Bromoperoxidase Producing Bacillus spp. Isolated from the Hypobranchial Glands of A Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography–mass spectrometry (LC–MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC–MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple

Putative role for bacterial symbionts in the biosynthetic origin of Tyrian purple in \u3cem\u3eDicathais orbita\u3c/em\u3e, a Muricidae mollusc

Muricidae molluscs are well known for the production of the historically important dye Tyrian purple, however the biosynthetic origin and the role of microbial symbionts in Tyrian purple production are not known. The broad objective of this thesis was to establish the biosynthetic origin and identify genes involved in the biosynthesis of bromoindole derivatives using culture and culture independent modern genomic approaches. This revealed the muricid Dicathais orbita and symbiotic Vibrios both have the capacity to produce indole precursors but specialised bacteria are required to produce the brominated precursor. This thesis offers new opportunities for sustainable production of Tyrian purple and has greatly increased the molecular understanding of Tyrian purple producing molluscs

Characterization of bacterial communities associated with the tyrian purple producing gland in a marine gastropod

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita

Bromoperoxidase Producing <i>Bacillus</i> spp. Isolated from the Hypobranchial Glands of A Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography&#8722;mass spectrometry (LC&#8722;MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC&#8722;MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple

Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod.

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita

Phylogenetic tree of <i>Dicathais orbita</i> samples generated from 16S rRNA sequences by MEGAN.

<p>A = Female hypobranchial gland (F2H); B = Male hypobranchial gland (M1H); C = Female foot (F3F); D = Male foot (M3F). All these sample types have more than 15,000 reads.</p

Similarity of percentages (SIMPER) analysis showing the bacterial genus that contribute most to the differences between hypobranchial gland and foot of <i>Dicathais orbita</i> (Average dissimilarity = 68.51).

<p>Similarity of percentages (SIMPER) analysis showing the bacterial genus that contribute most to the differences between hypobranchial gland and foot of <i>Dicathais orbita</i> (Average dissimilarity = 68.51).</p

Venn diagram showing shared and non-shared bacterial species between the hypobranchial gland and foot of <i>Dicathais orbita</i>.

<p>The number of species that have biosynthetic capabilities relevant to Tyrian purple production are highlighted in different colours (Orange = indole producers; Blue = brominating enzymes; Purple = indole producers and brominating capabilities).</p

Principal Coordinates Ordination (PCO) of bacterial genus composition, based on a Bray Curtis similarity matrix of the relative abundance of OTUs at 97% sequence similarity level for the hypobranchial gland (purple) and foot (orange) of female (F) and male (M) <i>Dicathais orbita</i>.

<p>Principal Coordinates Ordination (PCO) of bacterial genus composition, based on a Bray Curtis similarity matrix of the relative abundance of OTUs at 97% sequence similarity level for the hypobranchial gland (purple) and foot (orange) of female (F) and male (M) <i>Dicathais orbita</i>.</p

Mean (+s.e.) number of OTUs in the hypobranchial gland and foot tissue of <i>Dicathais orbita</i>, showing the mean proportion unique to individuals samples of foot and hypobranchial gland tissue.

<p>(A) = OTUs richness, (B) = H index/diversity.</p