance <bold>F</bold>actor, mean average of three biological replicates; the NSAF normalizes across samples and takes protein sizes into account; values range between 0 and 1, increasing values indicate higher abundance <xref rid="pone.0103326-Zybailov1" ref-type="bibr">[27]</xref>, stdev standard deviation of the means of three biological replicates; probability ranges associated with Students t-test (Scaffold 4.0.5).</p></fn><fn id="nt103"><label>3</label><p>ND not detected (NSAF 0 in at least two biological replicates and <0.005).</p></fn><fn id="nt104"><label></label><p>*due to high sequence similarity all peptide hits for “flagellin” (Q81FD3, Q81FD4, Q81FD5) were combined.</p></fn></table-wrap-foot></table-wrap><p>Due to frequent flagellar turnover, flagellum structural components are common constituents of bacterial secretomes <xref rid="pone.0103326-Clair1" ref-type="bibr">[28]</xref>–<xref rid="pone.0103326-Kaakoush2" ref-type="bibr">[31]</xref>. In agreement with the observed motility deficiency of the mutant and the highly reduced number of flagella seen in AFM experiments, levels of several flagellum structural proteins were reduced in the Δ<italic>secDF</italic> secretome. <italic>B. cereus</italic> ATCC 14579 encodes three highly similar flagellin proteins (Q81FD3, Q81FD4, Q81FD5), whose peptide fragments could not be distinguished from each other by the applied analysis method and were therefore analyzed together. In total, slightly less flagellin was detected in the Δ<italic>secDF</italic> mutant growth medium (60% of wild type level, p = 0.049). Furthermore, the cell-wall associated hook protein FlgE was detected in one of three mutant replicates only (4% of wild type level, p-value 0.12, table S3), and the three structural flagellar hook-associated proteins 1, 2 and 3 were present on average 18%, 40% and 52% of the wild type levels, respectively (p-values = 0.06, 0.35, 0.08, table S3).</p><p>Several cell wall-associated proteins were also found to be differentially secreted in the Δ<italic>secDF</italic> mutant, most prominently the putative murein hydrolases BC0991 and BC1991 which were absent in the medium of the Δ<italic>secDF</italic> mutant (<xref ref-type="table" rid="pone-0103326-t001">Table 1</xref>). Furthermore, EntB (BC2952), annotated as enterotoxin/cell-wall binding protein, was present at 33-fold lower levels (p<0.001) and was, in fact, not detectable in two out of three biological replicates. The similar proteins EntA (BC5239) and EntC (BC0813) did not show this trend, as the abundances varied across the samples.</p><p>In contrast to the less abundant proteins in the Δ<italic>secDF</italic> secretome, most of the 18 proteins found at higher levels in the growth medium of the mutant relative to wild type typically had intracellular functions, including ten ribosomal proteins, with sizes ranging between 11 and 25 kDa. Finally, it is also worth noticing that the so far uncharacterized putative enterotoxin BC1953 was among the most abundant proteins in the wild type secretome at the time of sampling (<xref ref-type="supplementary-material" rid="pone.0103326.s008">Table S3</xref>).</p></sec><sec id="s2e"><title>Toxin translocation is reduced in the ΔsecDF mutant</title><p>Mass spectrometry analysis of the Δ<italic>secDF</italic> secretome indicated a potentially important function for the SecDF moiety in translocation of <italic>B. cereus</italic> proteins, including toxins and other virulence factors. To further characterize this phenomenon, Western blot analyzes were conducted on both the growth medium and cell lysates using monoclonal antibodies against the Hbl toxin components L1 and L2 as well as against NheA and NheB <xref rid="pone.0103326-Dietrich1" ref-type="bibr">[32]</xref>, <xref rid="pone.0103326-Dietrich2" ref-type="bibr">[33]</xref> (<xref ref-type="fig" rid="pone-0103326-g004">Fig. 4</xref>). In the absence of added glucose, the level of toxin components in the growth medium was reduced in the Δ<italic>secDF</italic> mutant compared to the wild type after 3 h, 4 h and 6 h in LBG medium, but reached wild type levels after 6 h incubation in LB medium (<xref ref-type="fig" rid="pone-0103326-g004">Fig. 4</xref>). Over the same time period, cell-associated toxin components accumulated to a higher level in the Δ<italic>secDF</italic> mutant compared to the wild type (<xref ref-type="fig" rid="pone-0103326-g004">Fig. 4</xref>). Complementation assays in the Δ<italic>secDF</italic> mutant restored its ability to translocate and averted the cellular accumulation of the indicated toxin components (<xref ref-type="supplementary-material" rid="pone.0103326.s003">Fig. S3</xref>). Thus, differences in protein abundances in the Δ<italic>secDF</italic> mutant secretome are most likely due to inhibition of toxin translocation across the plasma membrane rather than downregulation of toxin gene transcription or translation. In general, extracellular toxin levels decreased both in wild type and Δ<italic>secDF</italic> mutant cells when grown in the presence of added glucose (<xref ref-type="fig" rid="pone-0103326-g004">Fig. 4</xref>), however, the difference in cellular accumulation of toxin components in the Δ<italic>secDF</italic> mutant relative to the wild type was most prominent in cultures grown in LBG rather than in LB (<xref ref-type="fig" rid="pone-0103326-g004">Fig. 4</xref>).</p><fig id="pone-0103326-g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.g004</object-id><label>Figure 4</label><caption><title>Comparison of NheA, NheB, Hbl_L1 and Hbl_L2 secretion by western-blot analysis.</title><p>Western-blot assay of secreted (<bold>left</bold>) and cell associated (<bold>right</bold>) toxin components NheA, NheB, Hbl_L1 and Hbl_L2. Samples of the growth medium were taken from the wild type (WT) and the Δ<italic>secDF</italic> mutant (Δ) from 3 h (exponential phase), 4 h (transition phase) and 6 h (stationary phase) cultures with and without added glucose. The blots are representative of at least two biological replicates. To visualize size differences between pre- and mature proteins, a supernatant wild type sample (SN6H) has also been applied to the blot showing cell associated protein.</p></caption><graphic xlink:href="pone.0103326.g004"></graphic></fig><p>While PC-PLC was the second most abundant protein in the wild type secretome, levels in the Δ<italic>secDF</italic> mutant culture medium were below the detection limit (<xref ref-type="table" rid="pone-0103326-t001">Table 1</xref>). To confirm the proteome data, both strains were grown in LB and LBG, and culture medium was collected periodically. The PC-PLC activity of sterile-filtered medium on egg yolk agar indicated reduced PC-PLC secretion by the Δ<italic>secDF</italic> mutant (<xref ref-type="fig" rid="pone-0103326-g005">Fig. 5B</xref>). Notably, growth in LBG resulted in hardly any visible PC-PLC activity in the mutant culture. Simultaneously, secretion of PC-PLC into the agar by actively growing cells was not detected on LBG agar containing egg yolk (<xref ref-type="supplementary-material" rid="pone.0103326.s002">Fig. S2A</xref>). In a more sensitive approach to determine PC-PLC activity, culture supernatants were incubated with egg yolk suspension and the substrate degradation measured photometrically (<xref ref-type="fig" rid="pone-0103326-g005">Fig. 5A</xref>). These experiments showed that extracellular PC-PLC activity from the <italic>secDF</italic> mutant grown in LBG remains at about 35% of the wild type activity over the studied time course. While the presence of glucose reduced PC-PLC activity in both strains, the effect was more pronounced for the mutant (<xref ref-type="fig" rid="pone-0103326-g005">Fig. 5</xref>).</p><fig id="pone-0103326-g005" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.g005</object-id><label>Figure 5</label><caption><title>Reduced PC-PLC activity in the Δ<italic>secDF</italic> mutant.</title><p>A PC-PLC activity assay indicates reduced enzyme activity in the Δ<italic>secDF</italic> mutant compared to the wild type strain. <bold>A</bold>) Filter-sterilized supernatant of cultures grown in LB (no glucose) or LBG (1% glucose) were assayed in a 2% egg-yolk solution. The results are the mean values of two independent experiments, and error bars represent standard deviations. <bold>B</bold>) Five µl of filter-sterilized supernatant of cultures grown in LB or LBG were spotted on 1% egg yolk agar plates. t0 marks the transition point of growth into stationary phase, and t<italic>n</italic> is the number of hours before (-) or after t0. The pictures represent one of two independent experiments.</p></caption><graphic xlink:href="pone.0103326.g005"></graphic></fig></sec><sec id="s2f"><title>Deletion of SecDF affects virulence in Galleria mellonella</title><p>In order to test if the observed reduction of secreted virulence factors in batch cultures is mirrored by diminished virulence of the Δ<italic>secDF</italic> mutant, <italic>in vivo</italic> infection assays using <italic>Galleria mellonella</italic> (<italic>G. mellonella</italic>) larvae were conducted <xref rid="pone.0103326-Ramarao1" ref-type="bibr">[34]</xref>. Survival of larvae 24 h and 72 h post infection either administered by oral feeding or by injection into the insect blood hemocoel was monitored and the LD<sub>50</sub>s of the wild type strain and of the <italic>ΔsecDF</italic> mutant were evaluated by Probit analysis (<xref ref-type="table" rid="pone-0103326-t002">Table 2</xref>). The confidence limits at the 95% interval of lower (LDL) and upper level (UDL) doses of mutant and wild type strains were not overlapping. Thus, the about 4-fold and 3-fold differences in the dose killing 50% of the exposed larvae at 24 h and 72 h post infection by direct injection into the hemocoel of various doses (2x10<sup>3</sup> to ≈1x10<sup>5</sup>) of vegetative bacteria, respectively, are significant (p-values ≤0.05). Estimation of virulence at 24 h and 72 h post oral infection revealed about 17-fold and 13-fold LD<sub>50</sub> difference, respectively. These results clearly indicate a reduced virulence of the Δ<italic>secDF</italic> mutant strain towards the insect model which is more pronounced if larvae are infected orally.</p><table-wrap id="pone-0103326-t002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.t002</object-id><label>Table 2</label><caption><title>Role of SecDF in virulence against <italic>Galleria mellonella</italic> insect larvae.