Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod
Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod
Ajit Kumar Ngangbam 0 1
Abdul Baten 0 1
Daniel L. E. Waters 0 1
Steve Whalan 0 1
Kirsten Benkendorff 0 1
0 1 Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University , Lismore, NSW 2480 , Australia , 2 Southern Cross Plant Science , Southern Cross University , Lismore, NSW 2480 , Australia
1 Editor: Jose Luis Balcazar, Catalan Institute for Water Research (ICRA) , SPAIN
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.
Competing Interests: The authors have declared
that no competing interests exist.
Tyrian purple is a dye of historical and religious importance [1, 2] and its indole precursors are
reported to have potential anticancer and antimicrobial properties [3–8]. Muricid molluscs are
the only natural source of Tyrian purple , which is formed as a secondary metabolite from
indoxyl sulfate precursors stored in the hypobranchial gland [4, 9]. The main pigment in
Tyrian purple (6, 6’ dibromoindigo) was the first marine natural product to be structurally
elucidated , however, even a century later, little information is available on its biosynthesis or
the potential role of endosymbiotic bacteria in its production . 6, 6’ Dibromoindigo is the
brominated derivative of the blue dye indigo, produced by plants [11, 12] and a range of
bacteria [13–15]. The formation of halogenated marine natural products (mostly containing
chlorine and bromine), requires enzymes, such as halogenases and haloperoxidases .
Bromoperoxidases are believed to be involved in the bromination of indoxyl sulfate precursors,
resulting in Tyrian purple biosynthesis in muricid molluscs [17, 18]. Several marine bacteria
such as Psychrobacter sp., Vibrio sp., Pseudomonas sp. and Streptomyces sp. produce
halogenases [19–23], while Pseudomonas sp. [24–26], Streptomyces sp. [27–30] and Synechococcus
sp.  are known to produce bromoperoxidases.
Bacterial profiling using pyrosequencing is an efficient approach for identifying the diversity
of endo-symbiotic bacteria and their interactions within marine invertebrates. For instance,
metagenome analysis has revealed the remarkable diversity of bacterial symbionts in sponges
[32, 33] and the presence of biosynthetic genes in sponge microbial symbionts [34, 35]. Other
studies have highlighted the host–symbiont biochemical interactions between Proteobacterium
sp. and the deep sea tube worm, Riftia sp. [36–38]. Thus, targeted metagenomic studies can
elucidate a range of species associations and functional relationships.
The Australian muricid, Dicathais orbita, provides a useful model for studying Tyrian
purple, and the biosynthesis of anticancer brominated indoles more generally . In preliminary
studies, three indole producing bacteria were cultured from the Tyrian purple producing
hypobranchial glands of D. orbita. However, these studies relied on traditional culture methods and
yielded a relatively low number of bacteria, with just 16 distinct strains isolated from all tissues,
only three of which were from the Tyrian purple producing gland . Culturing most marine
bacteria is difficult, with only an estimated 0.001–0.1% of marine microbes being successfully
cultured . Therefore, the aim of this study was to assess the diversity of bacteria associated
with the Tyrian purple producing hypobranchial gland using high-throughput sequencing
(454 GS FLX Titanium) of the variable region V1-V3 of the 16S rRNA bacterial gene.
Comparison of these sequences with equivalent sequences isolated from foot tissue will contribute to
the identification of bacteria specifically associated with, or more abundant in, the Tyrian
purple producing hypobranchial gland. A further aim was to identify bacteria with potential to
produce indoles and brominated compounds, based on their taxonomic affiliation.
Materials and Methods
Sample collection and maintenance
Adult specimens of D. orbita were collected under permit number F89/1171-6.0 and
P10/00691.0 issued by Department of Primary Industries, (NSW) Australia. Six live snails were collected
from the intertidal rocky reefs of Flat Rock, Ballina (28°840 S and 153°600 E), NSW, Australia,
during low tides in April and July 2014. Snails were held in aerated seawater tanks for a
maximum of 24 hours before processing.
