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.


Introduction
Many marine invertebrates produce secondary metabolites that contribute to a suite of ecological roles, including paralysing their prey and preventing predation [1], pathogens [2], surface fouling [3] and competitors [4]. Beyond the ecological roles of the secondary metabolites, these chemicals also provide opportunities that aid human society in the form of novel bio-products. A large number of marine natural products have been isolated and characterized from marine invertebrates [5], including antimicrobial, antifungal, antiviral, antiprotozoal, anthelminthic and anticancer compounds [6][7][8], as well as dyes and pigments [9][10][11][12].
symbionts and genes, some of which have the capacity of producing both indoles and brominated compounds [34,40]. This study aimed to culture bacteria potentially involved in Tyrian purple precursor synthesis, and then screen the isolates for bromoperoxidase genes, using molecular methods. Representative bacteria were then screened for their ability to produce tyrindoxyl sulphate in a potassium bromide supplemented media, using liquid chromatography-mass spectrometry (LC-MS).

Bacterial Isolation
Thirty-two distinct bacteria were isolated in total (Table 1). Six, ten and sixteen bacterial morphotypes were cultured from the hypobranchial gland homogenates, hypobranchial gland dorsal swabs, and hypobranchial gland ventral swabs, respectively ( Table 1). All of the distinct types of bacteria were recovered on marine agar at 7.2 pH, and no additional bacteria were cultured by incorporating the hypobranchial gland aqueous extract into the media. Despite the fact that the pH of the hypobranchial gland has been found to be highly acidic [41], no bacterial colonies were observed at pH 4.5 on marine agar, with or without a 10% aqueous gland extract, when incubated at 25 • C. A subset of the bacteria were recorded on the other growth media-25% of isolates grew on cetrimide agar (CA), 72% on blood agar (BA), 78% on marine agar with hypobranchial gland extracts (MAH) and 81% on thiosulphate citrate bile salts sucrose agar (TCBS) ( Table 1).
All 32 bacterial isolates were motile, with 29 identified as Gram negative and three as Gram positive (Bacillus sp., Bacillus thuringiensis and Halobacillus sp.) ( Table 1).
The sequence similarity to partial 16S rRNA gene sequences, available in the NCBI GenBank for isolates, ranged from 97% to 100% (Table 1).

Putative Bromoperoxidase Gene Screening by PCR
PCR of DNA derived from the 32 distinct bacterial isolates, using primer pairs BBFp (CCCATG TGG ACC ACC CTT TAT) and BBRp (TAA GTG GTC GAT CTT GGGAAT), amplified putative bromoperoxidase coding gene sequences from two Bacillus spp., but failed to amplify any DNA from the remaining 30 bacterial isolates. BLASTN comparison of the gene sequences amplified from the two Bacillus spp., against the NCBI database, revealed a 97% sequence similarity with Bacillus thuringiensis MC28-bromoperoxidase (CP003687.1) ( Table 2).