</title></caption><alternatives><graphic id="pone-0103326-t002-2" xlink:href="pone.0103326.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td colspan="4" align="left" rowspan="1">Oral force feeding<xref ref-type="table-fn" rid="nt106">b</xref></td><td colspan="4" align="left" rowspan="1">Hemocoel injection</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Hours post infection</td><td colspan="2" align="left" rowspan="1">24 h</td><td colspan="2" align="left" rowspan="1">72 h</td><td colspan="2" align="left" rowspan="1">24 h</td><td colspan="2" align="left" rowspan="1">72 h</td></tr><tr><td align="left" rowspan="1" colspan="1">Strain</td><td align="left" rowspan="1" colspan="1">WT</td><td align="left" rowspan="1" colspan="1">Δ<italic>secDF</italic></td><td align="left" rowspan="1" colspan="1">WT</td><td align="left" rowspan="1" colspan="1">Δ<italic>secDF</italic></td><td align="left" rowspan="1" colspan="1">WT</td><td align="left" rowspan="1" colspan="1">Δ<italic>secDF</italic></td><td align="left" rowspan="1" colspan="1">WT</td><td align="left" rowspan="1" colspan="1">Δ<italic>secDF</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt107">c</xref><bold>LD<sub>50</sub> cfu</bold></td><td align="left" rowspan="1" colspan="1"><bold>1.5x10<sup>6</sup></bold></td><td align="left" rowspan="1" colspan="1"><bold>26x10<sup>6</sup></bold></td><td align="left" rowspan="1" colspan="1"><bold>1.6x10<sup>6</sup></bold></td><td align="left" rowspan="1" colspan="1"><bold>21.1x10<sup>6</sup></bold></td><td align="left" rowspan="1" colspan="1"><bold>2.2x10<sup>4</sup></bold></td><td align="left" rowspan="1" colspan="1"><bold>9.9x10<sup>4</sup></bold></td><td align="left" rowspan="1" colspan="1"><bold>1.7x10<sup>4</sup></bold></td><td align="left" rowspan="1" colspan="1"><bold>4.6x10<sup>4</sup></bold></td></tr><tr><td align="left" rowspan="1" colspan="1"><xref ref-type="table-fn" rid="nt108">d</xref>LDL cfu</td><td align="left" rowspan="1" colspan="1">0.1x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">20.4x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">0.2x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">13.3x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">1.4x10<sup>4</sup></td><td align="left" rowspan="1" colspan="1">8.2x10<sup>4</sup></td><td align="left" rowspan="1" colspan="1">0.09x10<sup>4</sup></td><td align="left" rowspan="1" colspan="1">3.3x10<sup>4</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">UDL cfu</td><td align="left" rowspan="1" colspan="1">3.1x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">31.6x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">3.6x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">28.7x10<sup>6</sup></td><td align="left" rowspan="1" colspan="1">4.9x10<sup>4</sup></td><td align="left" rowspan="1" colspan="1">11.6x10<sup>4</sup></td><td align="left" rowspan="1" colspan="1">2.5x10<sup>4</sup></td><td align="left" rowspan="1" colspan="1">5.9x10<sup>4</sup></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Fold difference LD<sub>50</sub></bold></td><td colspan="2" align="left" rowspan="1"><bold>≈17.3</bold><xref ref-type="table-fn" rid="nt109">*</xref></td><td colspan="2" align="left" rowspan="1"><bold>≈13.2</bold><xref ref-type="table-fn" rid="nt109">*</xref></td><td colspan="2" align="left" rowspan="1"><bold>≈4.5</bold><xref ref-type="table-fn" rid="nt109">*</xref></td><td colspan="2" align="left" rowspan="1"><bold>≈2.7</bold><xref ref-type="table-fn" rid="nt109">*</xref></td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt105"><label>a</label><p>Infections with mid log phase (OD<sub>600nm</sub> = 1) vegetative bacteria cultured in LB medium.</p></fn><fn id="nt106"><label>b</label><p>For oral infection the bacteria are mixed with 3 µg/10 µl of Cry1C toxin. The toxin alone results in ≈10% mortality.</p></fn><fn id="nt107"><label>c</label><p>LC<sub>50</sub>, correspond to the bacterial dose (cfu, colony forming units) per larvae, killing 50% of the treated larvae.</p></fn><fn id="nt108"><label>d</label><p>confidence interval at 95% level.</p></fn><fn id="nt109"><label></label><p>*(<italic>P</italic>-value ≤0.05). LDL (lower dose limit), UDL (upper dose limit). Mortality was estimated by Probit analysis (StatPlus). based on at least two independent experiments. Control experiments were run with buffer (PBS) and no mortality occurred within 72 hours at 37°C.</p></fn></table-wrap-foot></table-wrap></sec><sec id="s2g"><title>Transcriptional profiling of the ΔsecDF mutant reveals the induction of multiple cellular stress responses</title><p>With the purpose of revealing molecular mechanisms linking the protein secretion defect and potential underlying processes to the observed phenotypic changes of the Δ<italic>secDF</italic> mutant, a global transcriptional profiling experiment was conducted. Custom-made microarray slides were hybridized with reverse transcribed RNA extracted from wild type and Δ<italic>secDF</italic> mutant cells at 3 h (two biological replicates) and 4 h (six biological replicates) of cultivation in LBG, on the basis that these time points mark the onset of morphological changes in the mutant compared to wild type. Significant differential expression was observed in more than 400 genes (>2-fold differential expression) during the transition phase (4 h). <xref ref-type="table" rid="pone-0103326-t003">Table 3</xref> lists 70 genes that exhibited confidently more than 5-fold differences in transcription levels at the 4 h time point. Quantitative RT-PCR confirmed the expression trend for 17 out of 18 selected genes (<xref ref-type="supplementary-material" rid="pone.0103326.s004">Fig. S4</xref>). In general, genes involved in metabolism and energy conversion processes, membrane transport, resistance and detoxification mechanisms, and motility, as well as several hypothetical genes, were most strongly affected. Furthermore, genes indicative of a cell wall stress response were stimulated in the Δ<italic>secDF</italic> mutant (<xref ref-type="table" rid="pone-0103326-t003">Table 3</xref>), including a phage shock response (<italic>pspA</italic>-like BC1436) gene, and an operon encoding a putative sigma W-type extracytoplasmatic function (ECF) sigma factor (BC5361-BC5363). Transcription of the genes <italic>entA</italic> (BC5239) and <italic>entC</italic> (BC0813) coding for putative cell-wall binding proteins were also upregulated. The <italic>entB</italic> gene (BC2952) showed a lower transcription level as well as a reduced amount of the EntB protein in the extracellular medium of the Δ<italic>secDF</italic> mutant.</p><table-wrap id="pone-0103326-t003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0103326.t003</object-id><label>Table 3</label><caption><title>Genes with at least a five-fold differential transcription level in the <italic>ΔsecDF</italic> mutant compared to the isogenic wild type strain <italic>B. cereus</italic> ATCC 14579.</title></caption><alternatives><graphic id="pone-0103326-t003-3" xlink:href="pone.0103326.t003"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Locus_tag<xref ref-type="table-fn" rid="nt110">1</xref></td><td align="left" rowspan="1" colspan="1">Genbank_annotation</td><td align="left" rowspan="1" colspan="1">FC<xref ref-type="table-fn" rid="nt111">2</xref></td><td align="left" rowspan="1" colspan="1">P–value<xref ref-type="table-fn" rid="nt112">3</xref></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Resistance/Detoxification</italic></bold></td><td align="left" rowspan="1" colspan="1">BC2984</td><td align="left" rowspan="1" colspan="1">Immune inhibitor A precursor</td><td align="left" rowspan="1" colspan="1">9.79</td><td align="left" rowspan="1" colspan="1">2.9E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC2985</td><td align="left" rowspan="1" colspan="1">Vancomycin B-type resistance protein vanW</td><td align="left" rowspan="1" colspan="1">8.63</td><td align="left" rowspan="1" colspan="1">1.6E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Transport</italic></bold></td><td align="left" rowspan="1" colspan="1">BC0816</td><td align="left" rowspan="1" colspan="1">periplasmic component of efflux system</td><td align="left" rowspan="1" colspan="1">5.01</td><td align="left" rowspan="1" colspan="1">2.9E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC3586</td><td align="left" rowspan="1" colspan="1">Oligopeptide-binding protein oppA</td><td align="left" rowspan="1" colspan="1">0.18</td><td align="left" rowspan="1" colspan="1">2.7E-03</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC3788</td><td align="left" rowspan="1" colspan="1">Nucleoside transport system permease protein</td><td align="left" rowspan="1" colspan="1">0.06</td><td align="left" rowspan="1" colspan="1">2.9E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC3790</td><td align="left" rowspan="1" colspan="1">Nucleoside transport ATP-binding protein</td><td align="left" rowspan="1" colspan="1">0.11</td><td align="left" rowspan="1" colspan="1">3.5E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC3791</td><td align="left" rowspan="1" colspan="1">Nucleoside-binding protein</td><td align="left" rowspan="1" colspan="1">0.06</td><td align="left" rowspan="1" colspan="1">1.7E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC3792</td><td align="left" rowspan="1" colspan="1">Transcriptional regulator, GntR family</td><td align="left" rowspan="1" colspan="1">0.09</td><td align="left" rowspan="1" colspan="1">1.4E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>BC4405</italic></td><td align="left" rowspan="1" colspan="1"><italic>Protein translocase subunit SecDF</italic></td><td align="left" rowspan="1" colspan="1">0.13</td><td align="left" rowspan="1" colspan="1">1.0E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC4831</td><td align="left" rowspan="1" colspan="1">ABC transporter ATP-binding protein</td><td align="left" rowspan="1" colspan="1">6.68</td><td align="left" rowspan="1" colspan="1">3.9E-08</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5117</td><td align="left" rowspan="1" colspan="1">ABC transporter permease protein</td><td align="left" rowspan="1" colspan="1">0.11</td><td align="left" rowspan="1" colspan="1">1.6E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5118</td><td align="left" rowspan="1" colspan="1">ABC transporter ATP-binding protein</td><td align="left" rowspan="1" colspan="1">0.12</td><td