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

Summary

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.

Abstract

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.

Protocol

1. Water sample processing

1. Filter 10L environmental water through 1 μm-pore-size membrane filters to remove large particles and bacteriovores. Collect the water filtrate in a carboy.
2. 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-μm-pore-size white membrane filters by vacuum filtration. Wash the filter by passing 10 ml phosphate-buffered saline (PBS) through the filter by vacuum filtration. Label the filters as negative controls and store them at -20°C.

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BrdU	Reagent	Sigma-Aldrich	B5002-5G
Lysozyme	Reagent	Sigma-Aldrich	L6876-5G
Proteinase K	Reagent	Sigma-Aldrich	P2308-25MG
In Situ Cell Proliferation Kit, FLUOS	Kit	Roche Group	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	EMD Millipore	FALP14250
Polycarbonate Membrane Filters (25-mm-diameter, 0.2 μm-pore-size)	Material	EMD 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 Biotechnologies	FS99060