Snail dissection and total DNA extraction
The hard shell of D. orbita was removed by applying pressure between the primary body whorl
and spire using a bench vice . The hypobranchial glands and the foot were carefully rinsed
by pipetting with sterile sea water to remove any sediment before dissecting. Total genomic
DNA from triplicate female and male hypobranchial glands, as well as the female and male
foot, was extracted using the QIAmp DNA mini kit (Qiagen) following the manufacturer’s
instructions. DNA quality and concentrations were determined with agarose gel
electrophoresis and a NanoDrop 2000 Spectrophotometer (Thermo Scientific) and then stored at –20°C
pending analysis. Only those samples that passed quality control checks were used in the 16s
rDNA bacterial profiling libraries, so in total only duplicate samples were obtained for each
gender and tissue combination.
Roche GS- FLX amplicon sequencing
Bacterial diversity of the biosynthetic organ (hypobranchial glands) and non-biosynthetic
tissues (foot) of D. orbita were characterised by high-throughput sequencing (454 GS FLX
amplicon sequencing)  using the primer pair of 27F/519R that targeted the variable region
V1-V3 of 16S rRNA bacterial gene [43, 44]. DNA samples were shipped to Macrogen Inc,
South Korea  for high-throughput sequencing. GS FLX data processing was performed
using Roche GS FLX software (v 2.9) in two stages, image processing and signal processing.
Image processing involves normalization of raw images and generation of raw signals. In the
signal processing stage, correction, filtering, and raw signal trimming were done prior to base
calling with corresponding quality score of reads. Sequence reads from each sample were
segregated with in-house script (Macrogen) using the tag (Barcode) sequences, and by matching the
initial and final bases of the reads to the known tag sequences used in the preparation of the
Sequences were filtered for low quality bases and chimeric sequences. Only sequences of 100
bp., or more, were selected for final analysis. All sequence analyses were performed using
QIIME version 1.8.0  and open-reference operational taxonomic units (OTUs) picking
strategy was employed. OTUs were picked based on 97% sequence similarity using UCLUST
algorithm  and taxonomies were assigned against the well curated Silva_119 database .
The parameters used for OTU picking and taxonomic assignments are as follows: pick_otus.py
-i all.merged.min100bp.fasta—-threads = 8 and assign_taxonomy.py -i rep_set.fna–r
/Silva119_for_Qiime/rep_set/97/Silva _119_ rep _set 97.fna -t/Silva119_for_Qiime/taxonomy
/97/ taxonomy_97_all_levels.txt -o taxonomy _results/ -e 0.01—uclust_similarity = 0.85.
Sample specific OTUs were retrieved from all the OTUs and aligned against the same database by
BLAST . Finally, the taxonomic classification were plotted using metagenome analyser
All 16S rRNA gene sequences were deposited in the European Nucleotide Archive
(ENAhttp://www.ebi.ac.uk/ena) under accession number PRJEB9174.
A full model two factor permutational analysis of variance was run using Primer v. 6 with
PERMANOVA add-on, to compare the bacterial communities between the hypobranchial gland
and foot tissue of male and female D. orbita. Bray Curtis similarity matrices with a dummy
value of 1 were generated from the untransformed OTU data at the genus level. Initial analyses
were performed using the number of reads as a covariate to establish whether the unequal
number of reads between samples influenced the outcomes. However, as the covariate was not
significant (Pseudo F = 9.83, p = 0.96), the covariate was removed and the results are presented
from the reduced two factor model. Additional analyses were also performed using a reduced
data set excluding the low read samples (i.e. F1H, F2F and M2H) and these produced
comparable results to the full data set (S1 Table). All PERMANOVA analyses were performed using
9999 permutations. Principal Coordinates Ordination (PCO) was undertaken to represent the
data graphically. Similarity of Percentages (SIMPER) was run to establish which bacterial taxa
contributed to the dissimilarity between the hypobranchial gland and foot tissue.
The DIVERSE function in PRIMER 6 was used to analyse the genus richness and diversity
(Shannon’s H index), which was calculated from the relative abundance (% of reads) for each
distinct OTU in each sample, but excluding the unassigned taxa. Univariate PERMANOVAs
of genus richness and diversity were performed using Euclidean distance similarity matrices.
