Ammonium availability affects the ratio between ammonia-oxidizing bacteria and ammonia-oxidizing archaea in simulated creek ecosystems

Martina Herrmann, Andrea Scheibe, Sharon Avrahami & Kirsten Küsel

Supplemental Material - Methods

Denaturing Gradient Gel Electrophoresis. Archaeal and bacterial amoA genes were amplified from biofilm samples using Taq Pol Polymerase (JenaBioscience) with primer sets Arch-AmoAF/Arch-AmoAR (1) and AmoA-1F*/AmoA-2R (6, 9) following published protocols (1, 5). PCR products were used as a template for a second PCR with primer sets AmoA-1F-GC and AmoA-2R-GG (5), or Arch-AmoAF-short (5’-TAATGGTCTGGCTT-3’) and Arch-AmoAR-GC, modified from Francis et al. (1), with a 40 bp-GC-clamp attached to the reverse primer (4). Cycling conditions were as given in (1, 5) but the number of PCR cycles was reduced to 19. PCR products were separated on 8 % polyacrylamide gels with a denaturant gradient of 40 % to 70 % for bacterial amoA and 30 % to 60 % for archaeal amoA at 100 V and 60°C for 15 h (DGGE-1001, C. B. S. Scientifc Corporation, CA.), followed by staining with SYBR Gold (Molecular Probes, Invitrogen). For analysis of DGGE band patterns, presence/absence matrices of DGGE band patterns obtained from water column and biofilm samples from each creek field site or flow channel, respectively, were turned into similarity matrices (Jaccard similarity) and dendrogrammes employing the online-tools Clustering calculator (http://www2.biology.ualberta.ca/jbrzusto/cluster.php) and Interactive Tree of Life (http://itol.embl.de).

Phylogenetic analysis. Archaeal and bacterial amoA genes were amplified from biofilm samples using Hotstar Mastermix (Qiagen) with primer sets Arch-AmoAF/Arch-AmoAR (1) and AmoA-1F*/AmoA-2R (6, 9), following published protocols (1, 5). Three clone libraries for AOA-amoA (Abf-AOA; LSbf-AOA; LNbf-AOA) or AOB-amoA (Abf-AOB; LSbf-AOB; LNbf-AOB) were generated (pGEM-T cloning kit, Promega) by pooling PCR products from the biofilms of duplicate or triplicate flow channels and all sampling times. Plasmids were extracted using Plasmid Mini-Prep Kit (JenaBioscience), and inserts were sequenced by Macrogen (Seoul, South Korea). Phylogenetic analysis was done using the ARB package (3). Phylogenetic trees of deduced AmoA protein sequences of AOA and AOB were constructed using neighbor-joining and parsimony analysis (1000 replicates), and finally a consensus tree was built based on the neighbor-joining and the parsimony tree. Analysis of 25 to 36 sequences per clone library yielded 3 to 6 Operational taxonomic units (OTUs) defined based on a 97 % sequence identity cut-off on amino acid level (DOTUR, 7) with a coverage of the libraries (8) between 94.4 and 100 %. In addition, one clone library for AOA-amoA or AOB-amoA was generated from pooled PCR products of all samples – water phase, biofilm, all flow channels and time points – to assign DGGE band positions of cloned amoA sequences to band positions in the DGGE patterns of the different samples. Sequences obtained from these two clone libraries have been deposited in GenBank under accession numbers GU561892 to GU561938 (archaeal amoA) and GU561939 to GU561986 (bacterial amoA).

Quantification of archaeal and bacterial amoA gene copies. Copy numbers of bacterial and archaeal amoA genes in water column samples from the creek field sites and in water column and biofilm samples from the different flow channels were determined by quantitative PCR (qPCR) using amoA primers Arch-AmoAF/Arch-AmoAR (1) and AmoA-1F/AmoA-2R (6), respectively, with SensiMixTM SYBR Low-ROX Mastermix (Quantace) on a Mx3000P® instrument (Agilent) with cycling conditions as described previously (2). qPCR reactions contained 12-20 ng environmental DNA. Standard curves were prepared from serial dilutions of plasmids containing an environmental archaeal amoA sequence obtained from freshwater sediment (clone AS_AOA_2, EU309881; 2) or the amoA sequence of Nitrosospira multiformis ATCC 25196, respectively, and were linear from 5∙108 to 5∙101 amoA gene copies with a detection limit of 5 copies per reaction (PCR efficiencies: 85 - 93 %). Standard curves prepared from a mixture of plasmids containing environmental sequences of bacterial or archaeal amoA genes obtained in this study gave similar results (data not shown). Specificity of PCR products was confirmed by melting curve analysis and agarose gel electrophoresis. Environmental DNA templates were spiked with standard plasmid DNA to test for the presence of PCR inhibitors.


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