align="left" rowspan="1" colspan="1">3.1E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5253</td><td align="left" rowspan="1" colspan="1">ABC transporter permease protein</td><td align="left" rowspan="1" colspan="1">0.08</td><td align="left" rowspan="1" colspan="1">9.1E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5254</td><td align="left" rowspan="1" colspan="1">ABC transporter ATP-binding protein</td><td align="left" rowspan="1" colspan="1">0.11</td><td align="left" rowspan="1" colspan="1">5.0E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5255</td><td align="left" rowspan="1" colspan="1">periplasmic component of efflux system</td><td align="left" rowspan="1" colspan="1">0.08</td><td align="left" rowspan="1" colspan="1">8.2E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Metabolism</italic></bold></td><td align="left" rowspan="1" colspan="1">BC0297</td><td align="left" rowspan="1" colspan="1">Guanine-hypoxanthine permease</td><td align="left" rowspan="1" colspan="1">0.08</td><td align="left" rowspan="1" colspan="1">9.2E-08</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0323<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">PRAI carboxylase catalytic subunit</td><td align="left" rowspan="1" colspan="1">0.04</td><td align="left" rowspan="1" colspan="1">2.4E-08</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0324<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">PRAI carboxylase ATPase subunit</td><td align="left" rowspan="1" colspan="1">0.07</td><td align="left" rowspan="1" colspan="1">2.1E-08</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0325<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">Adenylosuccinate lyase</td><td align="left" rowspan="1" colspan="1">0.07</td><td align="left" rowspan="1" colspan="1">3.7E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0326<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">PRAI-succinocarboxamide synthase</td><td align="left" rowspan="1" colspan="1">0.04</td><td align="left" rowspan="1" colspan="1">2.4E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0327<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">PRFGA synthetase, PurS component</td><td align="left" rowspan="1" colspan="1">0.04</td><td align="left" rowspan="1" colspan="1">4.6E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0328<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">PRFGA synthase</td><td align="left" rowspan="1" colspan="1">0.04</td><td align="left" rowspan="1" colspan="1">1.9E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0329<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">PRFGA synthase</td><td align="left" rowspan="1" colspan="1">0.04</td><td align="left" rowspan="1" colspan="1">1.6E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0330<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">Amidophosphoribosyltransferase</td><td align="left" rowspan="1" colspan="1">0.04</td><td align="left" rowspan="1" colspan="1">4.8E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0331<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">PRFGA cyclo-ligase</td><td align="left" rowspan="1" colspan="1">0.04</td><td align="left" rowspan="1" colspan="1">6.2E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0332<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">Phosphoribosylglycinamide formyltransferase</td><td align="left" rowspan="1" colspan="1">0.05</td><td align="left" rowspan="1" colspan="1">1.6E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0333<xref ref-type="table-fn" rid="nt113">§</xref></td><td align="left" rowspan="1" colspan="1">IMP cyclohydrolase</td><td align="left" rowspan="1" colspan="1">0.06</td><td align="left" rowspan="1" colspan="1">6.1E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0491</td><td align="left" rowspan="1" colspan="1">Formate acetyltransferase</td><td align="left" rowspan="1" colspan="1">0.18</td><td align="left" rowspan="1" colspan="1">2.1E-04</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0492</td><td align="left" rowspan="1" colspan="1">Pyruvate formate-lyase activating enzyme</td><td align="left" rowspan="1" colspan="1">0.15</td><td align="left" rowspan="1" colspan="1">7.7E-04</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Respiration</italic></bold></td><td align="left" rowspan="1" colspan="1">BC1939</td><td align="left" rowspan="1" colspan="1">Cytochrome d ubiquinol oxidase subunit II</td><td align="left" rowspan="1" colspan="1">6.31</td><td align="left" rowspan="1" colspan="1">2.3E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC2119</td><td align="left" rowspan="1" colspan="1">Respiratory nitrate reductase beta chain</td><td align="left" rowspan="1" colspan="1">0.07</td><td align="left" rowspan="1" colspan="1">2.2E-04</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC2120</td><td align="left" rowspan="1" colspan="1">Respiratory nitrate reductase delta chain</td><td align="left" rowspan="1" colspan="1">0.20</td><td align="left" rowspan="1" colspan="1">3.2E-02</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC4792</td><td align="left" rowspan="1" colspan="1">Cytochrome d ubiquinol oxidase subunit I</td><td align="left" rowspan="1" colspan="1">0.14</td><td align="left" rowspan="1" colspan="1">8.6E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC4793</td><td align="left" rowspan="1" colspan="1">Cytochrome d ubiquinol oxidase subunit II</td><td align="left" rowspan="1" colspan="1">0.11</td><td align="left" rowspan="1" colspan="1">8.1E-04</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Putative Cell Wall Stress Response</italic></bold></td><td align="left" rowspan="1" colspan="1">BC0813</td><td align="left" rowspan="1" colspan="1">enterotoxin/cell-wall binding protein entC</td><td align="left" rowspan="1" colspan="1">6.35</td><td align="left" rowspan="1" colspan="1">6.3E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1435</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">33.96</td><td align="left" rowspan="1" colspan="1">2.1E-08</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1436</td><td align="left" rowspan="1" colspan="1">Phage shock protein A</td><td align="left" rowspan="1" colspan="1">12.83</td><td align="left" rowspan="1" colspan="1">7.9E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5239</td><td align="left" rowspan="1" colspan="1">enterotoxin/cell-wall binding protein entA</td><td align="left" rowspan="1" colspan="1">5.60</td><td align="left" rowspan="1" colspan="1">7.9E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5361</td><td align="left" rowspan="1" colspan="1">ECF-type sigma factor negative effector</td><td align="left" rowspan="1" colspan="1">12.40</td><td align="left" rowspan="1" colspan="1">1.7E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5362</td><td align="left" rowspan="1" colspan="1">ECF-type sigma factor negative effector</td><td align="left" rowspan="1" colspan="1">8.26</td><td align="left" rowspan="1" colspan="1">2.4E-08</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5363</td><td align="left" rowspan="1" colspan="1">RNA polymerase ECF-type sigma factor</td><td align="left" rowspan="1" colspan="1">16.82</td><td align="left" rowspan="1" colspan="1">4.8E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Motility</italic></bold></td><td align="left" rowspan="1" colspan="1">BC1657</td><td align="left" rowspan="1" colspan="1">Flagellin</td><td align="left" rowspan="1" colspan="1">0.18</td><td align="left" rowspan="1" colspan="1">1.8E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1659</td><td align="left" rowspan="1" colspan="1">Flagellin</td><td align="left" rowspan="1" colspan="1">0.19</td><td align="left" rowspan="1" colspan="1">6.7E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Sigma B operon</italic></bold></td><td align="left" rowspan="1" colspan="1">BC0862</td><td align="left" rowspan="1" colspan="1">Protease I</td><td align="left" rowspan="1" colspan="1">15.77</td><td align="left" rowspan="1" colspan="1">1.3E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0863</td><td align="left" rowspan="1" colspan="1">Catalase</td><td align="left" rowspan="1" colspan="1">13.31</td><td align="left" rowspan="1" colspan="1">4.2E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0998</td><td align="left" rowspan="1" colspan="1">General stress protein 17M</td><td align="left" rowspan="1" colspan="1">11.41</td><td align="left" rowspan="1" colspan="1">2.1E-08</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC0999</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">12.27</td><td align="left" rowspan="1" colspan="1">2.8E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1000</td><td align="left" rowspan="1" colspan="1">hypothetical Membrane Spanning Protein</td><td align="left" rowspan="1" colspan="1">12.54</td><td align="left" rowspan="1" colspan="1">6.7E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1002</td><td align="left" rowspan="1" colspan="1">Anti-sigma B factor antagonist</td><td align="left" rowspan="1" colspan="1">5.36</td><td align="left" rowspan="1" colspan="1">2.5E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1003</td><td align="left" rowspan="1" colspan="1">Anti-sigma B factor</td><td align="left" rowspan="1" colspan="1">8.97</td><td align="left" rowspan="1" colspan="1">1.4E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1004</td><td align="left" rowspan="1" colspan="1">RNA polymerase sigma-B factor</td><td align="left" rowspan="1" colspan="1">7.84</td><td align="left" rowspan="1" colspan="1">1.8E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1010</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">10.61</td><td align="left" rowspan="1" colspan="1">4.5E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC3130</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">5.30</td><td align="left" rowspan="1" colspan="1">7.4E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>Others</italic></bold></td><td align="left" rowspan="1" colspan="1">BC0494</td><td align="left" rowspan="1" colspan="1">hypothetical Cytosolic Protein</td><td align="left" rowspan="1" colspan="1">0.19</td><td align="left" rowspan="1" colspan="1">6.7E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1760</td><td align="left" rowspan="1" colspan="1">3-oxoacyl-[acyl-carrier-protein] synthase III</td><td align="left" rowspan="1" colspan="1">5.06</td><td align="left" rowspan="1" colspan="1">2.6E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1852</td><td