mediterranea and H. salexigens. In order to determine if additional described

strains belonging to this clade have unrecognized phototrophic capabilities, extracted R788 in vivo DNAs of species that show no visible pigmentation under conditions of laboratory cultivation were used for a PCR screening with specific primers to detect pufLM genes. BChl a-containing species belonging to the OM60/NOR5 clade were used as positive control. In addition, primers for the detection of soxB (representative for a periplasmic enzyme complex oxidizing thiosulfate) and pop (gene encoding the opsin subunit of proteorhodopsin) were used to identify alternative potential mixotrophic pathways in described chemoheterotrophic species of the OM60/NOR5 clade and neighboring phylogenetic groups. Results obtained with the pufLM and soxB primers are depicted in the phylogenetic tree shown in Figure  1. It turned

out that the genomic DNA of all species described as non-pigmented (H. salexigens, H. mediterranea, “Oceanicoccus sagamiensis”, Dasania marina, Spongiibacter tropicus and Spongiibacter marinus) was negative in the amplification of pufLM genes, whereas a PCR product of the correct size was obtained from all strains supposed to encode genes for a photosynthetic apparatus, except H. rubra. It should be noted that application of the published primers pufLF1 und pufMR1 [5] failed to amplify pufLM genes from strain Rap1red, so that we designed the primers pufLF2 und pufMR2, which have a slightly modified sequence optimized 3-oxoacyl-(acyl-carrier-protein) reductase for members of the OM60/NOR5 selleck chemical clade. Application of the latter primer set allowed the amplification of the pufLM genes of Rap1red and all other available photoheterotrophic members of the OM60/NOR5 clade, but not from H. rubra and species described as non-pigmented. However, the pufLM nucleotide sequence of H. rubra could be finally obtained by the determination of a draft genome

sequence (unpublished data). It turned out that at least two mismatches at the binding site of the forward primer prevented a successful amplification of the pufL and pufM genes from this species. Figure 1 Phylogenetic tree based on almost complete 16S rRNA gene sequences showing the position of BChl a -containing strains within the OM60/NOR5 clade. The dendrogram was reconstructed with a neighbor-joining distance matrix program as implemented in the ARB package using phylogenetic distances calculated with the algorithm of Jukes and Cantor. No filter or weighting masks were used to constrain the used positions of the alignment. In addition, trees were reconstructed using the PHYLIP maximum parsimony program of ARB and the RAxML maximum likelihood program. Bootstrap values (as percentages of 1000 resamplings) are shown in front of each node, if at least with one reconstruction method a value of 80% or above was obtained.