Bacterial Extract Analysis for Brominated Compounds by Liquid Chromatography-Mass Spectrometry
Pure cultures of the bromoperoxidase containing Bacillus sp. and Bacillus thuringiensis and a subset of bromoperoxidase negative bacterial species (Vibrio chagasii, Pseudoalteromonas sp. and Phaeobacter sp.) were analysed for the possible production of brominated compounds using LC-MS. Evidence for the presence of tyrindoxyl sulphate was found in extracts from the two Bacillus spp., but not in the other three bacteria. An HPLC peak with a retention time of around 14 min was found in cell pellet extracts from Bacillus sp. (KR338869) and Bacillus thuringiensis (KR855712) cultures ( Figure 1B,C). This peak corresponded to a peak detected using selected ion monitoring (SIM) at m/z 224, 226, [M-H]− for 6 bromoisatin (C 8 H 2 BrNO 2 ), which is a stable rearrangement ion commonly detected in Tyrian purple precursors produced by D. orbita [67]. 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 1 H NMR (600 MHz, CD s CN, 25 o C aromatic protons δ 7.65 (1H, d), 7.55 (1H, d), 7.20, 1H, dd), methyl protons δ 2.5 (3H, s)) [67]. Tyrindoxyl sulphate did not produce a strong signal in the total ion current-mass spectrum (TIC-MS) in the negative or positive ion modes (Figures 1 and 2, respectively). Nevertheless, consistent with previous characterisations of tyrindoxyl sulphate from D. orbita hypobranchial gland extracts [67], major ions in the negative ion mode obtained at the apex of this peak, were m/z 336, 338 ( Figure  Using dianion resin extracts from the culture supernatant, we were again able to identify a peak corresponding to tyrindoxyl sulphate, at around 14 min, in a hypobranchial gland extract from D. orbita (Figure 2A We further analysed additional supernatant dianion extracts from the two Bacillus against the tyrindoxyl sulphate standard in the negative ion mode with SIM 224, 226. Despite eluting slightly later, at 15 min (Supplementary Figure S2), the relevant peaks were detected with molecular ion, confirmed at m/z 336, 338, although in the Bacillus thuringiensis supernatant, the peak was below the limit of detection for UV-Vis and, therefore, below the limit for accurate quantification. Quantification of tyrindoxyl sulphate in the broth extracts of Bacillus sp. was undertaken, using the procedure outlined by Valles-Regino et al. [67], and was estimated to be 1 mg/10 mL. We further analysed additional supernatant dianion extracts from the two Bacillus against the tyrindoxyl sulphate standard in the negative ion mode with SIM 224, 226. Despite eluting slightly later, at 15 min (Supplementary Figure S2), the relevant peaks were detected with molecular ion, confirmed at m/z 336, 338, although in the Bacillus thuringiensis supernatant, the peak was below the limit of detection for UV-Vis and, therefore, below the limit for accurate quantification. Quantification of tyrindoxyl sulphate in the broth extracts of Bacillus sp. was undertaken, using the procedure outlined by Valles-Regino et al. [68], and was estimated to be 1mg/ 10 mL.

Discussion
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. Tyrian purple is a dye of historical importance that traditionally could only be obtained by extraction from the Muricidae. Only 1g of dye is obtained from approximately 1,200 snails [31], highlighting the need for sustainable production methods, if the natural Tyrian purple dye is to be supplied on an industrial scale. Using small-scale culture without optimised conditions, we were able to obtain an estimated 1 mg of the precursor tyrindoxyl sulphate from 10 mL of Bacillus culture. Although 6,6 -dibromoindigo can be chemically synthesized [68][69][70] there is still a demand for the natural product. Targeting natural shellfish populations to supply the dye can place populations at risk-as demonstrated by the decline of the central American Muricidae Plicopurpura pansa populations-due to overharvesting [71]. Presently, P. pansa is considered a threatened species and is under special protection from the Mexican government [71]. However, there has been a renewed interest in natural shellfish dyes, from the Jewish community [16]. Bacteria that are capable of brominating indoxyl sulphate to generate Tyrian purple precursors, provide a potential alternative for sustainable production of this natural dye, if the culture and production can be scaled up in the future.
