The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny, Cell-Compartmentation, Eukaryote-Like Repeat Proteins, and Other Genomic Features

and Other Genomic Features. PLoS ONE 9(1): e87353. doi:10.1371/journal.pone.0087353 The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny, Cell- Compartmentation, Eukaryote-Like Repeat Proteins, and Other Genomic Features Janine Kamke 0 Christian Rinke 0 Patrick Schwientek 0 Kostas Mavromatis 0 Natalia Ivanova 0 Alexander Sczyrba 0 Tanja Woyke 0 Ute Hentschel 0 Matthias Horn, University of Vienna, Austria 0 1 Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Wuerzburg , Wuerzburg, Germany , 2 Department of Energy Joint Genome Institute , Walnut Creek , California, United States of America, 3 Center for Biotechnology, Bielefeld University , Bielefeld , Germany The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake. - Funding: This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing. URL: http://www.bibliothek.uni-wuerzburg.de/en/homepage/. Financial support to U.H. was provided by the SFB630-grant TPA5, the SFB567-grant TPC3, and by the Bavaria California Technology Center (BaCaTeC). T.W., C.R., P.S., N.I and K.M. were funded by the United States Department of Energy Joint Genome Institute, Office of Science of the United States Department of Energy under Contract No. DE-AC02-05CH11231. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Single-cell genomics is a powerful tool to describe genomes of as yet uncultivated organisms from diverse environments [1,2]. Recently it allowed a first glimpse into the vast functional diversity represented by genomes of previously largely uncharacterized candidate phyla [3]. This method further revealed the glycobiome of the candidate phylum Poribacteria, symbionts of marine sponges, based on six single-amplified genome (SAG) sequences [4]. In this study we further examined these SAGs for phylogenetic and additional functional features of Poribacteria. Poribacteria were first discovered as highly abundant symbionts of marine sponges [5] and as of now lack any cultivated representatives. Through amplicon sequencing studied based on 16S rRNA genes they were also detected in seawater albeit in low abundances [68]. Poribacteria are one of the most predominant taxa inhabiting the extracellular matrix (mesohyl) of sponge species around the world [911]. These symbionts are vertically transmitted over larval stages from the adult sponge to the next generation [7,12]. Initially, the candidate phylum Poribacteria showed a moderate phylogenetic relationship to Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC superphylum) based on monophyletic clustering in 16S rRNA gene analysis [5]. Later, Poribacteria were classified as members of the PVC superphylum although the exact position within the superphylum could not be completely resolved [13]. Similar to some members of the PVC superphylum Poribacteria were also suspected to have a compartmentalized cell plan [5]. In this study we revisited the features of phylogeny and cell compartmentalization based on the sequence data of six singlecell derived genomes from the candidate phylum Poribacteria. We further reveal a large abundance and diversity of eukaryote-like domain containing proteins as well as phyH-like proteins in Poribacteria. Materials and Methods Genome Annotation and Analysis Six poribacterial single-cell genome sequences were included in this study, these being Candidatus Poribacteria WGA 3A, 3G, 4C, 4CII, 4E and 4G with Genbank accession numbers ADFK02000000, ASZN01000000, APGO01000000, ASZM01000000, AQTV01000000, AQPC01000000, respectively. These genomes were previously obtained by our group from uncultivated bacteria inhabiting the marine sponge Aplysina aerophoba by fluorescence activated cell sorting (FACS), multiple displacement amplification (MDA), and next generation sequencing [14,4]. Please also note that the initial version of genome WGA 3A (first published as WGA A3 with accession number ADFK00000000 version ADFK01000000) [14] was found to be flawed. It was corrected accordingly and the submission to Genbank was updated (version ADFK02000000) [4]. All genomic information of WGA 3A in this manuscript is based on the latest version of the genome, which should be used for all future studies. For a detailed description of all steps from sample collection to genome assembly and annotation please refer to Kamke et al. [4]. Genome sequences were automatically annotated via the IMG pipeline [15] and manually curated in IMG/MER. All analyses were conducted using the tools in IMG/MER unless further specified. Clustering analysis of PhyH family genes. For clustering of pfam 05721-PhyH family proteins we used the fastclust algorithm in usearch [16] with an identity cutoff of 60% amino acidid. Phylogenetic 16S rRNA Gene Analysis Sequences for 16S rRNA gene based phylogenetic analysis were selected from the SILVA 16S rRNA database version 108 [17] in the ARB software package (V5.3) [18]. All poribacterial 16S rRNA sequences ($1100 bp) available in GenBank by June 2013 and the 16S rRNA sequences of poribacterial single-cell genomes were included. Additional sequences for the candidate phyla Aerophobetes (CD12) and Hydrogenedentes (NKB19) were obtained by blast searches [19] of reference sequences (accession number JN675971 for CD12 and CR933119 for NKB19) against Genbank nr/nt database in June 2013 and selecting the 100 best hits with .75% sequence ID and sequence length $1100 bp. All sequence added to the original database were aligned using the SINA aligner [20] and included into the ARB database for further (...truncated)