<i>Teredinibacter waterburyi</i> sp. nov., a marine, cellulolytic endosymbiotic bacterium isolated from the gills of the wood-boring mollusc <i>Bankia setacea</i> (Bivalvia: Teredinidae) and emended description of the genus <i>Teredinibacter</i>

A cellulolytic, aerobic, gammaproteobacterium, designated strain Bs02T, was isolated from the gills of a marine wood-boring mollusc, Bankia setacea (Bivalvia: Teredinidae). The cells are Gram-stain-negative, slightly curved motile rods (2-5×0.4-0.6 µm) that bear a single polar flagellum and are capable of heterotrophic growth in a simple mineral medium supplemented with cellulose as a sole source of carbon and energy. Cellulose, carboxymethylcellulose, xylan, cellobiose and a variety of sugars also support growth. Strain Bs02T requires combined nitrogen for growth. Temperature, pH and salinity optima (range) for growth were 20 °C (range, 10-30 °C), 8.0 (pH 6.5-8.5) and 0.5 M NaCl (range, 0.0-0.8 M), respectively when grown on 0.5 % (w/v) galactose. Strain Bs02T does not require magnesium and calcium ion concentrations reflecting the proportions found in seawater. The genome size is approximately 4.03 Mbp and the DNA G+C content of the genome is 47.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences, and on conserved protein-coding sequences, show that strain Bs02T forms a well-supported clade with Teredinibacter turnerae. Average nucleotide identity and percentage of conserved proteins differentiate strain Bs02T from Teredinibacter turnerae at threshold values exceeding those proposed to distinguish bacterial species but not genera. These results indicate that strain Bs02T represents a novel species in the previously monotypic genus Teredinibacter for which the name Teredinibacter waterburyi sp. nov. is proposed. The strain has been deposited under accession numbers ATCC TSD-120T and KCTC 62963T

Teredinibacter haidensis sp. nov., Teredinibacter purpureus sp. nov. and Teredinibacter franksiae sp. nov., marine, cellulolytic endosymbiotic bacteria isolated from the gills of the wood-boring mollusc Bankia setacea (Bivalvia: Teredinidae) and emended description of the genus Teredinibacter.

Here, we describe three endosymbiotic bacterial strains isolated from the gills of the shipworm, Bankia setacea (Teredinidae: Bivalvia). These strains, designated as Bs08T, Bs12T and Bsc2T, are Gram-stain-negative, microaerobic, gammaproteobacteria that grow on cellulose and a variety of substrates derived from lignocellulose. Phenotypic characterization, phylogeny based on 16S rRNA gene and whole genome sequence data, amino acid identity and percentage of conserved proteins analyses, show that these strains are novel and may be assigned to the genus Teredinibacter. The three strains may be differentiated and distinguished from other previously described Teredinibacter species based on a combination of four characteristics: colony colour (Bs12T, purple; others beige to brown), marine salt requirement (Bs12T, Bsc2T and Teredinibacter turnerae strains), the capacity for nitrogen fixation (Bs08T and T. turnerae strains) and the ability to respire nitrate (Bs08T). Based on these findings, we propose the names Teredinibacter haidensis sp. nov. (type strain Bs08T=ATCC TSD-121T=KCTC 62964T), Teredinibacter purpureus sp. nov. (type strain Bs12T=ATCC TSD-122T=KCTC 62965T) and Teredinibacter franksiae sp. nov. (type strain Bsc2T=ATCC TSD-123T=KCTC 62966T)

Teredinibacter haidensis sp. nov., Teredinibacter purpureus sp. nov. and Teredinibacter franksiae sp. nov., marine, cellulolytic endosymbiotic bacteria isolated from the gills of the wood-boring mollusc Bankia setacea (Bivalvia: Teredinidae) and emended description of the genus Teredinibacter.

Here, we describe three endosymbiotic bacterial strains isolated from the gills of the shipworm, Bankia setacea (Teredinidae: Bivalvia). These strains, designated as Bs08T, Bs12T and Bsc2T, are Gram-stain-negative, microaerobic, gammaproteobacteria that grow on cellulose and a variety of substrates derived from lignocellulose. Phenotypic characterization, phylogeny based on 16S rRNA gene and whole genome sequence data, amino acid identity and percentage of conserved proteins analyses, show that these strains are novel and may be assigned to the genus Teredinibacter. The three strains may be differentiated and distinguished from other previously described Teredinibacter species based on a combination of four characteristics: colony colour (Bs12T, purple; others beige to brown), marine salt requirement (Bs12T, Bsc2T and Teredinibacter turnerae strains), the capacity for nitrogen fixation (Bs08T and T. turnerae strains) and the ability to respire nitrate (Bs08T). Based on these findings, we propose the names Teredinibacter haidensis sp. nov. (type strain Bs08T=ATCC TSD-121T=KCTC 62964T), Teredinibacter purpureus sp. nov. (type strain Bs12T=ATCC TSD-122T=KCTC 62965T) and Teredinibacter franksiae sp. nov. (type strain Bsc2T=ATCC TSD-123T=KCTC 62966T)

Microbial distribution and abundance in the digestive system of five shipworm species (Bivalvia: Teredinidae).