A total of 149,804 reads, with an average length of 436.301 base pairs, were obtained from the
eight samples (four hypobranchial gland and four foot) of D. orbita (Table 1). Total acceptable
reads, for operational taxonomic unit (OTU) assignment for the eight samples, ranged from
637 to 36,728 in the hypobranchial gland and foot (Table 1). At least one replicate from each
sample type had > 15,000 reads. The total number of shared (non-overlapping) operational
taxonomic units (OTUs) resulting from the bacterial profiling data set was 3585. The foot
samples had a higher number of OTUs than the hypobranchial gland, across all taxonomic levels
Rarefaction curves indicated the richness of bacterial taxa had not peaked at the maximum
number of sequences read, with the exception of female hypobranchial gland 2, which reached
an asymptote of < 70 bacterial genera after ~ 10,000 sequences (Table 1). The number of
OTUs is likely to be highly under-represented in the other female hypobranchial gland sample
(F1H = 17) and male hypobranchial gland 2 (M2H = 71, Table 1), due to the low number of
sequence reads (Fig 1). The alpha diversity rarefaction plots also showed that the female
hypobranchial gland had lower bacterial diversity than the male hypobranchial gland and foot
samples of D. orbita (Fig 1).
Bacterial taxonomic diversity of the hypobranchial gland and foot of
Altogether, 28 different bacterial phyla were observed in the bacterial profiling data set;
however, only dominant phyla are presented (Fig 2). Bacterial groups that could not be assigned to
any phyla equated to 6.8%. The dominant phylum was Proteobacteria, representing 32.2% of
the bacterial abundance in all D. orbita samples (Fig 2). Bacteria from the phylum Tenericutes
were more abundant in the hypobranchial gland compared to the foot (Fig 2). Bacteriodetes
were more abundant in foot tissues than female hypobranchial glands (Fig 2). Bacteria from
the phylum Spirochaetes were also more abundant in the foot than the hypobranchial gland
samples (Fig 2).
1 The samples are labelled such that the first letter refers to the gender, the number to different replicate snails within each gender and the second letter to
the tissue type.
2 OTUs are shared among multiple samples and are based on 97% sequence similarity criteria in the Silva_119 database.
Phylogenetic analysis revealed male foot tissue (M3F) had greater taxonomic diversity than
the hypobranchial gland (Fig 3). Flavobacteriales, Sphingobacteriales and Rhodobacterales were
more common in the foot, while Vibrionales was more dominant in hypobranchial gland (Fig
3). Vibrionales was the dominant order in the female hypobranchial gland (Fig 3A) and
representatives from this order were observed in all samples of the foot and hypobranchial gland
(Fig 3). Mycoplasma, in the phyla Tenericutes, was found to be more dominant in the
hypobranchial gland when compared to D. orbita foot samples (Fig 3).
Altogether, 443 known bacterial genera were identified in the foot and hypobranchial gland
of the D. orbita bacterial profiling dataset, based on >97% sequence similarity. In total there
were 169 distinct bacterial genera present in the foot,52 in the hypobranchial gland and 222
common bacterial genera between the foot and hypobranchial gland of D. orbita. On average, a
higher number of distinct bacterial genera were recorded in the foot compared to
hypobranchial gland samples (Fig 4A). Univariate PERMANOVA analysis confirmed there was
significantly different genus richness between tissue types (Pseudo F = 8.54, p = 0.04). However,
genus richness was not significantly different between genders (Pseudo F = 6.33, p = 0.06), and
there was no interaction between gender and tissue type (Pseudo F = 2.49, p = 0.86).
Fig 1. Alpha diversity showing the richness of bacterial community diversity within Dicathais orbita foot (F2F, F3F, M2F and M3F) and
hypobranchial gland samples (F1H, F2H, M1H and M2H) (F = female; M = male). The phylogenetic diversity metric consists of genus richness based on
3585 observed OTUs at the 97% sequence similarity level and 443 possible observed genus. Sample with reads of more than 3000 are visible.
Using Shannon’s diversity index to assess richness and evenness of the bacterial
communities, higher diversity was consistently detected in the foot compared to the hypobranchial gland
of D. orbita (Fig 4B). Univariate PERMANOVA analysis revealed genus diversity (H0) was
significantly higher in the foot than the hypobranchial gland (Pseudo F = 18.44, p = 0.01). There
was no significant difference according to gender (Pseudo F = 3.71, p = 0.13), and no
interaction between gender and tissue (Pseudo F = 1.79, p = 0.25).
