Vol. 72: 269–280, 2014 AQUATIC MICROBIAL ECOLOGY Published online July 10 doi: 10.3354/ame01698 Aquat Microb Ecol FREE ACCESS Individual cell DNA synthesis within natural marine bacterial assemblages as detected by ‘click’ chemistry Steven Smriga1,2,*, Ty J. Samo1,3, Francesca Malfatti1,4, Joseph Villareal1, Farooq Azam1 1Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093-0202, USA 2Present address: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 3Present address: Center for Microbial Oceanography: Research & Education (C-MORE), Department of Oceanography, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA 4Present address: OGS (National Institute of Oceanography and Experimental Geophysics) Oceanography Section (OCE), 34151 Santa Croce, Trieste, Italy ABSTRACT: Individual cell growth rates enhance our understanding of microbial roles in regulat- ing organic matter flux in marine and other aquatic systems. We devised a protocol to microscop- ically detect and quantify bacteria undergoing replication in seawater using the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU), which becomes incorporated into bacterial DNA and is detected with a ‘click’ chemistry reaction in <1 h. Distinct EdU localization patterns were observed within individual labeled cells, e.g. some displayed 2 or more distinct EdU loci within a single DAPI- stained region, which likely indicated poleward migration of nascent DNA during the early phase of replication. Cell labeling ranged from 4.4 to 49%, comparable with cell labeling in parallel incubations for 3H-thymidine microautoradiography. Meanwhile, EdU signal intensities in cells ranged >3 orders of magnitude, wherein the most intensely labeled cells comprised most of a sam- ple’s sum community EdU signal, e.g. 26% of cells comprised 80% of the sum signal. This ability to rapidly detect and quantify signals in labeled DNA is an important step toward a robust approach for the determination of single-cell growth rates in natural assemblages and for linking growth rates with microscale biogeochemical dynamics. KEY WORDS: Single cell growth · Click chemistry · Marine bacteria · Microradiography · Epifluorescence microscopy · BrdU Resale or republication not permitted without written consent of the publisher INTRODUCTION growth at microscale resolution (del Giorgio & Gasol 2008). Metabolic activities that can currently Through individual cell interactions at the micro- be detected in individual cells include respiration scale, marine bacteria contribute to organic matter and esterase activity; and viable cells can be fluxes and elemental cycling at the ocean basin scale detected using live and dead stains, nalidixic acid, (Azam & Malfatti 2007). Our conceptual under- and observations of cultivatable microcolonies on standing of the role of microbial dynamics in regu- agar plates. These and other methods have revealed lating organic matter flux is continually strength- the potential for high variability in cell-specific ened by the development and use of techniques that metabolism within bacterioplankton assemblages detect and quantify individual-cell activities and (del Giorgio & Gasol 2008). *Corresponding author: [email protected] © Inter-Research 2014 · www.int-res.com 270 Aquat Microb Ecol 72: 269–280, 2014 A challenge to the field of microbial oceanography MATERIALS AND METHODS is the quantification of individual bacteria cell growth rates by methods that are applicable in a range of Application of click reaction chemistry field settings. One approach is the detection of incor- for detection of EdU porated tritiated thymidine (3H-TdR) via microau- toradiography (Fuhrman & Azam 1982, Douglas et al. Three types of filter membranes (25 mm diameter, 1987). While this method has been used in microbial 0.2 µm pore size) were tested for collecting formalde- oceanography for several decades and has yielded hyde-fixed seawater bacteria and subsequent treat- important insights, its use is often limited by regula- ment with click chemistry: white polycarbonate (Mil- tory restrictions, especially in remote locations and lipore, GTTP02500), black polycarbonate (Millipore, other field settings. An alternative non-radioisotopic GTBP02500), and Anodisc aluminum oxide (What- approach is based on incorporation of the TdR analog man, 6809-6022). The Anodisc membranes were 5-bromo-2’-deoxyuridine (BrdU) (Steward & Azam adopted for use (see supplementary ‘Materials and 1999). Some studies coupled BrdU immunochemical methods’ at www.int-res.com/articles/suppl/ a072 p269 detection with fluorescence in situ hybridization _supp.pdf for further details). After filtration, one- (FISH) to detect the phylogenetic affiliation