Marine bivalves of the family Teredinidae (shipworms) are voracious consumers of wood in marine environments. In several shipworm species, dense communities of intracellular bacterial endosymbionts have been observed within specialized cells (bacteriocytes) of the gills (ctenidia). These bacteria are proposed to contribute to digestion of wood by the host. While the microbes of shipworm gills have been studied extensively in several species, the abundance and distribution of microbes in the digestive system have not been adequately addressed. Here we use Fluorescence In-Situ Hybridization (FISH) and laser scanning confocal microscopy with 16S rRNA directed oligonucleotide probes targeting all domains, domains Bacteria and Archaea, and other taxonomic groups to examine the digestive microbiota of 17 specimens from 5 shipworm species (Bankia setacea, Lyrodus pedicellatus, Lyrodus massa, Lyrodus sp. and Teredo aff. triangularis). These data reveal that the caecum, a large sac-like appendage of the stomach that typically contains large quantities of wood particles and is considered the primary site of wood digestion, harbors only very sparse microbial populations. However, a significant number of bacterial cells were observed in fecal pellets within the intestines. These results suggest that due to low abundance, bacteria in the caecum may contribute little to lignocellulose degradation. In contrast, the comparatively high population density of bacteria in the intestine suggests a possible role for intestinal bacteria in the degradation of lignocellulose

Dual probe localization of bacteria in the gills and digestive system of <i>L. massa</i>.

<p>Confocal micrographs demonstrating patterns of hybridization of 16S rRNA directed oligonucleotide probes targeted to the domain bacteria (Bact338-cy5, red) and shipworm symbionts (SS1273-cy3, green) and negative control probes (Non338-cy5, red and SSnon-cy3, green to reveal autofluorescence or non-specific binding). Micrographs show tissue sections hybridized with Bact338-cy5 and SS1273-cy3 (A–C) or Non338-cy5 and SSnon-cy3 (D–F). These sections contain: gill bacteriocytes (A, D, dark voids are host nuclei and lysosomal residual bodies), a fecal pellet within the intestine (B,E), and caecum content (C,F). The combinatorial color yellow indicates hybridization of both bacteria- and symbiont-targeted probes within the same bacterial cells or bacteriocytes. The appearance of both red bacteriocytes (small arrows) and yellow bacteriocytes (large arrows) in (A) indicates that bacteria within some bacteriocytes contain the symbiont sequence targeted by probe SS1273 while others contain bacteria that lack this target sequence. Note that hybridization of the symbiont-targeted probe SS1273 was detected in gill bacteriocytes only, and not in other examined tissues. All scale bars are 10 µm.</p

Single probe localization of exogenous bacteria added to caecum contents.

<p>Confocal micrographs depicting hybridization of a bacteria-domain specific 16S rRNA directed oligonucleotide probe (Bact338-cy5) to samples on filters. Probe is labeled with the fluorochrome cy5 and displayed in red in images shown. (A) Filter with a mixture of caecum contents and <i>E. coli</i> cells. (B) An equivalent sample of <i>E. coli</i> cells without caecum contents. (C) Equivalent sample of caecum contents without added <i>E. coli</i> cells. Scale bar 10 µm.</p

Fluorescence labeled oligonucleotide probes used in Fluorescence <i>in situ</i> hybridizations.

<p>Fluorescence labeled oligonucleotide probes used in Fluorescence <i>in situ</i> hybridizations.</p

Anatomy of the shipworm <i>Bankia setacea</i>.

<p>a, anus; c, caecum; f, foot; g, gill; h, heart; i, intestine; v, valve (shell); aa, anterior adductor muscle; pa, posterior adductor muscle; st, stomach. Caecum and intestine are shown in brown and green respectively. Region boxed in blue corresponds to (A) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045309#pone-0045309-g002" target="_blank">Figure 2</a>.</p

Single probe localization of bacteria in the gills and digestive system of <i>L. pedicellatus</i>.

<p>Confocal micrographs and diagrams demonstrating patterns of hybridization of either a bacteria-domain targeted 16S rRNA directed oligonucleotide probe (Bact338-cy5) or a negative control probe (Non338-cy5, to reveal autofluorescence or non-specific binding) to adjacent tissue sections of <i>L. pedicellatus</i> containing both gill and digestive tissues. Both probes are labeled with the fluorochrome cy5 (red). (A) Tissue section showing gill filaments (upper right), caecum (lower left) and intestine (diagonal across center, upper left to lower right) probed with Bact338-cy5. Inset- Diagram showing detail of tissue boundaries in the tissue section shown in (A). This region corresponds to the blue box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045309#pone-0045309-g001" target="_blank">Figure 1</a>. (B–G) Enlarged views of tissue sections hybridized with bacteria specific probe (Bact338-cy5; B–D) and negative control probe (Non338-cy5; E–G). These sections contain: gill bacteriocytes, (B,E), a fecal pellet within intestine (C,F), and caecum contents (D,G). Note that fluorescence signal localizes bacteria in gill bacteriocytes (large arrows) and in fecal pellets (small arrows), but few bacteria are observed in caecum contents. c, caecum; f, fecal pellet; g, gill; i, lumen of intestine. Scale bar in (a), 50 µm; (B–G), 10 µm.</p

Shipworm specimens examined in this study.

1<p>captive breeding colony maintained at Ocean Genome Legacy, Ipswich MA, original collection location Long Beach, California.</p