Bacterial community structure in the hypobranchial gland and foot of
Principal Coordinates Ordination (PCO) revealed separation of bacterial communities, based
on genera level OTUs, between the hypobranchial gland and foot samples (Fig 5). The bacterial
communities of hypobranchial gland samples were more variable and also showed separation
between male and females, whereas foot samples clustered together on the left hand side of the
plot (Fig 5). Multivariate analyses of genera OTUs associated with the hypobranchial gland
and foot of D. orbita, revealed a significant difference between these tissues (Pseudo F = 5.46,
p = 0.02). However, there was no significant difference according to gender (Pseudo F = 0.58,
p = 0.67) and no interaction between gender and tissue (Pseudo F = 2.01, p = 0.17). Similar
results were found when just the presence and absence of bacteria in the samples are
considered (rather than relative abundance). Here, the PCO plot also revealed a general pattern of
foot samples clustering separately and hypobranchial gland samples being more variable
between the individual snails than foot samples (S1 Fig).
Fig 2. Phylum-level taxonomic diversity associated with the female (F) and male (M) hypobranchial gland (F1H, F2H, M1H and M2H) and foot (F2F,
F3F, M2F and M3F) of Dicathais orbita bacterial profiling. All the minor phyla and unnamed, but previously identified bacterial phyla (such as BD1-5,
CKC4, candidate division BRC1, OD1, OP8, SR1, TM7, SHA-109, and TM6) are grouped into “Bacteria Other”.
Fig 3. Phylogenetic tree of Dicathais orbita samples generated from 16S rRNA sequences by MEGAN. 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.
SIMPER (Similarity of percentages) analysis revealed high dissimilarity between the
bacterial communities of the hypobranchial gland and foot of D. orbita (Table 2, Average
disdissimilarity between the tissues. Vibrio and Mycoplasma were more abundant in the
hypobranchial gland, whereas Chitinophagaceae and Spirochaeta were more abundant in the foot
Fig 4. Mean (+s.e.) number of OTUs in the hypobranchial gland and foot tissue of Dicathais orbita, showing the mean proportion unique to
individuals samples of foot and hypobranchial gland tissue. (A) = OTUs richness, (B) = H index/diversity.
Fig 5. 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) Dicathais orbita.
(Table 2). A relatively small number of genera (e.g. Mycoplasma, Vibrio), along with an average
of approximately 8% unassigned bacteria contributed to the similarity between hypobranchial
gland samples of D. orbita (S2 Table). However, 30 diverse bacteria contributed to 90% of the
similarity between the foot samples (S2 Table).
Biosynthetic capabilities of the bacterial symbionts
Of the possible 443 bacterial genera identified from the tissues of D. orbita, only 22 bacterial
species are known to have biosynthetic capabilities directly relevant to Tyrian purple precursor
biosynthesis (Fig 6, S3 Table). A greater proportion of the bacteria found only in the
hypobranchial glands (9.6%) were found to have indole and/ or brominating capabilities compared to
those only found in the foot (0.6%, Fig 6). There were 21 indole producing species detected
across 9 genera (Fig 6) and the majority of these were Vibrio spp. common to both the foot and
hypobranchial gland samples (Fig 6). Three species were detected that are known to produce
both indoles and brominated secondary metabolites and a further three species produce
bromoperoxidase enzymes (Fig 6, S3 Table). More specifically, bacteria from three genera that
were detected more frequently in the hypobranchial gland, namely Bacillus, Pseudomonas and
Synechococcus, are known to produce bromoperoxidase (S3 Table). Pseudomonas spp., and
several other bacteria found in the hypobranchial gland, are also known to produce oxidised
sulphur metabolites, whereas three sulphur reducing bacteria were found exclusively in the foot
tissue (S3 Table).
This study determined differences in bacterial community composition in the Tyrian purple
producing hypobranchial gland and the non-biosynthetic foot tissues of the muricid mollusc,
D. orbita. Bacterial taxa representing 3585 OTUs from 28 different phyla, 243 families and 443
Table 2. Similarity of percentages (SIMPER) analysis showing the bacterial genus that contribute most to the differences between hypobranchial
gland and foot of Dicathais orbita (Average dissimilarity = 68.51).
genera (Table 1) were observed in the bacterial profiling data set of D. orbita. Phylogenetic
analysis highlighted the presence of more complex bacterial communities in the foot compared
to the hypobranchial gland, and this was supported by significantly lower OTU richness and
diversity in the gland than the foot. PCO and multivariate analysis revealed significantly
different bacterial community structure between the two tissues, and dissimilarity analysis revealed a
higher abundance of Vibrio, Mycoplasma and unassigned bacteria in the biosynthetic
hypobranchial gland. Consistent with previous culture studies , Vibrios were the dominant
indole producing bacteria detected. However, 16S rDNA bacterial profiling also revealed the
presence of bromoperoxidase producing bacteria such as Bacillus, Pseudomonas and
Synechococcus, and bacteria known to produce brominated secondary metabolites, such as
Pseudoalteromonas and Propionigenium, in the Tyrian purple producing gland.
