Refined NrfA Phylogeny Improves PCR-Based nrfA Gene Detection
Identifieur interne : 000494 ( Ncbi/Merge ); précédent : 000493; suivant : 000495Refined NrfA Phylogeny Improves PCR-Based nrfA Gene Detection
Auteurs : Allana Welsh [États-Unis] ; Joanne C. Chee-Sanford [États-Unis] ; Lynn M. Connor [États-Unis] ; Frank E. Löffler [États-Unis] ; Robert A. Sanford [États-Unis]Source :
- Applied and Environmental Microbiology [ 0099-2240 ] ; 2014.
Abstract
Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new
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DOI: 10.1128/AEM.03443-13
PubMed: 24463965
PubMed Central: 3993153
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Gene Detection</title>
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<front><div type="abstract" xml:lang="en"><p>Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new <italic>nrfA</italic>
-containing isolates. <italic>nrfA</italic>
amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.</p>
</div>
</front>
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<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">Appl Environ Microbiol</journal-id>
<journal-id journal-id-type="iso-abbrev">Appl. Environ. Microbiol</journal-id>
<journal-id journal-id-type="hwp">aem</journal-id>
<journal-id journal-id-type="pmc">aem</journal-id>
<journal-id journal-id-type="publisher-id">AEM</journal-id>
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<issn pub-type="ppub">0099-2240</issn>
<issn pub-type="epub">1098-5336</issn>
<publisher><publisher-name>American Society for Microbiology</publisher-name>
<publisher-loc>1752 N St., N.W., Washington, DC</publisher-loc>
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<article-id pub-id-type="pmc">3993153</article-id>
<article-id pub-id-type="publisher-id">03443-13</article-id>
<article-id pub-id-type="doi">10.1128/AEM.03443-13</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Microbial Ecology</subject>
</subj-group>
</article-categories>
<title-group><article-title>Refined NrfA Phylogeny Improves PCR-Based <italic>nrfA</italic>
Gene Detection</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Welsh</surname>
<given-names>Allana</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Chee-Sanford</surname>
<given-names>Joanne C.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Connor</surname>
<given-names>Lynn M.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>b</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Löffler</surname>
<given-names>Frank E.</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>c</sup>
</xref>
<xref ref-type="aff" rid="aff4"><sup>d</sup>
</xref>
<xref ref-type="aff" rid="aff5"><sup>e</sup>
</xref>
<xref ref-type="aff" rid="aff6"><sup>f</sup>
</xref>
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<contrib contrib-type="author"><name><surname>Sanford</surname>
<given-names>Robert A.</given-names>
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<xref ref-type="aff" rid="aff1"><sup>a</sup>
</xref>
</contrib>
<aff id="aff1"><label>a</label>
University of Illinois at Urbana Champaign, Urbana, Illinois, USA</aff>
<aff id="aff2"><label>b</label>
USDA-ARS, Urbana, Illinois, USA</aff>
<aff id="aff3"><label>c</label>
Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA</aff>
<aff id="aff4"><label>d</label>
Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, USA</aff>
<aff id="aff5"><label>e</label>
Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee, USA</aff>
<aff id="aff6"><label>f</label>
University of Tennessee and Oak Ridge National Laboratory (UT-ORNL) Joint Institute for Biological Sciences (JIBS) and Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA</aff>
</contrib-group>
<contrib-group><contrib contrib-type="editor"><name><surname>Lovell</surname>
<given-names>C. R.</given-names>
</name>
<role>Editor</role>
</contrib>
</contrib-group>
<author-notes><corresp id="cor1">Address correspondence to Allana Welsh, <email>welsh@illinois.edu</email>
.</corresp>
</author-notes>
<pub-date pub-type="ppub"><month>4</month>
<year>2014</year>
</pub-date>
<volume>80</volume>
<issue>7</issue>
<fpage>2110</fpage>
<lpage>2119</lpage>
<history><date date-type="received"><day>17</day>
<month>10</month>
<year>2013</year>
</date>
<date date-type="accepted"><day>16</day>
<month>1</month>
<year>2014</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2014, American Society for Microbiology. All Rights Reserved.</copyright-statement>
<copyright-year>2014</copyright-year>
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<self-uri xlink:title="pdf" xlink:type="simple" xlink:href="zam00714002110.pdf"></self-uri>
<abstract><p>Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new <italic>nrfA</italic>
-containing isolates. <italic>nrfA</italic>
amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.</p>
</abstract>
</article-meta>
</front>
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<name sortKey="Chee Sanford, Joanne C" sort="Chee Sanford, Joanne C" uniqKey="Chee Sanford J" first="Joanne C." last="Chee-Sanford">Joanne C. Chee-Sanford</name>
<name sortKey="Chee Sanford, Joanne C" sort="Chee Sanford, Joanne C" uniqKey="Chee Sanford J" first="Joanne C." last="Chee-Sanford">Joanne C. Chee-Sanford</name>
<name sortKey="Connor, Lynn M" sort="Connor, Lynn M" uniqKey="Connor L" first="Lynn M." last="Connor">Lynn M. Connor</name>
<name sortKey="Loffler, Frank E" sort="Loffler, Frank E" uniqKey="Loffler F" first="Frank E." last="Löffler">Frank E. Löffler</name>
<name sortKey="Loffler, Frank E" sort="Loffler, Frank E" uniqKey="Loffler F" first="Frank E." last="Löffler">Frank E. Löffler</name>
<name sortKey="Loffler, Frank E" sort="Loffler, Frank E" uniqKey="Loffler F" first="Frank E." last="Löffler">Frank E. Löffler</name>
<name sortKey="Loffler, Frank E" sort="Loffler, Frank E" uniqKey="Loffler F" first="Frank E." last="Löffler">Frank E. Löffler</name>
<name sortKey="Sanford, Robert A" sort="Sanford, Robert A" uniqKey="Sanford R" first="Robert A." last="Sanford">Robert A. Sanford</name>
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