The purpose if this was to obtain an overall picture of the planctomycete populations at each sampling time. Variation in OTU composition between individual kelp laminae is not captured by this approach, but has been addressed previously for the whole bacterial communities [18]. The pooled DNA extracts (from February 2007, July 2007 and September 2008) were selleck inhibitor used for the subsequent PCR amplification and clone library construction. PCR amplification and clone library construction The Planctomycetes specific forward primer Pla46f (5′-GGA TTA GGC ATG CAA GTC-3′) complementary to the Pla46 FISH probe [19] and the general bacterial reverse primer 1542r

(5′-AAG GAG GTG ATC CAG CCG CA-3′) [40] were used to amplify a near full length fragment of the 16S rRNA

gene of Planctomycetes. PCR conditions were: 94°C for 5 min, 25 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and final elongation at 72°C for 10 min. Each 25 μl PCR reaction contained Dorsomorphin order nuclease-free water, F511 buffer (Finnzymes), 0.1 mM of each dNTP (F506L, Finnzymes), 0.02% BSA, 0.5 μM of each primer, 0.02 U Dynazyme II F501-L (Finnzymes), and approximately 30 ng template DNA. Three clone libraries, one from each sampling occasion, were constructed using the TOPO TA cloning kit (Invitrogen). Ninety-six clones were picked from each clone library. Cloned fragments were reamplified using the supplied M13 primer pair according to the manufacturers instructions. Sequencing and sequence processing All cloned fragments were sequenced in one direction using the Pla46f primer. Sequencing find more was carried out with the BigDye Terminator v3.1 kit (Applied

Biosystems) at the Bergen Sequencing Facility http://​www.​seqlab.​uib.​no using an ABI 3700 sequencing system. Base calling from the chromatogram files was done using the Phred software [41] (version 0.020425.c). The resulting sequences representing partial fragments of the 16S rRNA gene were used to select a subset of clones to sequence in the reverse direction in order to obtain near complete length Coproporphyrinogen III oxidase 16S rRNA gene fragments. The sequences were trimmed to approximately 750 bp of good quality sequence and aligned against the Silva seed alignment (release 102) using the SINA web aligner [23]. The alignment was imported into the ARB software package [42] (version 5.0) and was manually edited to improve alignment quality. The resulting alignment was used to create a distance matrix in ARB, which was used to cluster the sequences into OTUs using the furthest neighbor algorithm in the Mothur software [43] (version 1.9.0). Rarefaction and overlap analysis were carried out in Mothur. The Shannon diversity index and the Chao1 richness estimate was calculated in the R statistical environment ([44], functions: diversity and estimateR, package: vegan). Based on the OTU clustering, one to six representatives of each OTU were sequenced in reverse using the 1542r primer.