align="left" rowspan="1" colspan="1">Exonuclease SbcC</td><td align="left" rowspan="1" colspan="1">0.20</td><td align="left" rowspan="1" colspan="1">3.4E-04</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1854</td><td align="left" rowspan="1" colspan="1">hypothetical Cytosolic Protein</td><td align="left" rowspan="1" colspan="1">0.20</td><td align="left" rowspan="1" colspan="1">1.4E-04</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC1861</td><td align="left" rowspan="1" colspan="1">DNA/RNA helicase (DEAD/DEAH box family)</td><td align="left" rowspan="1" colspan="1">0.20</td><td align="left" rowspan="1" colspan="1">3.2E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC2056</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">0.16</td><td align="left" rowspan="1" colspan="1">3.4E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC4482</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">5.32</td><td align="left" rowspan="1" colspan="1">6.5E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC4813</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">14.25</td><td align="left" rowspan="1" colspan="1">1.8E-07</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5116</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">0.16</td><td align="left" rowspan="1" colspan="1">1.3E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5119</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">0.12</td><td align="left" rowspan="1" colspan="1">2.8E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5120</td><td align="left" rowspan="1" colspan="1">hypothetical Cytosolic Protein</td><td align="left" rowspan="1" colspan="1">0.12</td><td align="left" rowspan="1" colspan="1">6.7E-06</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5121</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">0.12</td><td align="left" rowspan="1" colspan="1">1.7E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5122</td><td align="left" rowspan="1" colspan="1">hypothetical Cytosolic Protein</td><td align="left" rowspan="1" colspan="1">0.18</td><td align="left" rowspan="1" colspan="1">2.4E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5123</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">0.16</td><td align="left" rowspan="1" colspan="1">3.6E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5124</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">0.19</td><td align="left" rowspan="1" colspan="1">2.7E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5243</td><td align="left" rowspan="1" colspan="1">hypothetical protein</td><td align="left" rowspan="1" colspan="1">0.20</td><td align="left" rowspan="1" colspan="1">9.1E-05</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">BC5252</td><td align="left" rowspan="1" colspan="1">hypothetical Membrane Spanning Protein</td><td align="left" rowspan="1" colspan="1">0.11</td><td align="left" rowspan="1" colspan="1">2.3E-06</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt110"><label>1</label><p>data on the linear plasmid pBClin15 can be found in the supplementary file.</p></fn><fn id="nt111"><label>2</label><p>FC fold change of transcriptional expression in <italic>B. cereus</italic> Δ<italic>secDF</italic> compared to wild type.</p></fn><fn id="nt112"><label>3</label><p>P-values were computed using false discovery rate correction of 0.05 by an Bayesisn linear model as integrated in the Limma-package <xref rid="pone.0103326-Smyth1" ref-type="bibr">[90]</xref>; data represent six independent cultures.</p></fn><fn id="nt113"><label>§</label><p>purine operon under the control of PurA.</p></fn></table-wrap-foot></table-wrap><p>At the 4 h time point the Δ<italic>secDF</italic> mutant showed a highly activated sigma B stress response regulon compared to the wild type (<xref ref-type="table" rid="pone-0103326-t003">Table 3</xref>, <xref ref-type="supplementary-material" rid="pone.0103326.s009">Table S4</xref>). To confirm this, expression of <italic>sigB</italic> was followed over time by real-time quantitative PCR. While there were no significant changes (p<0.5) between the Δ<italic>secDF</italic> mutant and the wild type strain at early and mid-exponential growth phase, <italic>sigB</italic> was 3- to 17-fold induced in the mutant compared to the wild type at late-exponential and transition phase (p<0.01; data not shown). In total 14 out of 26 previously described heat-shock activated, sigma B-dependent genes <xref rid="pone.0103326-vanSchaik1" ref-type="bibr">[35]</xref>, were more than two-fold upregulated in the Δ<italic>secDF</italic> mutant (<xref ref-type="supplementary-material" rid="pone.0103326.s009">Table S4</xref>). The most strongly induced genes in the Δ<italic>secDF</italic> mutant were also among the highest heat-shock induced genes (e.g. those encoding KatE and Protease I). In order to test if these transcriptional changes translated into a cellular phenotype, the catalase activity of cultures grown for 6 h was measured. In support of the activation of the SigB operon, the Δ<italic>secDF</italic> mutant exhibited approximately 20% increased catalase activity (data not shown), however only when grown in the presence of glucose.</p><p>Secretome analysis of the <italic>B. cereus ΔsecDF</italic> mutant had revealed strongly reduced levels of virulence factors in the supernatant, which was confirmed by Western blot analyses of cell-accumulated and extracellular Hbl and Nhe toxin component levels, thus indicating SecDF-mediated export. Transcriptional levels were also altered for several (<italic>plcB, smase, colC</italic>, BC2552, <italic>nprB</italic>), but not all (<italic>cytK</italic>, <italic>nhe, hbl</italic>) PlcR-regulated virulence determinants (<xref ref-type="supplementary-material" rid="pone.0103326.s009">Table S4</xref>, <xref ref-type="supplementary-material" rid="pone.0103326.s010">S5</xref>). PlcR plays a key role in pathogenicity as it acts as a transcriptional regulator of many extracellular virulence factors. <italic>plcR</italic> transcription is autoregulated and the activity of the protein depends on the signaling peptide PapR <xref rid="pone.0103326-Slamti1" ref-type="bibr">[36]</xref>, <xref rid="pone.0103326-Grenha1" ref-type="bibr">[37]</xref>. However, PlcR was not differentially expressed over the course of 4 h growth (data not shown). It is nevertheless noteworthy, that the oligopeptide permease system, BC1179-BC1183, which is responsible for re-import of the PapR pheromone after extracellular cleavage <xref rid="pone.0103326-Gominet1" ref-type="bibr">[38]</xref>, was transcriptionally downregulated in the Δ<italic>secDF</italic> mutant (<xref ref-type="supplementary-material" rid="pone.0103326.s010">Table S5</xref>).</p><p>Interestingly, almost all motility-associated genes (BC1625-BC1671) were consistently downregulated two-fold or more in the mutant at the 4 h time point. Thus, the observed reduced flagellation and motility of the mutant was possibly due to reduced transcription of motility-associated genes encoding flagellar components and chemotaxis proteins.</p><p>Other prominent transcriptional responses due to <italic>secDF</italic> deletion were the stimulation of the cysteine regulon of CymRD, and downregulation of purine metabolism. Furthermore, seven uncharacterized ABC-transporters (out of a total of 111 <xref rid="pone.0103326-Ren1" ref-type="bibr">[39]</xref>) were more than 2-fold differentially regulated, as were 98 hypothetical protein-encoding genes (<xref ref-type="table" rid="pone-0103326-t003">Table 3</xref> and <xref ref-type="supplementary-material" rid="pone.0103326.s010">S5</xref>). Genes known to be activated by anaerobic conditions at low oxygen pressure or high culture densities <xref rid="pone.0103326-VanDerVoort1" ref-type="bibr">[40]</xref>–<xref rid="pone.0103326-Salvetti1" ref-type="bibr">[42]</xref> were downregulated in the Δ<italic>secDF</italic> mutant (<xref ref-type="table" rid="pone-0103326-t003">Table 3</xref> and <xref ref-type="supplementary-material" rid="pone.0103326.s010">Table S5</xref>). This encompassed factors involved in oxidative phosphorylation (operons BC3941-3944; BC0695-0698) including a cytochrome d ubiquinol oxidase (BC4792-4793), fermentation (BC0491-0492, BC2220), anaerobic respiration (BC2134, BC2128) and the regulator of the arginine deaminase operon arcABDC (BC0410).</p><p>The genome of <italic>B. cereus</italic> ATCC 14579 also contains a cryptic, linear plasmid pBClin15, encoding what appears to be a dormant prophage <xref rid="pone.0103326-Verheust1" ref-type="bibr">[43]</xref>. Most of the pBClin15 genes were found to be downregulated in the Δ<italic>secDF</italic> mutant (<xref ref-type="supplementary-material" rid="pone.0103326.s005">Fig. S5</xref>). This was not a result of loss of the pBClin15 plasmid, since (i) the presence of ORF 1–3 was detected via PCR using genomic DNA isolated from the bacterial culture used for the microarray analysis, and (ii) mRNA transcripts of BC_p0006 and BC_p0007 were detected by real-time qPCR from an independent culture.</p></sec></sec><sec id="s3"><title>Discussion</title><p>In the present study, deletion of <italic>secDF</italic> in <italic>B. cereus</italic> ATCC 14579 results in a pleiotropic phenotype which includes premature growth arrest and smaller colony size, aberrant cell morphology, reduced motility and reduced total protein in the bacterial secretome, consistent with previous reports on <italic>secDF</italic> mutants in other bacterial species <xref rid="pone.0103326-Pogliano1" ref-type="bibr">[6]</xref>, <xref rid="pone.0103326-Bolhuis1" ref-type="bibr">[9]</xref>, <xref rid="pone.0103326-Quiblier1" ref-type="bibr">[11]</xref>, <xref rid="pone.0103326-Gardel1" ref-type="bibr">[44]</xref>, <xref rid="pone.0103326-Nelson1" ref-type="bibr">[45]</xref>. In addition, our experiments demonstrated more pronounced pleiotropic effects in the presence of glucose.</p><p>Nhe, Hbl and Cytotoxin K are well-studied toxins from <italic>B. cereus</italic>, causing the diarrheal syndrome after ingestion of contaminated food <xref rid="pone.0103326-Beecher1" ref-type="bibr">[2]</xref>–<xref rid="pone.0103326-Lund2" ref-type="bibr">[4]</xref>. The <italic>nhe</italic> and <italic>hbl</italic> operons in the wild type and mutant strains were not found to be differentially transcribed, while Western blotting experiments using monoclonal antibodies showed accumulation of Hbl and Nhe toxin components in the Δ<italic>secDF</italic> mutant cells (<xref ref-type="fig" rid="pone-0103326-g004">Fig. 4</xref>, <xref ref-type="supplementary-material" rid="pone.0103326.s003">Fig. S3</xref>). A Sec-translocase - mediated export of these toxins has been advocated by Fagerlund <italic>et al.