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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Significant Role for <italic>ladC</italic> in Initiation of <italic>Legionella pneumophila</italic> Infection<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Newton, Hayley J" sort="Newton, Hayley J" uniqKey="Newton H" first="Hayley J." last="Newton">Hayley J. Newton</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sansom, Fiona M" sort="Sansom, Fiona M" uniqKey="Sansom F" first="Fiona M." last="Sansom">Fiona M. Sansom</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Dao, Jenny" sort="Dao, Jenny" uniqKey="Dao J" first="Jenny" last="Dao">Jenny Dao</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cazalet, Christel" sort="Cazalet, Christel" uniqKey="Cazalet C" first="Christel" last="Cazalet">Christel Cazalet</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Bruggemann, Holger" sort="Bruggemann, Holger" uniqKey="Bruggemann H" first="Holger" last="Bruggemann">Holger Bruggemann</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Albert Weissenberger, Christiane" sort="Albert Weissenberger, Christiane" uniqKey="Albert Weissenberger C" first="Christiane" last="Albert-Weissenberger">Christiane Albert-Weissenberger</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Buchrieser, Carmen" sort="Buchrieser, Carmen" uniqKey="Buchrieser C" first="Carmen" last="Buchrieser">Carmen Buchrieser</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cianciotto, Nicholas P" sort="Cianciotto, Nicholas P" uniqKey="Cianciotto N" first="Nicholas P." last="Cianciotto">Nicholas P. Cianciotto</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Hartland, Elizabeth L" sort="Hartland, Elizabeth L" uniqKey="Hartland E" first="Elizabeth L." last="Hartland">Elizabeth L. Hartland</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">18426879</idno><idno type="pmc">2446704</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446704</idno><idno type="RBID">PMC:2446704</idno><idno type="doi">10.1128/IAI.00209-08</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000F87</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000F87</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Significant Role for <italic>ladC</italic> in Initiation of <italic>Legionella pneumophila</italic> Infection<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Newton, Hayley J" sort="Newton, Hayley J" uniqKey="Newton H" first="Hayley J." last="Newton">Hayley J. Newton</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sansom, Fiona M" sort="Sansom, Fiona M" uniqKey="Sansom F" first="Fiona M." last="Sansom">Fiona M. Sansom</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Dao, Jenny" sort="Dao, Jenny" uniqKey="Dao J" first="Jenny" last="Dao">Jenny Dao</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cazalet, Christel" sort="Cazalet, Christel" uniqKey="Cazalet C" first="Christel" last="Cazalet">Christel Cazalet</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Bruggemann, Holger" sort="Bruggemann, Holger" uniqKey="Bruggemann H" first="Holger" last="Bruggemann">Holger Bruggemann</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Albert Weissenberger, Christiane" sort="Albert Weissenberger, Christiane" uniqKey="Albert Weissenberger C" first="Christiane" last="Albert-Weissenberger">Christiane Albert-Weissenberger</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Buchrieser, Carmen" sort="Buchrieser, Carmen" uniqKey="Buchrieser C" first="Carmen" last="Buchrieser">Carmen Buchrieser</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cianciotto, Nicholas P" sort="Cianciotto, Nicholas P" uniqKey="Cianciotto N" first="Nicholas P." last="Cianciotto">Nicholas P. Cianciotto</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Hartland, Elizabeth L" sort="Hartland, Elizabeth L" uniqKey="Hartland E" first="Elizabeth L." last="Hartland">Elizabeth L. Hartland</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">Infection and Immunity</title><idno type="ISSN">0019-9567</idno><idno type="eISSN">1098-5522</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Previously, we identified <italic>ladC</italic> in a cohort of genes that were present in <italic>Legionella pneumophila</italic> but absent in other <italic>Legionella</italic> species. Here we constructed a <italic>ladC</italic> mutant of <italic>L. pneumophila</italic> and assessed its ability to replicate in mammalian cell lines and <italic>Acanthamoeba castellanii</italic>. The <italic>ladC</italic> mutant was recovered in significantly lower numbers than wild-type <italic>L. pneumophila</italic> at early time points, which was reversed upon transcomplementation with <italic>ladC</italic> but not <italic>ladC</italic><sub>N430A/R434A</sub>, encoding a putative catalytically inactive derivative of the protein. In fact, complementation of <italic>ladC</italic>::Km with <italic>ladC</italic><sub>N430A/R434A</sub> resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the <italic>ladC</italic> mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the <italic>ladC</italic> mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type <italic>L. pneumophila</italic>. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that <italic>ladC</italic> does not influence the transcriptional profile of <italic>L. pneumophila</italic> in vitro or during <italic>A. castellanii</italic> infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in <italic>L. pneumophila</italic> infection.</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">Infect Immun</journal-id><journal-id journal-id-type="publisher-id">iai</journal-id><journal-title>Infection and Immunity</journal-title><issn pub-type="ppub">0019-9567</issn><issn pub-type="epub">1098-5522</issn><publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">18426879</article-id><article-id pub-id-type="pmc">2446704</article-id><article-id pub-id-type="publisher-id">0209-08</article-id><article-id pub-id-type="doi">10.1128/IAI.00209-08</article-id><article-categories><subj-group subj-group-type="heading"><subject>Molecular Pathogenesis</subject></subj-group></article-categories><title-group><article-title>Significant Role for <italic>ladC</italic> in Initiation of <italic>Legionella pneumophila</italic> Infection<xref ref-type="fn" rid="fn1">▿</xref> </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Newton</surname><given-names>Hayley J.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff1">4</xref></contrib><contrib contrib-type="author"><name><surname>Sansom</surname><given-names>Fiona M.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff1">4</xref></contrib><contrib contrib-type="author"><name><surname>Dao</surname><given-names>Jenny</given-names></name><xref ref-type="aff" rid="aff1">3</xref></contrib><contrib contrib-type="author"><name><surname>Cazalet</surname><given-names>Christel</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Bruggemann</surname><given-names>Holger</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Albert-Weissenberger</surname><given-names>Christiane</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Buchrieser</surname><given-names>Carmen</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Cianciotto</surname><given-names>Nicholas P.</given-names></name><xref ref-type="aff" rid="aff1">3</xref></contrib><contrib contrib-type="author"><name><surname>Hartland</surname><given-names>Elizabeth L.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff1">4</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1">Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Victoria 3800, Australia,<label>1</label> Biologie des Bactéries Intracellulaires, Institut Pasteur, and CNRS URA 2171, Paris Cedex 15, France,<label>2</label> Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois 60611,<label>3</label> Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia<label>4</label></aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia. Phone: (61) 3 8344 8041. Fax: (61) 3 9347 1540. E-mail: <email>Hartland@unimelb.edu.au</email></p></fn></author-notes><pub-date pub-type="ppub"><month>7</month><year>2008</year></pub-date><pub-date pub-type="epub"><day>21</day><month>4</month><year>2008</year></pub-date><volume>76</volume><issue>7</issue><fpage>3075</fpage><lpage>3085</lpage><history><date date-type="received"><day>14</day><month>2</month><year>2008</year></date><date date-type="rev-recd"><day>4</day><month>4</month><year>2008</year></date><date date-type="accepted"><day>9</day><month>4</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2008, American Society for Microbiology</copyright-statement></permissions><self-uri xlink:title="pdf" xlink:href="zii00708003075.pdf"></self-uri><abstract><p>Previously, we identified <italic>ladC</italic> in a cohort of genes that were present in <italic>Legionella pneumophila</italic> but absent in other <italic>Legionella</italic> species. Here we constructed a <italic>ladC</italic> mutant of <italic>L. pneumophila</italic> and assessed its ability to replicate in mammalian cell lines and <italic>Acanthamoeba castellanii</italic>. The <italic>ladC</italic> mutant was recovered in significantly lower numbers than wild-type <italic>L. pneumophila</italic> at early time points, which was reversed upon transcomplementation with <italic>ladC</italic> but not <italic>ladC</italic><sub>N430A/R434A</sub>, encoding a putative catalytically inactive derivative of the protein. In fact, complementation of <italic>ladC</italic>::Km with <italic>ladC</italic><sub>N430A/R434A</sub> resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the <italic>ladC</italic> mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the <italic>ladC</italic> mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type <italic>L. pneumophila</italic>. