Tyrian purple is a dye of historical importance that traditionally could only be obtained by extraction from the Muricidae. This study provides the first evidence of bromoperoxidase producing bacteria that are capable of biosynthesizing the brominated precursor of Tyrian purple in the hypobranchial gland of a muricid mollusc. The diode array revealed UV absorption maxima at 228 and 302 nm from the tyrindoxyl sulphate standard and Bacillus spp. orbita hypobranchial gland extracts, major ions in the negative ion mode obtained at the apex of this peak, were m/z 336, 338 ( Figure 1 A, B, C), which corresponds to the molecular ion − of tyrindoxyl sulphate (C 9H 7Br 79NO 4S 2 -, C 9H 7Br 81NO 4S 2 -). Nevertheless, consistent with previous characterisations of tyrindoxyl sulphate from D. Tyrindoxyl sulphate did not produce a strong signal in the total ion current–mass spectrum (TIC-MS) in the negative or positive ion modes ( Figure 1 and Figure 2, respectively). The peak detected using SIM at m/z 224, 226 was not detected in the broth control ( Figure 1D), but corresponded to the peak in a purified tyrindoxyl sulphate standard ( Figure 1A) isolated from the hypobranchial gland, and confirmed by proton nuclear magnetic resonance 1H NMR (600 MHz, CD sCN, 25 oC aromatic protons δ 7.65 (1H, d), 7.55 (1H, d), 7.20, 1H, dd), methyl protons δ 2.5 (3H, s)). This peak corresponded to a peak detected using selected ion monitoring (SIM) at m/z 224, 226, − for 6 bromoisatin (C 8H 2BrNO 2), which is a stable rearrangement ion commonly detected in Tyrian purple precursors produced by D. (KR338869) and Bacillus thuringiensis (KR855712) cultures ( Figure 1B,C). An HPLC peak with a retention time of around 14 min was found in cell pellet extracts from Bacillus sp. Evidence for the presence of tyrindoxyl sulphate was found in extracts from the two Bacillus spp., but not in the other three bacteria. and Phaeobacter sp.) were analysed for the possible production of brominated compounds using LC–MS. and Bacillus thuringiensis and a subset of bromoperoxidase negative bacterial species ( Vibrio chagasii, Pseudoalteromonas sp. Pure cultures of the bromoperoxidase containing Bacillus sp. Several other bacterial species are also known to produce bromoperoxidase and indoles orbita tissue, have been previously found to produce bromoperoxidase enzymes. Bacterial species, such as Bacillus sp., Synechococcus sp., and Pseudomonas putida, which were identified in D. orbita hypobranchial gland, suggests a potential role for bacterial symbionts in the biosynthesis of the precursor of Tyrian purple. The structural similarity between Tyrian purple and indigo, coupled with the culture-independent identification of bacteria capable of producing indole and brominating secondary metabolites, in the D. Structural homology between marine invertebrate natural products and microbial metabolites can provide an indication that these natural products originate from the microbes, rather than being synthesized by the host. There is a growing research interest in the involvement of bacteria–host invertebrate associations in the biosynthesis of marine secondary metabolites. This study supports a potential role for symbiotic Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. LC–MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. 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. In total, 32 bacterial isolates were cultured from D. 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). This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. 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. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans.
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