</italic><xref rid="pone.0103326-Fagerlund1" ref-type="bibr">[5]</xref>. However, it has also been indicated that the Hbl enterotoxin as well as the PC-PLC may be secreted via the flagellar apparatus <xref rid="pone.0103326-Ghelardi1" ref-type="bibr">[46]</xref>, <xref rid="pone.0103326-Ghelardi2" ref-type="bibr">[47]</xref>, similar to what is known in <italic>C. jejuni</italic><xref rid="pone.0103326-Konkel1" ref-type="bibr">[48]</xref> and <italic>C. difficile</italic><xref rid="pone.0103326-Aubry1" ref-type="bibr">[49]</xref>. Since the transcription of the flagellar machinery is downregulated in the Δ<italic>secDF</italic> mutant we cannot state explicitly whether the translocation defect of Hbl components is due to secondary effects on the flagellar system or to direct inhibition of the Sec-translocase pathway. For virulence factors other than Hbl and Nhe, such as cytotoxin K, PLC, SMase and collagenase C, a weak to moderate, yet statistically significant, transcriptional downregulation was observed. PlcR is a key transcriptional regulator involved in integration of a range of environmental signals such as cell-density and nutrient deprivation, and controls the expression of a range of extracellular <italic>B. cereus</italic> virulence factors, including Nhe, Hbl, CytK, PC-PLC and SMase. Interestingly, CytK, PLC, SMase, BC0991 and ColC were among the highest differentially detected proteins in the culture supernatants.</p><p>Knowing that the Δ<italic>secDF</italic> mutant has such a strong impact on secretion of known virulence factors and that the respective <italic>S. aureus</italic> and <italic>L. monocytogenes</italic> SecDF null mutants were affected in virulence <xref rid="pone.0103326-Quiblier2" ref-type="bibr">[12]</xref>, <xref rid="pone.0103326-BurgGolani1" ref-type="bibr">[50]</xref>, we sought to evaluate the role of <italic>B. cereus</italic> SecDF in its capacity to kill the insect larvae <italic>Galleria mellonella</italic>, which is currently used for infection studies of <italic>B. cereus</italic> or <italic>B. thuringiensis</italic> strains <xref rid="pone.0103326-Ramarao1" ref-type="bibr">[34]</xref>, <xref rid="pone.0103326-Bouillaut1" ref-type="bibr">[51]</xref>–<xref rid="pone.0103326-Fedhila1" ref-type="bibr">[53]</xref>. Virulence tests were performed by two routes of infection and the strongest effect was recorded following oral infection with about 17-fold more Δ<italic>secDF</italic> bacteria needed to kill 50% of the larvae at 24 hs compared to the wild type (<xref ref-type="table" rid="pone-0103326-t002">Table 2</xref>). In addition, the mutant strain was also 4.5-fold less virulent 24 h post infection when the bacteria were injected into the hemocoel. This indicates that the Δ<italic>secDF</italic> mutant is definitely affected in virulence but it is difficult to appoint the effect to a particular gene set because of the pleiotrophic effect of the mutation. Notably, the differences in virulence decreased after 74 h in both infection model experiments. This indicates that the reduced virulence of the Δ<italic>secDF</italic> mutant might only be of transient nature, a notion supported by Western Blot experiments showing Nhe and Hbl components adapting comparable extracellular levels in both strains over time. Meanwhile the results are in line with former work on the non-motile mutant <italic>B. thuringiensis</italic> 407 cry<sup>−</sup> Δ<italic>flhA</italic>, where a defective flagellar machinery assembly led to a decrease in virulence <xref rid="pone.0103326-Bouillaut1" ref-type="bibr">[51]</xref>. This was found to be partly due to a reduction of virulence gene expression, rather than direct involvement of the flagellar apparatus in virulence factor secretion <xref rid="pone.0103326-Fagerlund1" ref-type="bibr">[5]</xref>, <xref rid="pone.0103326-Bouillaut1" ref-type="bibr">[51]</xref>. Since flagellar gene expression is reduced in the Δ<italic>secDF</italic> mutant, the extent to which Hbl is transported via the Sec-translocase and the flagellar mechanism, respectively remains to be determined.</p><p>Out of the 96 proteins that could be identified in the <italic>B. cereus</italic> ATCC 14579 and isogenic Δ<italic>secDF</italic> mutant secretomes (<xref ref-type="supplementary-material" rid="pone.0103326.s008">Table S3</xref>), the majority (57%) were predicted to be of cytoplasmic origin. Other studies also frequently report a high percentage of non-secretory proteins in the medium <xref rid="pone.0103326-Kaakoush2" ref-type="bibr">[31]</xref>, <xref rid="pone.0103326-Hansmeier1" ref-type="bibr">[54]</xref>, <xref rid="pone.0103326-Gohar1" ref-type="bibr">[55]</xref>, and cell lysis has been determined to be of only minor contribution <xref rid="pone.0103326-Watt1" ref-type="bibr">[56]</xref>–<xref rid="pone.0103326-Lippolis1" ref-type="bibr">[58]</xref>. In LBG medium the Δ<italic>secDF</italic> mutant did not exhibit increased autolysis compared to the wild type (data not shown). Cytoplasmic proteins like enolase and pyruvate dehydrogenase were detected in the growth medium of <italic>B. cereus</italic> (<xref ref-type="supplementary-material" rid="pone.0103326.s008">Table S3</xref> and <xref rid="pone.0103326-Gilois1" ref-type="bibr">[29]</xref>), and these and other intracellular proteins have been reported to be secreted in <italic>B. subtilis</italic> during stationary phase by a non-classical translocation mechanism where protein domain structure appears to contribute <xref rid="pone.0103326-Yang1" ref-type="bibr">[59]</xref>. Although we did not find any indication of a stronger autolysis in the Δ<italic>secDF</italic> mutant compared to the wild type, an increased amount of small sized ribosomal proteins was identified in the growth medium of the mutant (<xref ref-type="table" rid="pone-0103326-t001">Table 1</xref>). No difference was seen at the transcriptional level of these genes between the wild type and the mutant. During co-translational insertion of proteins into the cell membrane the translocation channel protein SecY is bound to the ribosomal machinery <xref rid="pone.0103326-Mntret1" ref-type="bibr">[60]</xref>, <xref rid="pone.0103326-Mitra1" ref-type="bibr">[61]</xref> and in fact, it has been shown recently that this interaction opens the internal plug of SecY <xref rid="pone.0103326-Knyazev1" ref-type="bibr">[62]</xref>. Based on current knowledge, we cannot rule out the possibility that loss of SecDF could potentially result in a less specific translocation mechanism through a leaky SecYEG complex, feasibly affecting translocation of small sized proteins.</p><p>The Δ<italic>secDF</italic> mutant presented an aberrant cell morphology combined with an earlier growth arrest during cultivation (<xref ref-type="fig" rid="pone-0103326-g001">Fig. 1</xref>), phenotypes potentially caused by atypical activity of peptidoglycan remodeling enzymes. Murein hydrolases function during cell wall growth, peptidoglycan turnover, cell separation, and autolysis <xref rid="pone.0103326-Vollmer1" ref-type="bibr">[63]</xref>. Two uncharacterized putative murein hydrolases (BC0991 and BC1991) were absent in the Δ<italic>secDF</italic> mutant growth medium (<xref ref-type="table" rid="pone-0103326-t001">Table 1</xref>). Both contain a transglutaminase domain, known to facilitate intra- and interprotein crosslinks and to potentially play an important role in cell wall maturation <xref rid="pone.0103326-Milani1" ref-type="bibr">[64]</xref>. In addition the putative cell wall binding proteins EntA (BC5239), EntB (BC2952), and EntC (BC0813), identified in the secretome of <italic>B. cereus</italic><xref rid="pone.0103326-Clair1" ref-type="bibr">[28]</xref>, were affected at the transcriptional level (<xref ref-type="supplementary-material" rid="pone.0103326.s010">Table S5</xref>) and, in the case of EntB, also in the extracellular proteome in the <italic>secDF</italic> deletion mutant (<xref ref-type="supplementary-material" rid="pone.0103326.s008">Table S3</xref>). Secretome analyses for EntA and EntC were, however, not conclusive. EntA, EntB and EntC all contain two copies of the cell wall-binding SH3 domain, and are members of the resuscitation-promoting factor/stationary-phase survival (Rpf/Sps)-family identified in actinobacteria and firmicutes <xref rid="pone.0103326-Ravagnani1" ref-type="bibr">[65]</xref>. The <italic>B. subtilis</italic> muralytic enzyme YocH, which is a homolog of EntA, EntB and EntC, was induced by cell wall-turnover peptidoglycan fragments of growing cells and a null mutant displayed reduced survival after post-exponential phase <xref rid="pone.0103326-Shah1" ref-type="bibr">[66]</xref>. Crucial residues for enzyme activity in YocH <xref rid="pone.0103326-Shah1" ref-type="bibr">[66]</xref> are conserved in the three putative cell wall-binding proteins EntA, EntB and EntC. Clearly further analysis is required to understand the regulation and involvement of these and other muralytic enzymes in the phenotypic changes of the Δ<italic>secDF</italic> mutant (<xref ref-type="fig" rid="pone-0103326-g001">Fig 1</xref>).</p><p>AFM images clearly showed a reduction in cellular flagellation in the Δ<italic>secDF</italic> mutant (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2C</xref>), probably as a result of transcriptional deactivation of genes coding for flagella components (<xref ref-type="table" rid="pone-0103326-t003">Table 3</xref>). While intramembranous constituents of the flagellar body are generally believed to be inserted in a Sec-translocase dependent manner, the outer components are secreted via a flagellum-specific type III secretion system <xref rid="pone.0103326-Chevance1" ref-type="bibr">[67]</xref>–<xref rid="pone.0103326-Li1" ref-type="bibr">[69]</xref>. It is known from <italic>E. coli</italic> and <italic>S. enterica</italic> that the expression of flagellar genes is dependent on the state of assembly, in a step-wise manner (see reviews <xref rid="pone.0103326-Chilcott1" ref-type="bibr">[70]</xref>, <xref rid="pone.0103326-Terashima1" ref-type="bibr">[71]</xref>). Assuming a similar, energy-saving feedback loop in <italic>Bacillus</italic>, it is possible that the transcriptional downregulation of flagellar genes results from incomplete insertion and assembly of intramembrane flagellum body proteins. Thus, one could hypothesize that SecDF plays a role in early flagellum construction in <italic>B. cereus</italic> grown in the presence of glucose (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2</xref>).