of grow- quarter-sized membrane sections were removed for ing bacteria cells in coastal seawater (Pernthaler et click reactions, with the remainder stored at −20°C al. 2002, Tada et al. 2011). Another study optimized for future analyses. BrdU detection in marine bacteria and observed a Two different cell permeabilization treatments positive correlation between growth rates and fluo- were tested on filter membranes. One was lysozyme rescence intensity of growing cells (Hamasaki et al. incubation (50 mg ml−1 in TE buffer [10 mM Tris, 2004). Similar to microradiography, immunochemical 1 mM EDTA, pH 7.5], 30 min, 37°C). The other was detection of BrdU, as used in these studies, is a rela- pepsin incubation (2 mg ml−1 in 0.01N HCl, 2 h, 37°C) tively low throughput technique because it requires followed by lysozyme incubation (3 mg ml−1 in TE multiple processing steps, including cell permeabi- buffer, 15 min, ~21°C). The treatments were tested lization, DNA denaturation, probing with anti-BrdU because some fluorescent azides are not readily per- antibodies, and signal amplification via catalyzed meable in eukaryotic cells, and these cells required reporter deposition. additional permeabilization prior to incubation with Recently, a method that uses 5-ethynyl-2’-deoxy- fluorescent azides (Salic & Mitchison 2008). How- uridine (EdU) was developed as an alternative to 3H- ever, our tests demonstrated that a permeabilization TdR and BrdU in cell biology (Salic & Mitchison step was not necessary for marine bacteria (see sup- 2008). Similar to BrdU, EdU acts as an analog for plementary ‘Materials and methods’ for further TdR. Incorporated EdU is detected in cells by ‘ligat- details). ing’ it with a fluorescent azide through a copper(I)- All tests of EdU detection were performed using catalyzed alkyne-azide cycloaddition; this is a spe- the Click-iT EdU AlexaFluor High-Throughput Imag- cific type of ‘click’ reaction that we henceforth simply ing Assay kit (Invitrogen, Cat. No. A10027). The kit call ‘click reaction’ (Kolb et al. 2001, Rostovtsev et al. includes EdU, AlexaFluor-488 azide, reaction buffer, 2002). The relatively small sizes of molecules used in CuSO4, and reaction buffer additive. Reagents were click reactions permit penetration through many distributed and stored according to the manufac- eukaryotic cell membranes and eliminate the need turer’s recommendations. The ‘buffer additive’ com- for DNA denaturation, antibody probing, or signal ponent of the reaction mixture was added to the reac- amplification. tion cocktail just prior to use. AlexaFluor-488 azide Here we report the use of EdU to detect natural was used throughout this study, though other fluo- populations of DNA-synthesizing bacteria in coastal rophore azides are available. seawater. We optimized the method for microscopic Among 4 protocols tested for applying click reac- detection and summarize the parameters that were tion cocktail, a ‘coverslip-inversion’ method using compared during this optimization. We then quanti- ethanol-washed coverslips was adopted (for descrip- fied the dynamic range of single bacteria cell fluores- tion of non-adopted protocols see supplementary cence intensities in natural bacterial assemblages, a ‘Materials and methods’). For each sample, a portion critical step toward a robust approach in the future of filter membrane was placed onto a clean glass for the determination of single cell growth rates in slide with cell side facing up. Glass cover slips (Corn- marine microbes and for linking growth rates with ing, 25 × 25 mm, Part No. 2870-25) were washed with microscale biogeochemical dynamics in the ocean. 70% ethanol and thoroughly dried. The reaction Smriga et al.: DNA synthesis in marine bacterial assemblages 271 cocktail was prepared on ice according to the manu- labeled cells on membranes that had been stored dry facturer’s instructions, but volumes were adjusted for at least 18 mo at −20°C. accordingly depending on the number of filter pieces to be processed. For a one-quarter section of 25 mm membrane, 25 µl reaction cocktail was sufficient and Microscopy and image analysis more cost effective. (The cost per sample was $0.90 for the chemistry assuming 25 µl reaction cocktail per Image capture and analyses were controlled with sample.) The cocktail was spotted onto a cover slip the software package NIS Elements AR 3.0 (SP 1) and inverted onto the membrane piece on the slide; connected to a Nikon Eclipse TE-2000 inverted thus, cells were sandwiched between membrane and epifluorescence microscope equipped with a 100× cover slip. The slide was placed horizontally into an objective, a monochrome CoolSNAP HQCCD cam- incubation chamber that consisted