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that <italic>ladC</italic> does not influence the transcriptional profile of <italic>L. pneumophila</italic> in vitro or during <italic>A. castellanii</italic> infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in <italic>L. pneumophila</italic> infection.</p></abstract></article-meta><notes><fn-group><fn><p><italic>Editor:</italic> J. L. Flynn</p></fn></fn-group></notes></front><floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label><caption><p>Putative adenylate cyclases of <italic>L. pneumophila</italic>. A schematic representation of the domain structure of the five predicted adenylate cyclases of <italic>L. pneumophila</italic> (lpp1131/lpl1135/lpg1130, lpp0730/lpl0710/lpg0674, lpp1277/lpl1276/lpg1322, lpp1446/lpl1538/lpg1490, and lpp1704/lpl1703/lpg1739) is shown. Black rectangles at the N terminus represent predicted signal sequences for export. Shaded larger rectangles indicate predicted transmembrane domains. Other domains are labeled accordingly. The catalytic site of each adenylate cyclase is highlighted with the essential asparagine/arginine pair in bold. The absence of an arginine residue in lpp0730 suggests that this putative adenylate cyclase is catalytically inactive.</p></caption><graphic xlink:href="zii0070874010001"></graphic></fig><fig position="float" id="f2"><label>FIG. 2.</label><caption><p>(A to C) Replication of <italic>L. pneumophila</italic> 130b and the <italic>ladC</italic>::Km, <italic>ladC</italic>::Km(pLadC), and <italic>ladC</italic>::Km(pLadC<sub>N430A/R434A)</sub> mutants within the alveolar epithelial cell line A549 (A), the macrophage cell-line THP-1 (B), and <italic>A. castellanii</italic> (C). Results are expressed as the log<sub>10</sub> CFU of viable bacteria present in the extracellular medium and associated with cells (THP-1 and A549) at specific time points after inoculation; means and standard deviation from at least three independent experiments from duplicate wells are shown. (A) The <italic>ladC</italic>::Km mutant is significantly different from 130b at 3 h (<italic>P</italic> = 0.03, unpaired two-tailed <italic>t</italic> test). (B) The <italic>ladC</italic>::Km mutant is significantly different from 130b at 3 h (<italic>P</italic> = 0.0078, unpaired two-tailed <italic>t</italic> test). (C) The <italic>ladC</italic>::Km mutant is significantly different from 130b at 2 h (<italic>P</italic> = 0.002, unpaired two-tailed <italic>t</italic> test), 6 h (<italic>P</italic> = 0.0005), 12 h (<italic>P</italic> = 0.009), 24 h (<italic>P</italic> = 0.04), and 48 h (<italic>P</italic> = 0.03). (D to F) Ratios of 130b and the <italic>ladC</italic>::Km, <italic>ladC</italic>::Km(pLadC), and <italic>ladC</italic>::Km(pLadC<sub>N430A/R434A</sub>) mutants recovered from A549 epithelial cells (D), THP-1 macrophages (E), and <italic>A. castellanii</italic> (F) to the inoculum at specific time points after infection. Results are expressed as the mean log<sub>10</sub> ratio, <italic>T<sub>n</sub></italic>/<italic>T</italic><sub>0</sub>. *, significantly less than value for 130b (<italic>P</italic> < 0.05, unpaired two-tailed <italic>t</italic> test).</p></caption><graphic xlink:href="zii0070874010002"></graphic></fig><fig position="float" id="f3"><label>FIG. 3.</label><caption><p>Replication of <italic>L. pneumophila</italic> 130b and the <italic>ladC</italic>::Km mutant carrying <italic>ladC</italic> derivatives on pMMB2002, expressed as the ratio of recovered bacteria to the inoculum at 6 h (A) and 72 h (B) postinfection, from <italic>A. castellanii</italic>. Results are expressed as the mean log<sub>10</sub> ratio, <italic>T<sub>n</sub></italic>/<italic>T</italic><sub>0</sub>, and standard deviation from at least three independent infections. *, significantly less than value for 130b(pMMB2002) (<italic>P</italic> < 0.05, unpaired two-tailed <italic>t</italic> test).</p></caption><graphic xlink:href="zii0070874010003"></graphic></fig><fig position="float" id="f4"><label>FIG. 4.