The low microbial diversity observed in the hypobranchial gland homogenates and the identification of 25 Vibrio spp. is consistent with previous studies [40,41]. However, this study also identified two Bacillus species which have coding sequences for bromoperoxidase enzymes. Bacterial species belonging to the Bacillaceae family are known to produce bromoperoxidase [72], along with several other bacteria [45,46,73,74]. Bromoperoxidases produced by marine bacteria are often involved in the biosynthesis of halogenated natural products of pharmacological importance [75], and this enzyme has the capability of reacting with indole, specifically in the 6 position, for the production of 6-brominated indoles [75,76]. Bromoperoxidase activity has been previously reported in the hypobranchial glands of D. orbita [37] and other muricids [35]. Bacillus sp. have also been previously detected in the hypobranchial glands of D. orbita, using culture-independent bacterial profiling [40]. This study confirms that a bromoperoxidase associated with Bacillus in the hypobranchial gland of a Muricidae, is capable of brominating an indole precursor in the 6 position, on the aromatic ring, to form tyrindoxyl sulphate. Bacterial biosynthesis of tyrindoxyl sulphate provides opportunities for sustainable production of the anti-cancer and anti-inflammatory indole derivatives from Muricidae molluscs [13,25].
Other types of bacteria isolated from the hypobranchial gland, did not contain a bromoperoxidase gene, including Pseudoalteromonas sp., Phaeobacter sp. and Vibrio chagasii, and as expected, these failed to produce brominated indoxyl sulphate precursors in culture. Marine Pseudoaltermonas have previously been found to contain halogenase enzymes and produce small polyaromatic brominated secondary metabolites [77,78]. Given that no brominated compounds were detected from the Pseudoaltermonas cultures in this study, this bacterium is less likely to play a role in Tyrian purple precursor synthesis. On the other hand, Bacillus sp. have been isolated from the egg masses of Concholepas concholepas [79], another Muricidae species that produces Tyrian purple in its hypobranchial glands and egg masses [11]. The fact that these bacteria are associated with the egg masses indicates possible maternal transmission of the bacterial symbionts. Overall, this study identifies the potential association between Muricidae and Bacillus, for bioactive-brominated indole and Tyrian purple precursor production. This could be further tested by screening other Muricidae species for bromoperoxidase containing Bacillus species.
The majority of other bacteria cultured from the hypobranchial glands were identified as Vibrionaceae. The finding that Vibrio spp. are the dominant bacterial species in the hypobranchial glands of D. orbita is consistent with our previous metagenomic study on D. orbita hypobranchial glands [40]. Our previous culture study showed that the Vibrios isolated from D. orbita are capable of producing indole [41]. Indeed a range of Vibrio spp. isolated from marine invertebrates and fish, are known to produce indoles [47,48,[80][81][82], but to date, there are no reports of bromoperoxidase genes being isolated from marine Vibrio spp. Consistent with this, none of the Vibrio spp. isolated in our study contained coding sequences for putative bromoperoxidases and tyrindoxyl sulphate was not detected in culture extracts from a representative Vibrio sp. (V. chagasii). Nevertheless, it is possible that the high abundance of Vibrio spp. in the hypobranchial gland of D. orbita contribute indoxyl sulphate precursors, which are then brominated by Bacillus sp. for Tyrian purple precursor synthesis. This would involve a novel interaction between distinct endosymbiotic bacteria in the hypobranchial glands of Muricidae, which requires further investigation. Furthermore, a recent transcriptome study on D. orbita identified a tryptophanase gene, which can convert tryptophan to indole, as well as a number of genes involved in sulphur metabolism, indicating that the mollusc itself might have the capacity to produce indoxyl sulphate. Therefore, it is possible that at least part of the biogenic pathway for Tyrian purple precursors exists in the molluscs and other symbiotic bacteria, thus, potentially contributing to a mixed biosynthetic origin.
Overall, this study provides evidence that Bacillus spp. containing bromoperoxidase enzymes occur in the hypobranchial gland of D. orbita, and are capable of producing brominated precursors for Tyrian purple biosynthesis. However, there remains a possible role for marine Vibrio spp. in contributing non-brominated indoles, which provide the scaffold for bromination and generation of the ultimate precursor tyrindoxyl sulphate. Hence, the role of symbiotic bacteria in the biosynthesis of Tyrian purple precursors is highlighted and provides a scope for future studies on potential sustainable production of this natural dye and other bioactive 6-bromoindole derivatives, through the application of bacterial culture or genetic engineering.

