APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2009, p. 5695–5699 Vol. 75, No. 17 0099-2240/09/$08.00ϩ0 doi:10.1128/AEM.00035-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Widespread Distribution of Poribacteria in Demospongiaeᰔ Feras F. Lafi,1 John A. Fuerst,1* Lars Fieseler,2† Cecilia Engels,2 Winnie Wei Ling Goh,1 and Ute Hentschel2 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia,1 and Downloaded from Research Center for Infectious Diseases, University of Wu¨rzburg, Ro¨ntgenring 11, D-97070 Wu¨rzburg, Germany2 Received 7 January 2009/Accepted 21 June 2009 Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera. http://aem.asm.org/ Recently, a novel bacterial phylum, termed “Poribacteria,” until microbiological processing (9). The GBR marine sponges was discovered, and members of this phylum have been found were collected off Heron Island Research Station (23°27ЈS, exclusively in sponges (2). Phylogenetic analyses of 16S rRNA 151°5ЈE) in April 2002 (5). Pseudoceratina clavata was col- genes indicated that poribacteria are evolutionarily deeply lected by scuba divers at a depth of 14 m, and Rhabdastrella branching organisms and related to a superphylum composed globostellata was collected at a depth of ca. 0.5 m after a reef of Planctomycetes, Verrucomicrobia, and Chlamydia (11). Pori- walk consisting of a few hundred meters. The samples were bacterial 16S rRNA genes contain 13 of 15 planctomycete immediately placed in plastic bags and brought to Heron Is- signature nucleotides, but a level of sequence divergence of Ϫ land Research Station, where they were stored at 80°C until on November 11, 2015 by University of Queensland Library more than 25% compared to any other bacterial phylum, in- processing. Sponge DNA was extracted as described previously cluding the Planctomycetes, justifies the status of this taxon as (2, 5). an independent phylum. A consistent treeing pattern is difficult Total sponge-derived genomic DNA was screened by PCR to resolve in comparative phylogenetic sequence analyses, for the presence of poribacteria using a 16S rRNA gene primer making the poribacteria an unusual line of phylogenetic de- set. Poribacterial 16S rRNA genes were amplified by employ- scent. In addition to their divergent status as a separate phylum ing a pair of Poribacteria-specific primers, POR389f (5Ј-ACG on the basis of the 16S rRNA sequence, poribacteria are also ATG CGA CGC CGC GTG-3Ј) and POR1130r (5Ј-GGC divergent because they may have a compartmentalized cell TCG TCA CCA GCG GTC-3Ј) (2). The poribacterial PCR structure, a cell plan they share only with members of the phyla products that were ca. 740 bp long derived from one sponge Planctomycetes and Verrucomicrobia (2). They are also of in- individual were cloned into the pGEM-T Easy vector (Pro- terest for understanding the potential contribution of obligate mega, Madison, WI). Clone inserts were digested with restric- sponge-associated bacteria to the sponges harboring them and tion endonucleases MspI and HaeIII (New England Biolabs, as an example of a yet-to-be-cultured group of bacteria asso- Inc., United States), characterized to obtain restriction profiles ciated with invertebrate tissue apparently exclusively but for and unique profiles, and sequenced. The compiled partial 16S unknown reasons. This study aimed to further explore the rRNA gene sequences were then analyzed using BLASTN to presence and diversity of poribacteria in different marine select the most closely related poribacterial reference se- demosponge genera using samples from around the world. quences. The Mediterranean sponges were collected by scuba divers The sequences exhibiting levels of similarity of less than 97% Ј Ј offshore at Banyuls sur Mer, France (42°29 N, 03°08 E). The were used for further analysis. Poribacterial 16S rRNA gene Caribbean sponges were collected offshore at Little San Sal- sequences were aligned using the ARB software package (7). Ј Ј vador Island, Bahamas (24°32 N, 75°55 W). The eastern Pa- The resulting alignment was imported into PAUP (10) and cific sponge Aplysina fistularis was collected offshore at San analyzed by using distance, maximum parsimony, and maxi- Ј Ј Diego, CA (32°51 N, 117°15 W). The western Pacific sponge mum likelihood algorithms together with bootstrap resam- Ј Theonella swinhoei was collected offshore at Palau (07°23 N, plings (3,000, 3,000, and 200 resamplings, respectively), and the Ј 134°38 E). All non-Great Barrier Reef (non-GBR) sponges resulting bootstrap values were applied to nodes on the ARB were collected between May and July 2000, and once individ- neighbor-joining tree. Signature sequences were detected us- ual sponge specimens were brought to the surface, they were ing the ARB software package. A signature sequence is de- Ϫ frozen in liquid nitrogen on board ship and stored at 80°C fined here as a short sequence that is present in a group of poribacterial sequences in a phylogenetic clade but is not * Corresponding author. Mailing address: School of Chemistry and found in any other clade in the poribacterial tree. Molecular Biosciences, The University of Queensland, Brisbane, Analysis of the 16S rRNA gene clone library sequences Queensland 4072, Australia. Phone: (49) 931 312581. Fax: (49) 931 generated from sponge tissues revealed the presence of pori- 312578. E-mail: [email protected]. bacteria in sponge individuals belonging to the orders Verongida, † Present address: Institute for Food Science and Nutrition, ETH Zurich, Schmelzbergstrasse 7, CH-8092 Zurich, Switzerland. Astrophorida, Dictyoceratida, Haplosclerida, Lithistida, and ᰔ Published ahead of print on 26 June 2009. Homosclerophorida, while poribacteria could not be detected 5695 5696 LAFI ET AL. APPL.ENVIRON.MICROBIOL. TABLE 1. Distribution of poribacteria in different demosponge orders Presence of Sponge species or seawater Order Geographic locationa Reference poribacteriab Aplysina aerophoba Verongida MED ϩ 2 Aplysina lacunosa Verongida BAH ϩ 2 Aplysina fistularis Verongida EPAC or BAH ϩ 2 Aplysina insularis Verongida BAH ϩ 2 Verongula gigantea Verongida BAH ϩ 2 Smenospongia aurea Dictyoceratida BAH ϩ 2 Downloaded from Aplysina cauliformis Verongida BAH ϩ This study Aplysina archeri Verongida BAH ϩ This study Aplysina cavernicola Verongida MED ϩ This study Pseudoceratina clavata Verongida WPAC ϩ This study Rhabdastrella globostellata Astrophorida WPAC ϩ This study Ircinia sp. Dictyoceratida BAH ϩ This study Xestospongia muta Haplosclerida BAH ϩ This study Theonella swinhoei Lithistida EPAC ϩ This study Plakortis sp. Homosclerophorida BAH ϩ This study http://aem.asm.org/ Chondrilla nucula Hadromerida BAH Ϫ 2 Agelas wiedenmayeri Agelasida BAH Ϫ 2 Agelas cerebrum Agelasida BAH Ϫ This study Axinella polypoides Halichondrida MED Ϫ This study Ptilocaulis sp. Halichondrida BAH Ϫ 2 Dysidea avara Dictyoceratida MED Ϫ This study Haliclona sp. Haplosclerida MED Ϫ This study Ectyoplasia ferox Poecilosclerida BAH Ϫ 2 Seawater NAc MED Ϫ This study a MED, Mediterranean Sea; BAH, Bahamas; WPAC, western Pacific Ocean; EPAC, eastern Pacific Ocean. on November 11, 2015 by University of Queensland Library b The presence of poribacteria was evaluated by sequencing and phylogenetic analysis of amplified PCR products. ϩ, present; Ϫ, absent. c NA, not applicable. in sponges belonging to the orders Hadromerida and Agela- every tree-building algorithm applied (Fig. 1), and one clade sida. In the order Halichondrida, poribacteria were detected in (clade I) was supported by bootstrap values of 64, 98, and 71 in Xestospongia muta but not in Haliclona sp. Altogether, nine distance, maximum parsimony, and maximum likelihood trees, sponge species were added to the list of Poribacteria-containing respectively. Similarity calculations using approximately sponges (Table 1). Three distinct clades were observed that 740-bp amplified poribacterial 16S rRNA gene fragments and were clearly supported by bootstrap values greater than 75 with other poribacterial sequences from the NCBI database showed FIG. 1. Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing relationships of poribacterial clones from different global regions. The poribacterial clones on the right are additional clones belonging to the same clades as strains in the tree at the same level. Bootstrap confidence values of Ͼ75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba;C,Aplysina cavernicola;F,Aplysina fistularis;L,Aplysina lacunosa;S,Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata;T,Theonella swinhoei;X,Xestospongia muta. Scale bar ϭ 0.1 nucleotide substitution per site. VOL. 75, 2009 DISTRIBUTION OF PORIBACTERIA 5697 Downloaded from http://aem.asm.org/ FIG. 2. Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing the internal