Figure 5. Type I PKS members per genome equivalent for the Minnesota farm soil, whale falls, Sargasso Sea and acid mine drainage sample estimated by Raes et al. [31]. The soil sample has the highest density of type I PKS per genome. doi:10.1371/journal.pone.0003515.g005 are metagenomic sequences that seem to have a closer relationship to eukaryotic type I PKS members. For example six Sargasso Sea sequences can be found close to C. elegans and Alveolata proteins in the AT domain tree. The originating species of these sequences is unfortunately unclear. Despite the fragmentation of the metagenomic sequences we were able to find proteins with multiple domains in some of the six environments. In the Sargasso sea sample, 15 of these with a maximum number of seven domains were detected. The farm soil collection hosted nine multidomain proteins but none extended beyond two domains. The phosphorus removal sludge set contained six (up to three domains) and the whale fall one (two domains) of such sequences. The small number of multi domain hits found reflects the low coverage of the samples. But the fact that at least some are found give high confidence that we have detected real PKS I members and that these communities might be useful as sourced for further and more focused sequencing and screenings. Distribution of potential type I PKS members in the different Sargasso Sea samples The Sargasso Sea data set is composed of seven samples. It has been suggested that sample 1 of the Sargasso Sea data set was contaminated with Burkholderia and Shewanella species [32]. To exclude the possibility that this contamination biased the identification of PKS I proteins, the sample of origin of each of protein identified was examined. Additionally, their closest relatives in UniRef were determined by using BLAST. We found that seven of the 15 proteins with multiple domain hits were encoded by contigs mainly built from sample 1 reads, four from Burkholderia and two from Shewanella. Of the 171 single-domain hit proteins in the seven Sargasso samples, only 27 are found in contigs with contributions of sample 1 and none of these seems to be close related to Burkholderia proteins or Shewanella proteins. The high number of multi-domain protein hits coming from potential contaminations may be a result of the better coverage of these genomes in the first sample. However, the remaining single- domain hit proteins provide enough evidence that type I PKS proteins are not solely due to the contaminating species but that the uncontaminated ocean sample also hosts type I PKS producing organisms. PKS I Screen in Metagenomes Detection of non-annotated PKS I members in UniRef The screening and tree based refinement of UniRef proteins revealed type I PKS members that were so far not annotated as PKS I or PKS at all. This includes 971 proteins with multiple PKS I domain HMM hits and 760 proteins (mostly short, fragmented ones) with only one such hit. Additionally we could confirm the proposed annotation of further proteins, 197 proteins with multiple domain hits and 146 proteins with single domain hits, that were marked as hypothetical, putative, probable or predicted PKS or PKS I. The classification and functionality of PKS proteins in animals is still unclear. Based on the analysis of AT and KS domains Jenke- Kodama et al. [12] placed the animal FAS into the type I PKS family which makes them a subfamily of PKS. Castoe et al. [10] showed that sea urchins (Strongylocentrotus purpuratus and Lytechinus variegatus), birds (Gallus gallus), and fish (Danio rerio and Tetraodon nigroviridis) harbour PKS-like proteins with uncertain functionality, which are closely related to PKS members of Dictyostelium. In our study, the Metazoa contributed proteins with AT and KS domains (in some cases also the ER domain) that were placed in the PKS I branches of the trees while the remaining domains were found in non-PKS I branches. This distribution was the case for some insects, amphibia fish, echinodermata and mammals. In contrast all detected six domains of a protein in Caenorhabditis briggsae and eleven domains (except one DH domain) in Caenorhabditis elegans seem to be true type I PKS domains. The proteins in the Alveolata Cryptosporidium hominis, Cryptospo- ridium parvum, Toxoplasma gondii are very large and contain only PKS I annotated domains. It confirms the described occurrence of PKS I in the protozoan pathogen Cryptosporidium parvum [9]. The detection of type I PKS members in Ostreococcus tauri and Ostreococcus lucimarinus sequences in UniRef supports a study that reported type I PKS proteins in unicellular green algae based on a KS domain tree [33]. The PKS I of these protists are described to be different from the currently known PKS proteins and might have a long separated evolution. The different domains detected were found to be placed close to disparate taxonomic groups (within bacteria and eukaryotes) in the trees generated. Indication of horizontal gene transfer The constructed phylogenetic trees also revealed some cases of potential horizontal gene transfers. An example is a small group of 3 fungal protein taxa in the AT domain tree that is placed in the Actinobacteria. In the DH domain tree, four Danio rerio (zebra fish) sequences are nested in a small group of fungal sequences that is surrounded by sequences from Actinobacteria. All proteins have the same domain structure including a KS, AT and KR domain in addition to the DH domain. It cannot be excluded that the detected protein originated from a genome contamination though. Protein identifiers of the described cases are listed in the Metods S3. Discussion Because of their size, modular structure, complicated evolution and similarity to type I FAS and other enzymes, PKS members are a challenging group of enzymes to identify and to classify. We were able to detect type I PKS proteins – one subgroup of the PKS group - in almost all the samples studied (Fig. 3). The Minnesota farm soil sample shows the highest density of PKS I which is not surprising as this environment has the highest species density which leads to strong competition and an ‘‘arms race’’ between species. The enormous potential for soil as source of useful secondary metabolites was already discussed earlier [34] and our results support these statements. PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3515