Quantifying Substrate Uptake by Individual Cells of Marine Bacterioplankton by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Microautoradiography

Abstract

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up 3 H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucineassimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.In situ hybridization with fluorescently labeled oligonucleotide probes (FISH) allows enumeration of specific prokaryotic groups or individual phylotypes with a microscope. Recently, this technique was modified to enhance the detection efficiency, and the new method was called catalyzed reporter deposition FISH (CARD-FISH) (25). CARD-FISH uses oligonucleotide probes labeled with horseradish peroxidase, which results in a sensitivity comparable to that obtained with FISH with polynucleotide probes (26). The CARD-FISH protocol includes a permeabilization step for bacterial cells using lysozyme (25) or for Archaea using proteinase K (28). This CARD-FISH protocol combined with microautoradiography (MICRO-CARD-FISH) was used to determine the number of bacterial and archaeal cells taking up the enantiomeric amino acid aspartic acid in waters of the North Atlantic down to a depth of 4,000 m (29).Microautoradiography combined with FISH is increasingly being used to obtain qualitative information on the substrate utilization by specific prokaryotic populations in complex natural communities (6, 24). Cottrell and Kirchman (6) demonstrated that the method commonly used to assess the prokaryotic activity of entire communities (leucine and thymidine incorporation) indeed covers the entire prokaryotic community. Using MICRO-FISH, it has been shown that some groups in a bacterial community are more active than expected based on their relative abundance (7,11,(17)(18)(19). Also, it has been shown that Cytophaga utilizes high-molecular-weight material more efficiently (7), while members of the SAR11 cluster and Roseobacter sp. apparently also efficiently utilize dimethylsulfoniopropionate (18,19).Generally, however, our knowledge about the phylogenetic composition of prokaryotic communities is increasing much faster than our understanding of the function of these phylotypes in their natural environments. Thus, methods that more closely link phylogenetic information with functional aspects are required, and one of these methods is...

Abundance and single‐cell activity of bacterial groups in Antarctic coastal waters

“…We examined the area of the silver grain clusters around bacteria in microautoradiographic preparations in order to estimate single-cell activity more quantitatively (Sintes and Herndl 2006). Mean silver grain area around cells taking up leucine in WAP samples (1.18 6 0.18 mm 2 ; n 5 17; Table 1) was significantly larger (t-test, t 5 2.86, df 5 32, p , 0.05) than the area around cells in MAB waters (0.65 6 0.06 mm 2 , n 5 17; Table 4 mostly at the class or phylum level, as is the case for most previous studies in other systems (del Giorgio and Gasol 2008).…”

Dissolved organic matter assimilation by heterotrophic bacterial groups in the western Arctic Ocean

“…However, the distribution of per-cell assimilation is similar among the broad groups we examined (Cottrell & Kirchman 2003), and there is a high correlation between percent of DOMactive bacteria belonging to a group and the percent of total silver grain area attributable to that group (Cottrell & Kirchman 2003, the present study). There is also a high correlation between silver grain area and bulk leucine incorporation rates (Sintes & Herndl 2006).…”

“…However, the distribution of per-cell assimilation is similar among the broad groups we examined (Cottrell & Kirchman 2003), and there is a high correlation between percent of DOMactive bacteria belonging to a group and the percent of total silver grain area attributable to that group (Cottrell & Kirchman 2003, the present study). There is also a high correlation between silver grain area and bulk leucine incorporation rates (Sintes & Herndl 2006).The second problem is that the sum of contribution to DOM assimilation by the 3 bacterial groups did not equal 100%. In the shelf and shelf-break the sum of the averaged contribution to DOM assimilation was significantly greater than 100% (Student's t-test, 1-sided p < 0.05), but in the slope and basin it was not.…”

Who eats what, where and when? Isotope-labelling experiments are coming of age

“…In oligotrophic environments, physiologically active cells may contain low rRNA copy numbers and be undetectable with standard FISH techniques. This limitation can be overcome by use of catalysed reporter deposition with horseradish peroxidase-labelled probes (CARD-FISH), which can also be combined with MAR (MICRO-CARD-FISH) (Sintes and Herndl, 2006). Recently, the uptake of radiolabelled substrate into FISH-identified bacterial populations was monitored by beta microimaging instead of MAR .…”