Sample Collection, Preparation and Culturing
D. orbita (n = 15 snails) were collected from subtidal and intertidal rocky reefs near Ballina (28 • 84 S and 153 • 60 E), Northern NSW, Australia. Samples were collected during low tide on 11 December 2014 under permit number F89/1171-6.0 issued by Primary Industries, NSW Government, Australia. Snails were transferred live to the Southern Cross University and processed immediately. The hard shells were removed and the snails dissected, according to Westley and Benkendorff [10]. Hypobranchial glands ( Figure 3) were removed aseptically, under laminar flow, and rinsed three times with sterile seawater to remove any external bacteria loosely associated with the gland. An aqueous extract of the hypobranchial gland was prepared separately for incorporation into bacterial culture media, by homogenising 2 g hypobranchial gland (15 snails) with 35 mL of phosphate buffer saline (PBS) solution in a blender. The extract solution was filter sterilized through a 0.25 µm syringe filter (Minisart, Sartorius, Sigma-Aldrich, Castle Hill, NSW Australia), before adding to the autoclaved marine agar. The pH of the hypobranchial gland was measured, using a pH microprobe (Orion, pH Micro Electrode, Thermo Scientific, Brisbane, Qld, Australia) and was found to have a mean of 4.5 (± 0.08 st. dev, n = 3).
The culture of potential D. orbita hypobranchial gland microbial symbionts was undertaken using five different growth media-marine agar (pH 7.2), marine agar (pH 4.5), marine agar and hypobranchial gland extract (pH 7.2), marine agar and hypobranchial gland extract (pH 4.5), blood agar, TCBS (thiosulphate citrate bile salts sucrose) agar and cetrimide agar. These media were chosen on the basis of their potential to provide favourable conditions, which might not be provided by the standard growth media. Marine agar with hypobranchial gland extract was used to mimic the natural environment of the D. orbita hypobranchial gland. TCBS and cetrimide agar was used as a selective media for isolating Vibrio sp. and Pseudomonas sp., respectively [83][84][85]. Blood agar was used as an enriched media to isolate fastidious bacterial symbionts [86]. Marine agar and marine agar supplemented with 10% aqueous gland extract plates were used at pH 7.2 and adjusted to pH 4.5, using small amounts of HCl, in order to match the pH of the hypobranchial gland lumen. culture media, by homogenising 2 g hypobranchial gland (15 snails) with 35 mL of phosphate buffer saline (PBS) solution in a blender. The extract solution was filter sterilized through a 0.25 μm syringe filter (Minisart, Sartorius, Sigma-Aldrich, Castle Hill, NSW Australia), before adding to the autoclaved marine agar. The pH of the hypobranchial gland was measured, using a pH microprobe (Orion, pH Micro Electrode, Thermo Scientific, Brisbane, Qld, Australia) and was found to have a mean of 4.5 (± 0.08 st. dev, n = 3). Three approaches were used to isolate and culture bacteria from the hypobranchial glands. The sampling approaches included: (1) sampling homogenates of whole hypobranchial glands; (2) taking dorsal swabs of glands and (3) taking ventral swabs of glands. In all cases, three hypobranchial glands, with an approximate total weight of 0.25 g, were used. Homogenates were prepared using a sterile mortar and pestle, whereas, swab samples were taken using sterile cotton swabs. Each of the samples was diluted in 9 mL of sterile sea water, mixed thoroughly by vortexing, and three-fold dilutions were prepared with sterile seawater. Additional concentrated homogenates were also prepared from six individual hypobranchial glands. Following Marinho et al. [87], these were homogenised, separately, in marine broth at a concentration of 1 g/mL, then directly plated onto marine agar and cetrimide agar, for maximum recovery of the bacterial symbionts, including Pseudomonas sp.
A 100 µL aliquot from each homogenate and swab sample was spread onto the duplicate agar plates. The agar plates were incubated for 14 days at 25 • C. Agar plates were observed daily for bacterial colonies and the colony size and morphology were recorded. Morphologically distinct colonies were selected for Gram-staining, using standard procedures [41], and molecular identification of the isolates. All genetically distinct isolates were screened for bromoperoxidase genes and a subset of these were analysed for brominated indole production.