The taxonomic diversity of symbiotic bacteria, with a dominance of Proteobacteria in D.
orbita, is comparable with previous 16S rDNA analyses of marine mollusc associated bacteria.
Metagenomic (16S rDNA) bacterial diversity studies of the molluscan sea slug, Elysia
chlorotica, from a total number of reads among samples ranging from 4601 to 11374, produced 199
to 889 OTUs derived from 5 to 9 distinct phyla . Another metagenomic study of the 16S
rRNA gene of a bivalve mollusc (internal body parts without the shell) resulted in the discovery
of 3553 OTUs from 44 phyla, in which Proteobacteria was found to be the most abundant
phylum . The metagenome of the digestive tract of a marine limpet also revealed diverse
microbial communities with the most dominant phylum being Proteobacteria . Indeed,
Proteobacteria accounts for more than 40% of all known prokaryotic genera  and is the
Fig 6. Venn diagram showing shared and non-shared bacterial species between the hypobranchial gland and foot of Dicathais orbita. 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).
dominant bacterial phyla reported from other marine invertebrate taxa, including the sponge
Halichondria sp. , the coral Porites astreoides  and the oyster Crassostrea sp..
Significantly different bacterial community structure, with a higher richness and diversity of
OTUs, was observed in the foot relative to the hypobranchial gland samples of D. orbita (Figs
3–5). This is consistent with a previous study on heterotrophic culturable bacteria, where no
bacterial biochemical activity was detected in the homogenised glands and significantly fewer
species were isolated from swabs of the hypobranchial gland in comparison to the foot tissue of
D. orbita . The lower Shannon’s diversity index from the hypobranchial gland coupled
with the SIMPER analyses imply the bacterial community in this gland is dominated by two
abundant symbiotic genera (i.e. Mycoplasma, Vibrio), some unassigned, possibly novel bacteria
and a larger number of genera detected in low abundance, many of which may be rare
opportunists or contaminants. This pattern of bacterial diversity is consistent with a highly
specialised internal environment; indeed the hypobranchial gland has a low pH  and produces
secretions containing antibacterial sulphated mucopolysaccharides and brominated indoles
that would be expected to kill the majority of opportunistic bacteria [41, 58]. Mycoplasma, the
best known genus in the class Mollicutes, are common parasites in marine organisms and can
persist in extreme environments including low pH and low oxygen [59, 60]. They lack a cell
wall and are unaffected by many antibiotics . They are also common laboratory
contaminants , but may exist as parasites or commensals within the hypobranchial gland, which
provides a rich source of carbohydrates. To establish the potential for vertical or horizontal
transmission of the bacterial symbionts in D. orbita, future bacterial profiling studies could
include samples of the egg capsules, water and benthic substrate for comparison. Interestingly,
a preliminary study on the culturable heterotrophic bacteria from D. orbita tissues identified at
least one indole producing bacteria common to the hypobranchial gland and egg capsules .
The precursors of Tyrian purple are found in the egg capsules , as well as the female capsule
gland, which lies adjacent to the hypobranchical gland in D. orbita [1, 63] suggesting a
potential role of vertical transmission of biosynthetic symbiotic bacteria.
The dominance of Vibrionaceae in the hypobranchial glands of D. orbita implies these
bacteria are selectively retained or are able to multiply within this unusual mucus producing
organ. Vibrios are commonly associated with marine organisms [64–66] and species such as V.
parahaemolyticus, V. orientalis and V. campbellii are pathogens of marine invertebrates [67–
69] with V. orientalis, V. harveyi, V. coralliilyticus and V. splendidus being specific mollusc
pathogens [68, 70–72]. However, Vibrio species can also be endosymbionts such as those
found in the viscera of the muricid, Nucella lapillus, and mucus of the sea slug, Elysia rufescens
[73, 74], as well as V. fischeri, which is found in squid bioluminescent organs . Vibrio
species are known, not only for their symbiotic relationships with marine molluscs, but also for
the production of important secondary metabolites [75–77]. A previous study successfully
cultured three indole producing Vibrio species from the biosynthetic organs of D. orbita .