</p><p>A global transcriptional profiling experiment revealed profound transcriptional changes in the Δ<italic>secDF</italic> mutant, a phenomenon seen previously for selected genes in a <italic>S. aureus secDF</italic> mutant <xref rid="pone.0103326-Quiblier1" ref-type="bibr">[11]</xref>. Among the genes most highly upregulated by <italic>secDF</italic> deletion were a range of genes thought to respond to disturbances in cell envelope structures: the phage shock response system, the sigma B regulon, an extracytoplasmatic function (ECF) sigma factor and the putative murein hydrolase BC1991 (<xref ref-type="table" rid="pone-0103326-t003">Table 3</xref>). The PspA-like gene (BC1436) is similar to <italic>liaH</italic> of <italic>B. subtilis</italic>. The Lia operon (LiaIHFSR) is highly conserved in Firmicutes, and the system is a cell envelope stress response activated by peptide antibiotics <xref rid="pone.0103326-Wolf1" ref-type="bibr">[72]</xref>, <xref rid="pone.0103326-Kesel1" ref-type="bibr">[73]</xref>. PspA is particularly well studied in <italic>E. coli</italic> and is induced by a wide range of cell envelope stress conditions and thought to maintain the energetic state of cells under stress (for review see <xref rid="pone.0103326-Joly1" ref-type="bibr">[74]</xref>). In <italic>E. coli</italic> it has been shown that single gene deletions of Sec-translocase components such as SecA, SecD and SecF, lead to PspA overexpression <xref rid="pone.0103326-Kleerebezem1" ref-type="bibr">[75]</xref>, and that PspA supports the efficient translocation of Sec- and TAT-dependent proteins <xref rid="pone.0103326-DeLisa1" ref-type="bibr">[76]</xref>. In our study of the <italic>B. cereus</italic> Δ<italic>secDF</italic> mutant, the strong induction of the <italic>pspA</italic>-like gene may be a result of sensing the secretion defect as well as of an internal accumulation of proteins. In addition, the sigma B regulon known to provide a non-specific stress response to a range of different stress signals affecting cell envelope integrity <xref rid="pone.0103326-vanSchaik2" ref-type="bibr">[77]</xref>–<xref rid="pone.0103326-vanSchaik3" ref-type="bibr">[79]</xref> is moderately upregulated. Among the ten ECF-type sigma factors identified in <italic>B. cereus</italic><xref rid="pone.0103326-Ulrich1" ref-type="bibr">[80]</xref>, recognizing environmental signals <xref rid="pone.0103326-Helmann1" ref-type="bibr">[81]</xref>, the so far uncharacterized BC5363 exhibits similarity to the <italic>B. subtilis</italic> SigW sigma factor (34% identity at the protein level). Interestingly, <italic>sigW</italic> is induced by cell envelope stress factors (for review see <xref rid="pone.0103326-Ho1" ref-type="bibr">[82]</xref>).</p><p>While the addition of glucose to the growth medium resulted in general in more pronounced phenotypes (<xref ref-type="fig" rid="pone-0103326-g001">Fig. 1</xref>, <xref ref-type="fig" rid="pone-0103326-g004">Fig. 4</xref>, <xref ref-type="fig" rid="pone-0103326-g005">Fig. 5</xref>), it is noteworthy that only sugars consisting of at least one glucose component profoundly inhibited motility of the <italic>ΔsecDF</italic> mutant (<xref ref-type="fig" rid="pone-0103326-g002">Fig. 2A</xref>). Although the rationale and mechanism behind the effects of glucose on the phenotype of the Δ<italic>secDF</italic> mutant remain to be elucidated, this study confirms previous reports showing that glucose exerts more functions than only being an important nutrient. Recent research indicates for instance a direct involvement of glucose in expression of the toxin hemolysin II in <italic>B. cereus</italic> by activation of HlyIIR by glucose 6P which resulted in repression of <italic>hlyII</italic> gene expression <xref rid="pone.0103326-Guillemet1" ref-type="bibr">[83]</xref>.</p><p>The present study shows that some toxins and other virulence factors produced by the pathogenic Gram-positive, spore-forming bacterium <italic>B. cereus</italic> are dependent on SecDF for proper translocation across the cell membrane, confirming a role for SecDF in protein secretion in general and efflux of some toxins, directly or indirectly, in particular. It could be assumed that the ubiquitous SecDF protein fills similar functions also in other bacteria, as it has been reported for <italic>S. aureus</italic> and <italic>L. monocytogenes</italic><xref rid="pone.0103326-Quiblier2" ref-type="bibr">[12]</xref>, <xref rid="pone.0103326-BurgGolani1" ref-type="bibr">[50]</xref>. Finally, although we cannot explain the phenomenon at this moment, this study shows clearly an exacerbating effect of glucose on the phenotype of the Δ<italic>secDF</italic> mutant.</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and methods</title><sec id="s4a"><title>Growth conditions</title><p>Unless otherwise stated, <italic>B. cereus</italic> and <italic>E. coli</italic> strains were streaked on LB agar plates and incubated at 30°C and 37°C, respectively. Liquid cultures were inoculated from a single colony, incubated overnight and then diluted 1∶100 in LB medium. These starter cultures were grown at 30°C or 37°C, respectively, at 200 rpm. After reaching an OD<sub>600nm</sub> of approximately 0.5, experimental cultures were inoculated from the starter culture to an initial OD<sub>600nm</sub> of 0.02, and grown as above. If applicable, 1% glucose was added to LB (LBG). When relevant, erythromycin 5 µg/ml (with pHT304 plasmid) or ampicillin 100 µg/ml (with pTTQ18 plasmid) was added to the culture. For assessment of glucose fermentation the strains were streaked on <italic>Bacillus cereus</italic> agar (Oxoid) supplemented with 1% glucose. Acidic by-products of glucose fermentation were monitored by color change of the pH indicator bromothymol blue.</p></sec><sec id="s4b"><title>Construction of the Δ<italic>secDF</italic>-mutant</title><p>The markerless Δ<italic>secDF</italic> mutant of the type strain <italic>B. cereus</italic> ATCC 14579 was constructed by the method of Janes & Stibitz <xref rid="pone.0103326-Janes1" ref-type="bibr">[84]</xref>. A deletion construct consisting of overlapping flanking regions of the target gene is cloned into a temperature-sensitive shuttle vector carrying the homing endonuclease restriction site <italic>I-SceI</italic>. Under replication non-permissive temperatures and selection pressure the vector integrates either up- or downstream of the target gene. To enforce a double-strand break of the chromosomal DNA, a second plasmid encoding I-SceI is introduced into the organism. Repair of the break by cross-over leads to either wild type or knock-out genotypes. Mutants are then selected by PCR and the vector sporadically lost during non-selection. Oligonucleotides used for making the gene deletion construct, substituted the BC4405 ORF in frame with <named-content content-type="gene">ATGGTCGACTAA</named-content> and thus introduced a <italic>SalI</italic> restriction site (<xref ref-type="supplementary-material" rid="pone.0103326.s011">supplemental information S1</xref>). After cloning of the gene deletion construct with about 500 bp flanking regions into the suicide shuttle vector pBKJ236 and electroporation into <italic>B. cereus</italic> ATCC 14579, the protocol was followed as previously described <xref rid="pone.0103326-Janes1" ref-type="bibr">[84]</xref>. Successful gene deletion was confirmed by PCR using genomic DNA as template and oligonucleotides binding outside of the deleted region, and by DNA sequencing. The presence of the plasmid pBClin15 was confirmed by PCR as reported previously <xref rid="pone.0103326-Vrs1" ref-type="bibr">[85]</xref>.</p></sec><sec id="s4c"><title>Assessment of phospholipase C activity</title><p>The activity of secreted Phospholipase C (PC-PLC) was measured for cells growing on agar and in liquid cultures. For the first test, bacteria were grown in LB medium for 16 h at 30°C and 220 rpm, washed in 0.9% NaCl and resuspended to an OD<sub>600nm</sub> of 8.5. Five µl of the bacterial suspension was spotted onto LB and LBG agar plates supplemented with 5% egg yolk suspension (Oxoid). The phospholipase C activity was analyzed by visual inspection after 7 hours incubation at 30°C. PC-PLC activity of filter-sterilized supernatant sampled at different time points, from cultures grown in LB and LBG, was measured by spotting 5 µl on 1% egg yolk agar plates, and incubating them at 30°C for 24 h. In addition, 100 µl of these supernatants were incubated with 900 µl 2% egg yolk saline suspension at room temperature for 75 min after which the OD<sub>600nm</sub> was measured. Variations in growth between the wild type and the mutant strains were accounted for when necessary by diluting the wild type supernatant with fresh LB after filter-sterilization.</p></sec><sec id="s4d"><title>Light microscopy and atomic force microscopy (AFM)</title><p>Micrographs were made using 3 µl sample of a fresh culture with 400-fold magnification. Pictures were obtained with a Nikon Labophot-2 microscope coupled to a Leica DFC320 camera and assessed with the LAS v3.6 program. For AFM, <italic>B. cereus</italic> ATCC 14579 wild type and Δ<italic>secDF</italic> mutant strains were grown in LBG as detailed under “growth conditions” and one ml samples were collected after 4 h growth. Following 3 min centrifugation at 2400xg the cells were washed and resuspended in 1 ml 0.9% saline. Ten µl of the suspension was diluted to a final volume of 50 µl in 10 mM magnesium/Tris buffer, pH 7.5, ten µl of which was applied to a freshly cleaved muscovite mica (Agar Scientific, Norway) mounted on a glass slide, and incubated for 10 min at room temperature. After ten washing steps with 100 µl sterile filtered MQ water, the samples were dried under a gentle N<sub>2</sub> stream. AFM images were recorded in intermittent contact mode in air using a NanoWizard I atomic force microscope (JPK, Berlin, Germany). To quantify the number of flagella, a total of 103 cells for the Δ<italic>secDF</italic> mutant and 26 cells of the wild type were analyzed, from two independent cultures.