Discovery of the novel candidate phylum "Poribacteria" in marine sponges

Abstract

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.

FIG. 1.

Phylogenetic maximum likelihood dendrograms calculated with nearly complete 16S rRNA gene sequences (>1,300 bp) of the candidate phylum “Poribacteria” (a) and of the Deltaproteobacteria and Chloroflexi (b) that were recovered from marine verongid and dictyoceratid sponges. Sequences obtained in this study are indicated in bold. The specificities of newly designed, “Poribacteria”-specific FISH probes POR389, POR600, and POR1130 are indicated in square brackets. Neighbor-joining or maximum parsimony (100 resamplings) bootstrap values are provided for relevant groups. Bar, 10% divergence. The arrow indicates the outgroup.

FIG. 2.

FISH of bacterial preparations from A. aerophoba. (a) Hybridization with EUB338 probe mix (FITC-labeled) and “Poribacteria”-specific probe POR1130 (Cy3-labeled). (b and c) Hybridization with probe POR1130 (FITC-labeled) and probes POR389 (b) and POR600 (c) (Cy3-labeled), specific for the “Poribacteria” subgroups. Yellow signals result from cohybridization. Note the ring-shaped fluorescent signal in the inset in panel c. (d) Hybridization with probe GNS934, specific for the Chloroflexi I cluster (FITC-labeled), and probe POR1130 (Cy3-labeled).

FIG. 3.

Transmission electron microscopy of bacteria associated with A. aerophoba. The nucleoid-containing bacterial morphotypes, according to the work of Fuerst et al. (17), are described in Table 2.