16S rRNA Sequencing of Bacterial Isolates
Morphologically distinct colonies were subjected to 16S rRNA sequence analysis. DNA was extracted using Qiagen DNA extraction kits (QIAmp DNA mini kit, Qiagen, Chadstone, Vic, Australia).

Bromoperoxidase Gene Screening
The genomic DNA of the 32 bacterial isolates were screened for the bromoperoxidase genes, using primer pair BBFp (CCCATG TGG ACC CTT TAT) and BBRp (TAA GTG GTC GAT CTT GGGAAT). These primers were designed on the basis of bromoperoxidase consensus sequence, derived from five Bacillus strains [88]. The PCR reaction was composed of 2.5 µL of 10× PCR buffer; 4 µL of dNTPs (2mM), 1 µL of 50 mM MgCl 2 , 2 µL genomic DNA, 0.5 µL Taq polymerase, 1.5 µL forward primer (FP) (10 µM), 1.5 µL reverse primer (RP) (10 µM), 12 µL Milli-Q water in a final volume of 25 µL. PCR cycle conditions comprised an initial denaturation at 94 • C, for 5 min, followed by 30 cycles of 1 min at 94 • C, 1.30 min at 58 • C and 1 min at 72 • C. PCR amplicons were separated and visualised by agarose gel electrophoresis and GelRed staining, under UV irradiation. Positive bromoperoxidase fragments, approximately 700 bp in size, were purified and sequenced by Applied Biosystems 3730 and 3730xl capillary sequencers (Australian Genome Research Facility, Brisbane, Australia), were further analysed using the sequence scanner software v1.0 (ThermoFisher, Brisbane, Australia) and the sequences were compared with the NCBI GenBank database by BLASTN. Nucleotide sequences for putative bromoperoxidase genes were submitted to the NCBI GenBank under accession number KT180165 and KT180166.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of Bacterial Extracts
Extracts from the pure subcultures of Bacillus sp., Bacillus thuringiensis, Vibrio chagasii, Pseudoalteromonas sp. and Phaeobacter sp. were analysed for the possible production of any brominated compounds. Bacteria were grown in sterilized Schott bottles containing 60 mL marine broth and tryptone broth, since these contain tryptophan which is suspected to be the ultimate precursor for Tyrian purple [23], and were supplemented with 0.2 g potassium bromide (KBr). The bacterial growth was maintained for 24 h at 25 • C. A control of tryptone broth without bacterial inoculation was also maintained with KBr, for comparison. Exponentially growing cultures were centrifuged at 6000 rpm (Heraeus Instruments Biofuge pico, ThermoFisher, Brisbane, Australia) for 10 min, to separate the cells and the supernatant. Extraction of the supernatant was performed by ion exchange chromatography, by passing the supernatant through a dianion resin (Dianion HP 20, Supelco, Bellefonte, PA, USA), then washing the column with methanol, before drying the methanol extract using a stream of high purity nitrogen gas. The cell pellet was also extracted in chloroform-methanol (1:1) and dried under nitrogen gas. These extracts were analysed using LC-MS.
LC-MS analysis was undertaken using an Agilent 1260 infinity (Santa Clara, CA, USA) High Performance Liquid Chromatography (HPLC) system, coupled with a 6120 Quad mass spectrometer (MS), according to the procedure described by Valles-Regino et al. [67]. Tyrindoxyl sulphate in the bacterial extracts was quantified against a standard calibration curve, using HPLC, by integrating the area under the curve for absorbance at 210 nm [67]. The HPLC utilized a Phenomenex luna C18 reversed phase column (100 × 4.6 mm), with a solvent gradient from 10% to 95% acetonitrile (ACN) with 0.005% trifluoroacetic acid (TFA) over 18 min, at a flow rate of 0.75 mL/min. Peak absorption was monitored using parallel UV-Vis diode-array detection (DAD). Electrospray ionisation (ESI) mass spectrometry was used in the positive and negative ion modes. Selected ion monitoring at m/z 224, 226 was also used in negative mode to detect these common fragment ions that are typically generated in the mass spectrum, from the Tyrian purple precursors in the extracts from D. orbita [67]. Agilent ChemStation (Agilent Technologies Australia, Mulgrave, Vic) was used to analyze the LC-MS data. Characteristic ion cluster patterns from Br 79 and Br 81 in the mass spectrum were used for identifying the presence of any brominated compounds [10]. To confirm the identity of the purified tyrindoxyl sulphate standard H 1 MNR was undertaken, and was recorded on a Bruker Advance III HD 500 MHz spectrometer (Bruker Biospin, Alexandria, NSW, Australia) in CD 3 CN and D 2 O (Novachem, Cambridge Isotopes Laboratories, Tewksbury, MA, USA). 1 H chemical shifts were referenced to either CD 3 CN (1.96 ppm) or D 2 O (4.80 ppm).

Supplementary Materials:
The following are available online at http://www.mdpi.com/1660-3397/17/5/264/s1, Figure S1: Liquid chromatography-mass spectrometry analysis of dianion resin extracts of Dicathais orbita hyprobranchial glands and culture supernatant from two Bacillus spp. cultured from the hypobranchial gland. Left panels show the HPLC chromatograms from the diode array detector at 280 nm (blue lines) and total ion current (red lines) in positive ion mode. The UV-Vis spectra is inset for the major peak at 14mins and the right panels show the positive ion mass spectra for the major peaks; Figure S2. Tyrindoxyl sulfate control (A) and dianion resin extracts from the supernatant of two Bacillus species (B, C) cultured from the hypobranchial glands of Dicthais orbita and a corresponding tryptone -KBr supplmentaed broth control (D). Left panels show the HPLC scan at 280nm in the diode array (blue), total ion current (TIC) in negative ion mode (red) and selected ion monitoring for major fragment ions at 224, 226 (green). Right panels show the UV Vis spectra and mass spectrum obtained from the apex of the major peak obtained at 15.16 min.
Author Contributions: K.B. initiated the study and all authors contributed to the experimental design. A.K.N. did the sampling, dissections, culturing, DNA extraction and quality check, PCR and submission of samples for genetic sequencing. J.S. assisted in designing the bacterial culture conditions. P.M. assisted in LC-MS analyses on the extracts from the bacterial cultures. A.K.N. and K.B. wrote the paper, with editorial input from D.W. K.B. revised the paper in response to reviews.
Funding: This research received no external funding.