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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A unique <italic>Coxiella burnetii</italic> lipoprotein involved in metal binding (LimB)</title><author><name sortKey="Battisti, James M" sort="Battisti, James M" uniqKey="Battisti J" first="James M." last="Battisti">James M. Battisti</name></author><author><name sortKey="Hicks, Linda D" sort="Hicks, Linda D" uniqKey="Hicks L" first="Linda D." last="Hicks">Linda D. Hicks</name></author><author><name sortKey="Minnick, Michael F" sort="Minnick, Michael F" uniqKey="Minnick M" first="Michael F." last="Minnick">Michael F. Minnick</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21212117</idno><idno type="pmc">3139440</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139440</idno><idno type="RBID">PMC:3139440</idno><idno type="doi">10.1099/mic.0.046649-0</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">000134</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000134</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A unique <italic>Coxiella burnetii</italic> lipoprotein involved in metal binding (LimB)</title><author><name sortKey="Battisti, James M" sort="Battisti, James M" uniqKey="Battisti J" first="James M." last="Battisti">James M. Battisti</name></author><author><name sortKey="Hicks, Linda D" sort="Hicks, Linda D" uniqKey="Hicks L" first="Linda D." last="Hicks">Linda D. Hicks</name></author><author><name sortKey="Minnick, Michael F" sort="Minnick, Michael F" uniqKey="Minnick M" first="Michael F." last="Minnick">Michael F. Minnick</name></author></analytic><series><title level="j">Microbiology</title><idno type="ISSN">1350-0872</idno><idno type="eISSN">1465-2080</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Coxiella burnetii</italic> is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. <sc>blast</sc> analyses with CBU1224a showed no significant similarity to sequences outside strains of <italic>C. burnetii</italic>. Additional <italic>in silico</italic> analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni<sup>2+</sup> or Zn<sup>2+</sup>, and roughly halved in the presence of Co<sup>2+</sup> or Fe<sup>3+</sup>. The <italic>limB</italic> gene was maximally expressed at 3–7 days post-infection in <italic>Coxiella-</italic>infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique <italic>Coxiella</italic> lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Microbiology</journal-id><journal-id journal-id-type="iso-abbrev">Microbiology (Reading, Engl.)</journal-id><journal-id journal-id-type="publisher-id">mic</journal-id><journal-title-group><journal-title>Microbiology</journal-title></journal-title-group><issn pub-type="ppub">1350-0872</issn><issn pub-type="epub">1465-2080</issn><publisher><publisher-name>Society for General Microbiology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">21212117</article-id><article-id pub-id-type="pmc">3139440</article-id><article-id pub-id-type="publisher-id">966</article-id><article-id pub-id-type="doi">10.1099/mic.0.046649-0</article-id><article-categories><subj-group subj-group-type="heading"><subject>Cell and Molecular Biology of Microbes</subject></subj-group></article-categories><title-group><article-title>A unique <italic>Coxiella burnetii</italic> lipoprotein involved in metal binding (LimB)</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Battisti</surname><given-names>James M.</given-names></name></contrib><contrib contrib-type="author"><name><surname>Hicks</surname><given-names>Linda D.</given-names></name></contrib><contrib contrib-type="author"><name><surname>Minnick</surname><given-names>Michael F.</given-names></name></contrib></contrib-group><aff id="d32e41">Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA</aff><author-notes><corresp><bold>Correspondence</bold>: James M. Battisti: <email>jim.battisti@mso.umt.edu</email></corresp></author-notes><pub-date pub-type="ppub"><month>4</month><year>2011</year></pub-date><pub-date pub-type="pmc-release"><day>1</day><month>4</month><year>2012</year></pub-date><pmc-comment> PMC Release delay is 12 months and 0 days and was based on the copyright element. </pmc-comment>
<volume>157</volume><issue>Pt 4</issue><fpage>966</fpage><lpage>976</lpage><history><date date-type="received"><day>4</day><month>11</month><year>2010</year></date><date date-type="rev-recd"><day>4</day><month>1</month><year>2011</year></date><date date-type="accepted"><day>6</day><month>1</month><year>2011</year></date></history><permissions><copyright-statement>Copyright © 2011, SGM</copyright-statement><copyright-year>2011</copyright-year></permissions><self-uri xlink:title="pdf" xlink:href="966.pdf"></self-uri><abstract><p><italic>Coxiella burnetii</italic> is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. <sc>blast</sc> analyses with CBU1224a showed no significant similarity to sequences outside strains of <italic>C. burnetii</italic>. Additional <italic>in silico</italic> analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni<sup>2+</sup> or Zn<sup>2+</sup>, and roughly halved in the presence of Co<sup>2+</sup> or Fe<sup>3+</sup>. The <italic>limB</italic> gene was maximally expressed at 3–7 days post-infection in <italic>Coxiella-</italic>infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique <italic>Coxiella</italic> lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.</p></abstract></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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