</p></sec><sec id="s4e"><title>Motility assays</title><p>To assess motility, 0.3% and 0.7% LB agar plates were used. Five µl of overnight cultures (OD<sub>600nm</sub> between 7 and 10) of the wild type and mutant strains grown in 5 ml LB at 30°C at 220 rpm were spotted on the agar surface of the same plate, with two technical replicates per biological sample. The diameter of the culture was measured after 7–9 h incubation, the start diameter of the drop was subtracted and the ratio of the recorded motility for wild type and mutant was calculated. Every experiment was done at least four times, and the motility of the wild type strain in each condition was set to 100% (unpaired, two-tailed Student's t-Test for wild type vs. mutant, <italic>P</italic><0.05). Statistical significance of differences between the mutant's motility compared to the wild type in pure LB and LB + additives was evaluated using the MS Office Excel unpaired t-test function with a two-tailed distribution. Additives were supplemented with the following final concentrations: glucose 0.4%, other sugars 1%, Tween-80; 0.02%.</p></sec><sec id="s4f"><title>Expression of SecDF</title><p>For expression of SecDF in <italic>B. cereus</italic> ATCC 14579, the native gene was cloned into the low-copy number <italic>E. coli</italic>/<italic>Bacillus</italic> plasmid shuttle vector pHT304-Pxyl <xref rid="pone.0103326-Arantes1" ref-type="bibr">[86]</xref>. pHT304-Pxyl contains the <italic>xylR</italic> and <italic>xylA</italic> promoters from <italic>B. subtilis</italic>, allowing xylose-inducible expression of SecDF fused with a C-terminal 6x histidine tag. For heterologous overexpression of SecDF in <italic>E. coli</italic>, the <italic>secDF</italic> gene from <italic>B. cereus</italic> ATCC 14579 was cloned into a modified version of the high copy number, IPTG inducible vector pTTQ18 <xref rid="pone.0103326-Stark1" ref-type="bibr">[87]</xref>. Expression of <italic>secDF</italic> from this plasmid resulted in a recombinant protein carrying a C-terminal 6x histidine tag. The plasmid was introduced into <italic>E. coli</italic> BW25112 Δ<italic>acrB</italic>. This strain lacks the RND-type transporter AcrB, which has been shown to be the major xenobiotic efflux transporter in <italic>E. coli</italic> (for recent reviews see <xref rid="pone.0103326-Nikaido1" ref-type="bibr">[88]</xref>, <xref rid="pone.0103326-Pos1" ref-type="bibr">[89]</xref>). Correct cloning of the gene was in both cases confirmed by sequencing, and protein expression in both host organisms was measured using the histidine tags for detection by specific antibodies. Induction of protein expression by 20 mM xylose and 0.05 mM IPTG, respectively, resulted in a protein band of approximately 82 kDa on a Western blot, in both cases (data not shown).</p></sec><sec id="s4g"><title>Determination of minimum inhibitory concentrations (MICs)</title><p>To identify the susceptibility of <italic>B. cereus</italic> and <italic>E. coli ΔacrB</italic> to a range of xenobiotics, bacterial suspensions were incubated in LB and LBG, respectively, with 2-fold serial dilutions of the tested compounds. Pre-cultures grown in LB were diluted to an OD<sub>600nm</sub> value of 0.02 and aliquoted into 96-well plates (final volume 150 µl). The plates were incubated in a humidified chamber at 30°C, 200 rpm for 22 h. The lowest concentration of xenobiotics that resulted in no visual growth was considered as the MIC. Experiments were done in technical duplicates and with at least two biological replicates. If protein overexpression strains were tested, xylose (20 mM) or IPTG (0.05 mM) was added to the medium for pHT304-Pxyl and pTTQ18 vector constructs, respectively. Alternatively, the susceptibility of <italic>B. cereus</italic> strains was examined by disk diffusion on LB or LBG agar plates. Mid-logarithmic precultures were diluted to an OD<sub>600nm</sub> of 0.05 in 0.9% NaCl, and 1 ml of this cell suspension was spread out on agar plates and air-dried. Thereafter, 6 mm paper disks applied on the surface were impregnated with 10 µl of each tested compound. Inhibition zones were examined after 16 h incubation at 30°C, for the following compounds: ethanol 100%, spectinomycin 100 mg/ml, phosphomycin 25 mg/ml, ciprofloxacin 10 mg/ml, norfloxacin 10 mg/ml, chloramphenicol 25 mg/ml, tetracycline 10 mg/ml, oxytetracycline 0.8 mg/ml, gentamicin 50 mg/ml, ampicillin 50 mg/ml, oxacillin-5 (BD), SDS 20%, DOC 80 mg/ml, chlorhexidin 1.6 mg/ml, ethidium bromide 5 mg/ml, CCCP 7.5 mM, sodium lactate 50%, polymyxin B 25 mg/ml, sodium benzoate 0.5 g/ml, erythromycin 100 mg/ml, kanamycin 10 mg/ml, plant extracts: tea tree (<italic>Melaleuca alternifolia</italic>); steam distillates of peppermint leaves (<italic>Mentha piperita</italic>) and calabash (<italic>Melaleuca leucadendron var. cajaputi</italic>) (Primavera Life).</p></sec><sec id="s4h"><title>Microarray analysis</title><p>Cells were grown in LBG in 50 ml cultures in 500 ml non-baffled Erlenmeyer flasks at 30°C, 220 rpm for 3 h (two biological replicates) and 4 h (six biological replicates), respectively. Five ml culture was then mixed with equal amounts of ice-cold methanol, followed by harvesting by a short centrifugation. Cells were lysed by beadbeating and the RNA was isolated using the RNA Mini Kit (Qiagen), including the on-column DNase treatment step. cDNA conversion and labelling, microarray hybridization and data analysis using Bayesian linear modelling (Limma-package <xref rid="pone.0103326-Smyth1" ref-type="bibr">[90]</xref> was basically performed as described previously by Gohar <italic>et al.</italic><xref rid="pone.0103326-Gohar2" ref-type="bibr">[91]</xref> and detailed procedures and raw data were deposited according to MIAME guidelines in the Arrayexpress database (accession number E-MTAB-1759).</p></sec><sec id="s4i"><title>Validation of gene expression by real-time RT-PCR analysis</title><p>Quantitative real-time PCR (qRT-PCR) was used to validate the microarray results <xref rid="pone.0103326-Provenzano1" ref-type="bibr">[92]</xref>, <xref rid="pone.0103326-Chuaqui1" ref-type="bibr">[93]</xref>. qRT-PCR was carried out following the MIQE guidelines (<xref ref-type="supplementary-material" rid="pone.0103326.s011">supplemental information S1</xref>). The genes tested included non-differentially (FC<1.5: <italic>BC_p006, BC2271, ccpA, plcR, hlyR, nheB</italic>), moderately (1.5<italic>BC1991, BC5239, Flagellin, cytK, hlbB, hlyII</italic>) and highly differentially (FC>5: <italic>BC_p007, BC0862, BC1436, BC2119, ECF-type sigma factor, sigB</italic>) expressed genes from the microarray experiment, in order to best mirror the expression pattern observed in the microarray experiments.</p></sec><sec id="s4j"><title>Analysis of secreted proteins</title><p>For the analysis of secreted proteins, mid-logarithmic cultures of the <italic>B. cereus</italic> ATCC 14579 wild type and the isogenic Δ<italic>secDF</italic> mutant strains grown in LB were transferred into fresh LB or LBG medium. Following 3 h, 4 h and 6 h aerated growth at 30°C and 220 rpm, PBS-adjusted volumes (by dilution according to 1 ml culture with lowest OD<sub>600nm</sub>) of each culture were harvested by centrifugation. Sterile-filtered (0.2 µm) culture supernatant was mixed 1∶4 with ice-cold methanol:acetone (1∶1) and proteins were precipitated overnight at −20°C. Proteins were harvested by centrifugation at 12,000xg for 30 min at 4°C. For gel electrophoresis, 8 ml of duplicate, independent and normalized supernatants of cultures grown in LBG were concentrated 40-fold by methanol:acetone precipitation, resuspended in 250 µl TES (20 mM Tris pH 7.5, 0.8% NaCl, 1 mM EDTA), and 12 µl of each sample was analyzed on a 4–20% SDS-polyacrylamide gel (Pierce) by silver staining (Sigma-Aldrich). For label-free mass spectrometry analyses, triplicate independent cultures (from individual colonies) were grown for 4 h in LB added 1% glucose, as described above. Using acid-cleaned glassware, the PBS-adjusted culture supernatants (according to the culture with lowest OD<sub>600nm</sub>; final volume of 2.5 ml) were subjected to methanol:acetone precipitation at −20°C overnight. After centrifugation at 12.000xg for 30 min at 4°C, proteins were resuspended in 50 mM ammoniumbicarbonate/1 M urea. After protein concentration determination using the Bradford Assay with BSA as a standard, 20 µg of each sample was used for analyses.</p><sec id="s4j1"><title>Sample Preparation</title><p>Proteins were reduced with 10 mM DTT (1 h at 70°C, pH 9), alkylated for 1 h using 25 mM iodoacetamide, and digested with trypsin (1 µg) at 37°C for 16 h. Digested protein samples were analysed using a TripleTOF 5600 mass spectrometer (AB SCIEX Foster City, CA, USA) coupled to an Eksigent NanoLC-Ultra 2Dplus system (Eksigent Technologies, Dublin, CA, USA). Peptides were separated as described previously <xref rid="pone.0103326-McKay1" ref-type="bibr">[94]</xref>, and the LC eluent subjected to positive ion nanoflow analysis using an ion spray voltage, heater interface temperature, curtain gas flow and nebulizing gas flow of 2.5 kV, 150°C, 20°C and 16°C, respectively. Information dependent acquisition-experiments utilized a survey scan (350–1500 amu) with an accumulation time of 100 ms, followed by 15 MS/MS product ion scans (350–1600 amu) with an accumulation time of 100 ms each.</p></sec><sec id="s4j2"><title>Protein Identification</title><p>Proteins were identified using the Paragon search algorithm <xref rid="pone.0103326-Tang1" ref-type="bibr">[95]</xref>, <xref rid="pone.0103326-Shilov1" ref-type="bibr">[96]</xref> in ProteinPilot Version 4.0.8085 (AB SCIEX Foster City, CA, USA). Searches were carried out against the reference proteome of <italic>B. cereus</italic> ATCC 14579, extracted from the Universal Protein Resource (UniProt) (4) using the thorough search mode and included biological modifications, trypsin-cleaved peptides and iodoacetamide-modification of cysteine residues. False discovery rates were determined in ProteinPilot using a detected protein threshold of 0.05 and the decoy database searching strategy, and only proteins at 1% global FDR and distinct peptides at 5% local FDR were reported. For further data analysis of all three biological replicates, Scaffold (version Scaffold_4.0.5, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Equal amount of total protein was used for tryptic digestion and comparative analyses were conducted after normalization of the data sets accordingly to the <bold>N</bold>ormalized <bold>S</bold>pectral <bold>A</bold>bundance <bold>F</bold>actor (NSAF) approach using total spectral counts <xref rid="pone.0103326-Zybailov1" ref-type="bibr">[27]</xref>. A Student's T-test comparing total spectral counts was performed to determine statistical significances of protein abundances in wild type and mutant strain samples.