Name: bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton
Uploaded: 2011-09-10T14:00:00
Description: Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Funding of this project was provided by the National Science Foundation grants OCE1029607 (to X.M.) and MCB0702125 (to M.A.M.) and the Gordon and Betty Moore Foundation (to M.A.M.).

1. Water sample processing
<ol>
 <li>Filter 10L environmental water through 1 &mu;m-pore-size membrane filters to remove large particles and bacteriovores. Collect the water filtrate in a carboy. </li>
 <li>Transfer 36 ml filtrate each into 3 sterile Eppendorf tubes (50 ml) containing 4 ml freshly prepared paraformaldehyde solution (PFA; 10%). Incubate for 2 hrs at room temperature to preserve cells. Collect cells onto 0.22-&mu;m-pore-size white membrane filters by vacuum filtration. Wash the filter by passing 10 ml ph...

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometric+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

Our approach couples BrdU incorporation, FACS and 16S rDNA analysis to allow species-level identification of bacterioplankton that metabolize individual DOC components in aquatic environments. The BrdU incorporation assay labels bacterial cells based on metabolic activities, which allows analysis only on active bacteria and thus does not include dormant cells. In our approach, BrdU incorporation is in situ immunodetected and bacteria that have different levels of BrdU incorporation are subsequently visualized, g...

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>BrdU</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>B5002-5G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lysozyme</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>L6876-5G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>P2308-25MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>In Situ Cell Proliferation Kit, FLUOS</td>
 <td>Kit</td>
 <td>Roche</td>
 <td>11810740001</td>
 <td>Consume more of Anti-BrdU-FLUOS and Incubation buffer per reaction than suggested by the manufacturer.</td>
 </tr>
 <tr>
 <td>Frame-Seal Incubation Chambers</td>
 <td>Material</td>
 <td>Bio-Rad</td>
 <td>SLF-1201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polycarbonate Membrane Filters (142-mm-diameter, 1.0 &mu;m-pore-size)</td>
 <td>Material</td>
 <td>Millipore</td>
 <td>FALP14250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polycarbonate Membrane Filters (25-mm-diameter, 0.2 &mu;m-pore-size)</td>
 <td>Material</td>
 <td>Millipore</td>
 <td>FGLP02500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>illustra PuReTaq Ready-To-Go PCR Beads</td>
 <td>Kit</td>
 <td>GE Healthcare</td>
 <td>27-9559-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>QIAquick gel extraction kit</td>
 <td>Kit</td>
 <td>QIAGEN</td>
 <td>28704</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FailSafe PCR System</td>
 <td>Kit</td>
 <td>EPICENTRE</td>
 <td>FS99060</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

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