</label><caption><p>Quantification of differential fluorescence staining to determine the ratio of intracellular and cell-associated bacteria at 15 min (A and B) and 1 h (C and D) after infection of THP-1 cells with <italic>L. pneumophila</italic> 130b and the <italic>ladC</italic>::Km, <italic>ladC</italic>::Km(pLadC), and <italic>ladC</italic>::Km(pLadC<sub>N430A/R434A</sub>) mutants. Results are expressed as the mean percentage ± standard deviation from at least three independent infections for intracellular bacteria as a percentage of total cell-associated bacteria (representing bacterial uptake) (A and C) and for cell-associated bacteria as a percentage of the inoculum (representing bacterial adherence) (B and D). *, significantly greater than values for both the <italic>ladC</italic>::Km and <italic>ladC</italic>::Km(pLadC<sub>N430A/R434A</sub>) mutants (<italic>P</italic> < 0.05, unpaired two-tailed <italic>t</italic> test).</p></caption><graphic xlink:href="zii0070874010004"></graphic></fig><fig position="float" id="f5"><label>FIG. 5.</label><caption><p>CI values from mixed infections of A/J mice. Mice were inoculated with approximately 10<sup>5</sup> CFU of each strain under investigation and were sacrificed at 24 or 72 h after infection to examine the bacterial content of their lungs. (A, B, and C) Three independent competition experiments between <italic>L. pneumophila</italic> 130b and the <italic>ladC</italic>::Km mutant. (D and E) Two independent competition experiments between <italic>L. pneumophila</italic> 130b and the complemented <italic>ladC</italic>::Km(pLadC) mutant. At 72 h the CI of the <italic>ladC</italic> mutant was significantly lower than that at 24 h (A, <italic>P</italic> = 0.00051; B, <italic>P</italic> = 0.004; C, <italic>P</italic> = 0.0000012 [unpaired two-tailed <italic>t</italic> test]), and at 72 h the CI of the <italic>ladC</italic>::Km(pLadC) mutant was similar to that at 24 h (D, <italic>P</italic> = 0.31; E, <italic>P</italic> = 0.86 [unpaired two-tailed <italic>t</italic> test]).</p></caption><graphic xlink:href="zii0070874010005"></graphic></fig><fig position="float" id="f6"><label>FIG. 6.</label><caption><p>Localization and topology of LadC in the bacterial inner membrane. (A) Immunoblot analysis of fractions from the <italic>ladC</italic>::Km(pLadC) mutant with anti-LadC and anti-DotA antibodies. Bacteria were grown to stationary phase and induced with IPTG, and proteins were fractionated into the cytoplasmic phase (lane 1), Triton X-100-soluble phase (lane 2), Triton X-100-insoluble phase (lane 3), and culture supernatant phase (lane 4). Anti-LadC antibodies specifically detected a Triton X-100-soluble protein, of the correct size (54.7 kDa), that is absent in the <italic>ladC</italic>::Km strain (arrow). Anti-DotA antibodies detected DotA in the inner membrane and secreted fractions of the bacteria and to a lesser extent in the Triton X-100-insoluble fraction (arrowheads) (B). Expression of LadC:PhoA fusions by <italic>L. pneumophila</italic> 130b carrying pLadC-PhoA (lane 1) or LadC<sub>1-184</sub>-PhoA (lane 2). Rabbit anti-PhoA antibodies diluted 1:10,000 were used to detect LadC-PhoA (∼100 kDa) and LadC<sub>1-184</sub>-PhoA (∼70 kDa) by immunoblotting of whole-cell lysates. (C) Alkaline phosphatase activity of <italic>L. pneumophila</italic> 130b carrying pLadC-PhoA (showing no measurable activity) or pLadC<sub>1-184</sub>-PhoA (9.51 ± 1.02 units). Results represent the mean and standard deviation from three independent assays. *, significantly greater than LadC-PhoA (<italic>P</italic> = 0.00009, unpaired two-tailed <italic>t</italic> test).</p></caption><graphic xlink:href="zii0070874010006"></graphic></fig><table-wrap position="float" id="t1"><label>TABLE 1.</label><caption><p>Bacterial strains and plasmids used in this study</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Bacterial strain or plasmid</th><th colspan="1" rowspan="1" align="center" valign="bottom">Serogroup or characteristics</th><th colspan="1" rowspan="1" align="center" valign="bottom">Source or reference(s)</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>L. pneumophila</italic> strains</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> 130b (ATCC BAA-74)</td><td colspan="1" rowspan="1" align="left" valign="top">O1; clinical isolate</td><td colspan="1" rowspan="1" align="left" valign="top"><xref ref-type="bibr" rid="r16">16</xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> 130b <italic>ladC</italic>::Km</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>ladC</italic> disrupted within its native EcoRI site with a