</p></sec></sec><sec id="s4k"><title>Toxin detection</title><p>Cell lysates were prepared by harvesting 2 ml of <italic>B. cereus</italic> wild type and Δ<italic>secDF</italic> mutant cultures by centrifugation at 4500xg for 5 min. The pellets were washed once in cold PBS and stored over night at −20°C. Cell pellets were then resuspended in TES containing 2 mg/ml lysozyme and the volume was adjusted according to the original culture OD. The bacterial suspensions were incubated at 37°C for 1 h. After partial cell wall degradation, cell lysis was achieved by six rounds of freezing and thawing in liquid nitrogen and a 37°C water bath respectively. Cell debris was removed by centrifugation and the supernatant was stored on ice for no more than 4 h. Twenty µl of normalized, sterile-filtered supernatants and 2 µl of cell lysates were separated on 10% SDS polyacrylamide gels and blotted onto a nitrocellulose membrane. Toxin components were detected using 1∶20 dilutions of the following monoclonal antibodies: 1A8 and 1E11, against NheA and NheB, respectively <xref rid="pone.0103326-Dietrich2" ref-type="bibr">[33]</xref>; and 1E9 and 8B12, specific for the L1 and L2-subunits of Hbl <xref rid="pone.0103326-Dietrich1" ref-type="bibr">[32]</xref>. 1∶10,000 dilution of HRP-conjugated anti-mouse antibody (Sigma) was used for chemiluminescent signal development.</p></sec><sec id="s4l"><title>Analyses of proteolytic activity</title><p>Experiments analyzing milk and gelatin proteolytic activities in the secretomes of the wild type and Δ<italic>secDF</italic> mutant strains did not reveal significant differences (data not shown). Skim milk agar plates were prepared by dissolving skim milk powder and agar separately in Milli-Q water, to a concentration of 75 mg/ml and 15 mg/ml, respectively. Following autoclaving for 15 min at 110°C and cooling to 50°C, the skim milk and agar solutions were mixed (1∶1). Overnight cultures grown in LB broth were normalized to an OD<sub>600nm</sub> of 1 with 0.9% NaCl and 50 µl was added into punched holes (5 mm) in skim milk plates and incubated at 37°C, 30°C and 20°C, respectively. Proteolytic activity was visible as change in opacity of the milk around the bacterial spots. Total gelatinase activity was carried out as described in Millipores technical publication on gelatin zymography (<ext-link ext-link-type="uri" xlink:href="http://www.millipore.com/userguides/tech1/mcproto009">http://www.millipore.com/userguides/tech1/mcproto009</ext-link>). Ten µl of sterile-filtered and normalized culture of <italic>B. cereus</italic> strains grown for 4 h in LBG at 220 rpm were loaded on an 8% acrylamide gel co-polymerized with 0.1% gelatin, using non-reductive SDS sample buffer. Gelatinase activity appeared as clear bands in the turbid gel background.</p></sec><sec id="s4m"><title>Autolysis</title><p>In order to determine if the Δ<italic>secDF</italic> mutant displayed a higher autolysis rate than the wild type strain, two different tests were conducted: (<bold>i</bold>) cell lysis activity of <italic>B. cereus</italic> cell lysates was investigated by performing zymograms using whole <italic>B. cereus</italic> cells as substrate, according to the method of Raddadi <italic>et. al.</italic><xref rid="pone.0103326-Raddadi1" ref-type="bibr">[97]</xref>; (<bold>ii</bold>) spontaneous autolysis was determined as described by Quiblier <italic>et al.</italic><xref rid="pone.0103326-Quiblier1" ref-type="bibr">[11]</xref>. Cells grown in LB or LBG, were harvested 4 h after inoculation, washed in 0.9% NaCl, and resuspended in 0.01 M Na-phosphate buffer, pH 7.4 to a final OD<sub>600nm</sub> of 1. The resulting bacterial suspensions were incubated at 30°C, 200 rpm for 90 min and the decrease in optical density (600 nm) was measured at regular intervals. Neither of the experiments supported a higher autolysis rate in the <italic>secDF</italic> deletion strain compared to wild type when grown in LBG (data not shown).</p></sec><sec id="s4n"><title>Catalase test</title><p>The catalase test, based on a stable yellow complex-formation of hydrogen peroxide with molybdate, was carried out basically as described by Góth 1991 <xref rid="pone.0103326-Gth1" ref-type="bibr">[98]</xref>. Briefly, <italic>B. cereus</italic> strains were grown in LB or LBG medium at 30°C and 220 rpm. After 3 h and 4 h growth, OD<sub>600nm</sub> was measured in duplicate, and a volume corresponding to an optical density of 14 per ml was pelleted and resuspended in 100 µl of 6 mM phosphate buffer, pH 7.4. Samples were mixed with 500 µl preheated substrate solution (65 µM H<sub>2</sub>O<sub>2</sub> in 6 mM phosphate buffer) and incubated at 37°C for 120 sec. The reaction was stopped by adding 500 µl of 32.4 mM ammonium molybdate in 6 mM phosphate buffer. After pelleting the cells, the color change was measured spectrophotometrically in a microplate reader at 405 nm. Each sample was analyzed in triplicate. Absorbance values were subtracted by values of the blank non-reactive wells containing 100 µl of 6 mM phosphate buffer and no bacterial cells. As a loading control, pelleted cells were lysed as described above, and equal volumes of cell lysates were applied on a 12% SDS-polyacrylamide gel. Proteins were stained with Bradford reagent.</p></sec><sec id="s4o"><title>Insect infection experiments</title><p>The virulence-related properties of Δ<italic>secDF</italic> were assessed by comparing the killing effect of the <italic>B. cereus</italic> wild type and the Δ<italic>secDF</italic> mutant strains by both oral infection and direct injection into the hemocoel of 5<sup>th</sup> instar <italic>Galleria mellonella</italic> larvae <xref rid="pone.0103326-Ramarao1" ref-type="bibr">[34]</xref>, <xref rid="pone.0103326-Guillemet2" ref-type="bibr">[99]</xref>. <italic>G. mellonella</italic> eggs were hatched at 25°C and the larvae reared on beeswax and pollen. In each experiment, groups of 20 to 30 <italic>G. mellonella</italic> larvae, weighing about 200 mg, were used. For oral infection, the larvae were force-fed with 10 µl of a mixture containing various doses (1.5x10<sup>5</sup> to 2.5x10<sup>7</sup>) of vegetative bacteria (exponential growth OD≈1in LB medium) and 3 µg of activated Cry1C toxin, prepared as previously described <xref rid="pone.0103326-Fedhila2" ref-type="bibr">[100]</xref>. For injection experiments, the larvae were also infected with vegetative bacteria at various doses, from ≈2,000 to ≈100,000 cfu (colony forming units). Experiments were repeated at least twice. Infected larvae were kept at 37°C and mortality was recorded at 24 h and 72 h post infection. The larvae in the control group were fed PBS buffer. The 50% lethal doses (LD<sub>50s</sub>) values, as estimated using the Probit analysis StatPlus program, corresponds to the cfu killing 50% of the treated larvae.</p></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0103326.s001"><label>Figure S1</label><caption><p><bold>Susceptibility of the </bold><bold><italic>ΔsecDF</italic></bold><bold> mutant towards selected compounds.</bold></p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s001.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s002"><label>Figure S2</label><caption><p><bold>Determination of lecithinase activity.</bold></p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s002.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s003"><label>Figure S3</label><caption><p><bold>Complementation of the </bold><bold><italic>ΔsecDF</italic></bold><bold> mutant.</bold></p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s003.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s004"><label>Figure S4</label><caption><p><bold>Validation of microarray results by qRT-PCR.</bold></p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s004.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s005"><label>Figure S5</label><caption><p><bold>Regulation of pBClin15 ORFs.</bold></p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s005.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s006"><label>Table S1</label><caption><p>Susceptibility to toxic compounds of <italic>B. cereus</italic> ATCC 14579 wild type strain and its isogenic <italic>ΔsecDF</italic> variant.</p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s006.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s007"><label>Table S2</label><caption><p>Susceptibility to toxic compounds of <italic>E. coli</italic> BW25113_ΔacrB expressing SecDF.</p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s007.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s008"><label>Table S3</label><caption><p>Secretome of <italic>B. cereus</italic> ATCC 14579 wild type and <italic>ΔsecDF</italic> mutant.</p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s008.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s009"><label>Table S4</label><caption><p>Transcriptional activation of the SigB regulon in the <italic>B. cereus</italic> ATCC 14579 <italic>ΔsecDF</italic> mutant compared to its wild type strain.</p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s009.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s010"><label>Table S5</label><caption><p>Complete list of microarray results. The list shows at least 2-fold differentially regulated genes in the <italic>B. cereus</italic> ATCC 1459 <italic>ΔsecDF</italic> mutant compared to wild type (P-value <0.05).</p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s010.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0103326.s011"><label>Supplemental information S1</label><caption><p><bold>Materials and methods.</bold></p><p>(PDF)</p></caption><media xlink:href="pone.0103326.s011.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>The <italic>E. coli</italic> overexpression vector pTTQ18 was a kind gift from P. Henderson (Leeds, UK) and the <italic>E. coli</italic> BW25113 Δ<italic>acrB</italic> strain was generously allocated by K.M. Pos (Frankfurt/M., Germany). We are very grateful to R. Dietrich and E. 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