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.
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.
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…
The authors have nothing to disclose.
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.).
Name | Type | Company | Catalog number | Comments |
BrdU | Reagent | Sigma | B5002-5G | |
Lysozyme | Reagent | Sigma | L6876-5G | |
Proteinase K | Reagent | Sigma | P2308-25MG | |
In Situ Cell Proliferation Kit, FLUOS | Kit | Roche | 11810740001 | Consume more of Anti-BrdU-FLUOS and Incubation buffer per reaction than suggested by the manufacturer. |
Frame-Seal Incubation Chambers | Material | Bio-Rad | SLF-1201 | |
Polycarbonate Membrane Filters (142-mm-diameter, 1.0 μm-pore-size) | Material | Millipore | FALP14250 | |
Polycarbonate Membrane Filters (25-mm-diameter, 0.2 μm-pore-size) | Material | Millipore | FGLP02500 | |
illustra PuReTaq Ready-To-Go PCR Beads | Kit | GE Healthcare | 27-9559-01 | |
QIAquick gel extraction kit | Kit | QIAGEN | 28704 | |
FailSafe PCR System | Kit | EPICENTRE | FS99060 |