Km<sup>r</sup> gene</td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> 130b <italic>dotA</italic>::Cm</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>dotA</italic> disrupted with a Cm<sup>r</sup> gene</td><td colspan="1" rowspan="1" align="left" valign="top"><xref ref-type="bibr" rid="r32">32</xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>E. coli</italic> strains</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> XL1-Blue</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac</italic>-F′ [<italic>proAB<sup>+</sup> lacI</italic><sup>q</sup><italic>lacZ</italic>ΔM15 Tn<italic>10</italic>] (Tet<sup>r</sup>)</td><td colspan="1" rowspan="1" align="left" valign="top">Stratagene</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> BL21(DE3)</td><td colspan="1" rowspan="1" align="left" valign="top">F<sup>−</sup><italic>omp</italic><italic>T hsdS</italic><sub>B</sub> (r<sub>B</sub><sup>−</sup> m<sub>B</sub><sup>−</sup>) <italic>gal dcm</italic> (DE3)</td><td colspan="1" rowspan="1" align="left" valign="top">Novagen (Darmstadt, Germany)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Plasmids</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pCR-Script</td><td colspan="1" rowspan="1" align="left" valign="top">High-copy-number cloning vector (Amp<sup>r</sup>)</td><td colspan="1" rowspan="1" align="left" valign="top">Stratagene</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pMMB2002</td><td colspan="1" rowspan="1" align="left" valign="top">pMMB207 with 401-bp deletion in <italic>mobA</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><xref ref-type="bibr" rid="r33">33</xref>, <xref ref-type="bibr" rid="r42">42</xref></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pLadC</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>L. pneumophila</italic> 130b <italic>ladC</italic>, including promoter region, cloned into XbaI/PstI of pMMB2002</td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pLadC<sub>N430A/R434A</sub></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>L. pneumophila</italic> 130b <italic>ladC</italic> with site-directed changes to the active site, including promoter region, cloned into XbaI/PstI of pMMB2002</td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pLadC<sub>1-296</sub></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>L. pneumophila</italic> 130b <italic>ladC</italic> encoding promoter region to amino acid 296, cloned into XbaI/PstI of pMMB2002; this truncation removes the CycC domain</td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pCYC</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>L. pneumophila</italic> 130b <italic>ladC</italic> fragment encoding amino acids 297 to 483, cloned into XbaI/PstI of pMMB2002; encodes the adenylate cyclase catalytic domain</td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pCYC<sub>N430A/R434A</sub></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>L. pneumophila</italic> 130b <italic>ladC</italic> fragment encoding amino acids 297 to 483 with site-directed changes to the active site, cloned into XbaI/PstI of pMMB2002; encodes the adenylate cyclase catalytic domain</td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pPhoA</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>E. coli phoA</italic>, lacking the N-terminal signal sequence cloned into PstI/HindIII sites of pMMB2002</td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pLadC-PhoA</td><td colspan="1" rowspan="1" align="left" valign="top">Full-length <italic>ladC</italic> cloned into XbaI/PstI sites of pPhoA to create an N-terminal fusion with <italic>phoA</italic></td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pLadC<sub>1-184</sub>-PhoA</td><td colspan="1" rowspan="1" align="left" valign="top">Truncated <italic>ladC</italic>, encoding for the first 184 amino acids, cloned into XbaI/PstI sites of pPhoA to create an N-terminal fusion with <italic>phoA</italic></td><td colspan="1" rowspan="1" align="left" valign="top">This study</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pRSET C</td><td colspan="1" rowspan="1" align="left" valign="top">pUC-derived expression vector with a polyhistidine tag and T7 promoter</td><td colspan="1" rowspan="1" align="left" valign="top">Invitrogen</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> pGEM-T Easy</td><td colspan="1" rowspan="1" align="left" valign="top">High-copy-number cloning vector (Amp<sup>r</sup>)</td><td colspan="1" rowspan="1" align="left" valign="top">Promega</td></tr></tbody></table></table-wrap></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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