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Supplementary material
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Materials and methods for BrdU immunodetection improvements
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For BrdU method improvements, water samples from Lake Stechlin were collected
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on 25th and 27th January as well as on 17th March 2012 by using 2 l pre-combusted glass
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bottles. Samples were incubated with BrdU (final 1µM) for 24 hours at 4°C (in situ
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temperature ±2°C) to label the DNA of actively growing bacteria (AGB). After
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incubation, 5 ml samples were filtered onto 0.1 µm pore-size polycarbonate membrane
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filters and fixed with 50% ethanol for 1 h. Filters were stored at –30 ºC until further
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analysis.
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To optimize the antibody reaction buffer, samples were processed for BrdU
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immunodetection according to Tada et al. (2010), whereby antibody reaction buffers
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were slightly modified and used at different concentrations. The antibody reaction
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buffers consisted of freshly prepared PBS+BSA+Tween 20 (0.1% tween 20, and 0.5%
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acetylated BSA in 1 × PBS buffer), PBS+blocking (1% blocking reagent in 1 × PBS
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buffer), and TNB (0.5% blocking reagent, 100mM Tris HCl, 150 mM NaCl). For
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reducing the ‘false’ BrdU-positive cells (Supplementary Figure S1), the antibody
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concentration was modified (the ratio of antibody: PBS+BSA+Tween20 buffer = 1:50,
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1:100, 1:200, and 1:500; Supplementary Figure S2). After these modifications, we
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compared results of our TSA protocol with those of the previously developed method of
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Mou et al. (2007) using a FITC-labeled antibody (In Situ Cell Proliferation Kit, FLUOS,
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1810740, Roche, Mannheim, Germany). All reagents were filtered with 0.1-µm
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membrane filter to remove the junk of reagents. To examine the loss of cells after each
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methodological step during direct application of the BrdU detection assay in the glass
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vacuum filter holders, both cells of the no addition control and the BrdU amendments
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were stained with DAPI (1 µg ml-1) and counted with epifluorescence microscope.
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References
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Tada Y, Taniguchi A, Hamasaki K. (2010). Phylotype-specific growth rates of marine
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bacteria measured by bromodeoxyuridine immunocytochemistry and fluorescence in
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situ hybridization. Aquat Microb Ecol 59: 229–238.
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Materials and methods for DGGE analysis
Partial 16S RNA gene amplification for DGGE analysis was carried out with the
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eubacterial primer set.consisting of 0.4 µM of the forward primer with a 40-bp GC
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clamp (341f-GC, 5’-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA
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CGG GGG GCC TAC GGG AGG CAG CAG-3’) and the reverse primer (907r, 5’-CCG
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TCA ATT C[A/C]T TTG AGT TT-3’) (Schäfer and Muyzer, 2001). A touchdown PCR
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program was performed with annealing temperatures sequentially decreasing from 62 to
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52°C by 1°C per cycle, followed by 15 cycles at 52°C. In each cycle, denaturing at
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95°C, annealing at 62 to 52°C, and extension at 72°C lasted for 1 min each. An initial 3
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min denaturation and a final 10 min extension step were added to the PCR program.
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PCR amplification products were confirmed by electrophoresis on ethidium
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bromide-stained 1% agarose gels.
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For DGGE, PCR products (about 500 ng) were loaded onto 8% polyacrylamide
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gels in 0.5 × TAE with a denaturing gradient from 20 to 70% (from top to bottom).
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Electrophoresis was performed at 85 V for 16 h at 60°C in a hot-bath DGGE unit
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(Ingeny PhorU-2, Ingeny International BV, Goes, Netherlands). Gels were stained with
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SYBR gold in 0.5 × TAE for 30 min and destained with 0.5 × TAE for 10 min. The gels
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were then visualized and documented by using a digital camera imaging system
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(VersaDoc 5000, BioLad). The Jaccard coefficient was calculated for cluster analysis of
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DGGE profiles, and the distance matrix was analyzed by using the between-group
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average linkage method for clustering with the R software (R Development Core Team,
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2008).
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Partial 16S RNA gene amplification for cloning analysis was carried out with 0.4 µM of
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forward primer (341f, 5’-CCT ACG GGA GGC AGC AG-3’) and reverse primer (907r,
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5’-CCG TCA ATT C[A/C]T TTG AGT TT-3’). Our PCR program included an initial
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denaturation step for 3 min at 95°C, followed by 30 cycles of denaturation (96°C, 1
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min), annealing (56°C, 1 min), and extension (72°C, 1 min). A final extension of 10 min
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at 72°C completed the reaction. PCR amplification products were confirmed by
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electrophoresis on ethidium bromide-stained 1% agarose gels. All amplicons were
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excised from the agarose gel and cleaned with the QIAquick gel extraction kit (28704,
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Qiagen, Hilden, Germany) before subsequent cloning analysis. The amplicons were
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cloned with a TOPO-TA cloning kit with pCR2.1-TOPO vector (45-0030, Invitrogen,
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CA, USA) according to the manufacturer’s instructions and commercially sequenced
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(Solgent, Korea).
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References:
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Schäfer H, Muyzer G. (2001). Denaturing Gradient Gel Electrophoresis in Marine
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Microbial Ecology. San Diego, CA, USA: Academic Press.
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R-Development-Core-Team (2008) R: A language and environment for statistical
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computing. R Foundation for Statistical Computing: Vienna, Austria.
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