Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Abstract

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G؉C gram-positive bacteria, the ␤-and ␥-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.

The Significance of Syncytial Tissues for the Position of the Hexactinellida in the Metazoa

“…Individual sponge species have a highly specific com− plement of bacterial taxa within their tissues, which are passed on through reproduction and are sufficiently constrained to al− low phylogenetic reconstructions based on the associated bac− teria (Erpenbeck et al 2002;Steger et al 2008). In some taxa, the associated microbiota is extremely diverse, and also in− cludes some groups distributed around the choanocyte cham− bers, suggesting some degree of symbiosis in food gathering (Webster et al 2001). In the tetractinellid Geodia barretti, in− ternal sulphate−reducing bacteria (especially Desulfovibrio) facilitate a sequence of sulphate reduction within the sponge tissues in response to oxic−anoxic cycles that relate to pump− ing of water through the aquiferous system (Hoffmann 2003).…”

High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms

“…The mixture was autoclaved (121 1C, 20 min) and cooled to 45 1C. As bacteria using cyanobacterial biomass for their growth, such as actinobacteria (Webster et al, 2001;Eiler and Bertilsson, 2004) often grow slowly, 10 mg l À1 cycloheximide, nystatin and nalidixic acid were added to prevent the growth of yeasts, fungi and fast-growing Gram-negative bacteria. These additions were used to allow longer incubation times for obtaining slowly growing bacteria.…”

Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons

“…The analysis of 16S rRNA genes through clone libraries and fingerprinting approaches such as denaturing gradient gel electrophoresis (DGGE) has greatly extended our knowledge about the phylogenetic richness of sponge-associated bacteria and archaea (Webster et al, 2001(Webster et al, , 2004Hentschel et al, 2002;Taylor et al, 2004Taylor et al, , 2007bHolmes and Blanch, 2006;Longford et al, 2007;Schmitt et al, 2007Schmitt et al, , 2008Thiel et al, 2007;Mohamed et al, 2008b;Zhu et al, 2008). Researchers in other systems have taken this approach one step further, yielding insights into both richness and activity by comparing 16S rRNA gene-and 16S rRNA-derived sequences, respectively (Moeseneder et al, , 2005Winter et al, 2001;Troussellier et al, 2002;Mills et al, 2005;Gentile et al, 2006;Martinez et al, 2006;Brinkmann et al, 2008;McIlroy et al, 2008;West et al, 2008;Rodriguez-Blanco et al, 2009).…”