Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton

Abstract

We describe a method for microscopic identification of DNA-synthesizing cells in bacterioplankton samples. After incubation with the halogenated thymidine analogue bromodeoxyuridine (BrdU), environmental bacteria were identified by fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-linked oligonucleotide probes. Tyramide signal amplification was used to preserve the FISH staining during the subsequent immunocytochemical detection of BrdU incorporation. DNA-synthesizing cells were visualized by means of an HRP-labeled antibody Fab fragment and a second tyramide signal amplification step. We applied our protocol to samples of prefiltered (pore size, 1.2 micro m) North Sea surface water collected during early autumn. After 4 h of incubation, BrdU incorporation was detected in 3% of all bacterial cells. Within 20 h the detectable DNA-synthesizing fraction increased to >14%. During this period, the cell numbers of members of the Roseobacter lineage remained constant, but the fraction of BrdU-incorporating Roseobacter sp. cells doubled, from 24 to 42%. In Alteromonas sp. high BrdU labeling rates after 4 to 8 h were followed by a 10-fold increase in abundance. Rapid BrdU incorporation was also observed in members of the SAR86 lineage. After 4 h of incubation, cells affiliated with this clade constituted 8% of the total bacteria but almost 50% of the visibly DNA-synthesizing bacterial fraction. Thus, this clade might be an important contributor to total bacterioplankton activity in coastal North Sea water during periods of low phytoplankton primary production. The small size and low ribosome content of SAR86 cells are probably not indications of inactivity or dormancy.

FIG. 1.

Numbers of bacterioplankton cells in filtrates (pore size, 1.2 μm) of North Sea surface water detected by probe EUB338-HRP during 20 h of incubation. The bars indicate the percentages of anti-BrdU-HRP-positive Bacteria cells, and the error bars indicate the total ranges for duplicates.

FIG. 2.

Photomicrographs of FISH- and anti-BrdU-HRP-stained marine bacteria. Each pair of panels shows the FISH signal (green) (left) and the anti-BrdU-HRP signal (red) (right). (a and b) Probe EUB338-HRP and anti-BrdU-HRP staining of preparations incubated without BrdU; (c and d) probe EUB338-HRP and anti-BrdU-HRP staining of preparations supplemented with BrdU; (e and f) probe ALT1413-HRP, specific for members of the Alteromonas lineage, and anti-BrdU-HRP staining; (g and h) probe ROS537-HRP, specific for members of the Roseobacter lineage, and anti-BrdU-HRP staining; (i and j) probe SAR86-1249-HRP, specific for members of the SAR86 clade, and anti-BrdU-HRP staining. The arrows indicate cells that show both FISH staining and anti-BrdU-HRP staining. Bar, 10 μm.

FIG. 3.

Cell numbers of members of the Roseobacter lineage detected by probe ROS537-HRP. The bars indicate the percentages of anti-BrdU-HRP-positive Roseobacter cells, and the error bars indicate the total ranges for duplicates.

FIG. 4.

Cell numbers of members of the Alteromonas lineage detected by probe ALT1413-HRP. The bars indicate the percentages of anti-BrdU-HRP-positive Alteromonas cells, and the error bars indicate the total ranges for duplicates.

FIG. 5.

Cell numbers of members of the SAR86 clade detected by probe SAR86. The bars indicate the percentages of anti-BrdU-HRP-positive SAR86 cells, and the error bars indicate the total ranges for duplicates.