Many additional indole producing Vibrio species from the hypobranchial gland of D. orbita
were identified in this study (S3 Table). The relatively high concentration of these Vibrios in
the hypobranchial gland, and their capacity for indole synthesis, suggest they may contribute
to Tyrian purple precursor synthesis in the hypobranchial glands of Muricidae. Indole
producing Vibrio species, including V. orientalis and V. coralliilyticus were found exclusively in the
hypobranchial gland, but not in the foot. A range of other bacterial genera that produce indoles
were also detected in the hypobranchial gland. These include Bacillus,  Propionigenium,
 and Pseudomonas, which produce indoles such as indole-3-acetic acid . Hence, indole
precursors may be opportunistically acquired from more than one bacterial species for Tyrian
purple production in muricids.
It has been suggested bromoperoxidase plays a role in Tyrian purple biosynthesis through
the addition of bromine to the 6-position of tyrindoxyl sulphate [81, 82]. This is supported by
evidence of bromoperoxidase activity in Trunculariopsis (Murex) trunculus hypobranchial
gland homogenates  and histochemical sections from D. orbita hypobranchial and rectal
glands [18, 83]. Several bacterial genera were detected in our bacterial profiling studies that are
known to produce bromoperoxidase (S3 Table), including Pseudomonas  and bacteria of
the Bacillaceae family . The cyanobacterium, Synechococcus produce vanadium dependent
bromoperoxidase ; this enzyme is implicated in the biosynthesis of marine halogenated
natural products of pharmacological importance  and can also react with indole to produce
region specific brominated indole products [85, 86]. Bacillus, Pseudomonas and Synechococcus
were all present in the hypobranchial gland of D. orbita and these bromoperoxidase producing
bacterial genera are the priorities for future targeted culture work to further investigate their
role in Tyrian purple production in D. orbita. Future studies could also apply functional
metagenomics approaches to uncover the brominating enzymology associated with the
hypobranchial glands of Muricidae by screening specifically for bromoperoxidase and brominase genes
Several other marine bacteria found in the hypobranchial glands are known to produce
halogenases and could provide an alternative path for brominating indole precursors of Tyrian
purple. Halogenating enzymes such as brominases, responsible for the synthesis of
polybrominated metabolites, including phenol and imidazole structures, have been identified in marine
bacteria . Tribromoimidazole, a brominated secondary metabolite found within the eggs of
muricid molluscs, may be produced by brominase activity . Pseudomonas sp. also produce
halogenase enzymes  and we detected Pseudomonas spp. in the hypobranchical glands of
D. orbita. Other marine studies have isolated a tryptophan 6-halogenase with brominating
activity from Streptomyces sp.  and a novel halogenase gene from Psychrobacter sp.
(associated with the marine sponge Crambe crambe) , a genus that was also detected in our D.
orbita study. Other bacteria detected in the hypobranchial gland of D. orbita, including Vibrio,
and Pseudoalteromonas, have previously been found to produce brominated secondary
metabolites (S3 Table). For example, Vibrio sp. (strain KMM-81-1) associated with the marine
sponge (Dysidea sp.) produces brominated secondary metabolites , and several species of
Pseudomonas produce brominated nitrophenyl pyrrole compounds [90, 91], while
Pseudoalteromonas sp. can produce pentabromopseudilin and bromophene . However, these bacteria
were not specifically associated with the hypobranchial glands of D. orbita and thus appear less
likely candidates for providing tissue localised precursors to Tyrian purple.
It is possible bacteria may be responsible for several steps which occur early in the Tyrian
purple biosynthetic pathway. Enzymes such as sulphur transferase and sulphur reductase may
be involved in contributing the methane thiol group on the indole ring of tyrindoxyl sulfate.
High concentrations of mercaptan and dimethyl disulfide are present in muricid
hypobranchial glands that produce Tyrian purple [1, 4, 93]. Pseudomonas found in the hypobranchial
gland is known to utilize dimethyl disulfide . Several bacteria that metabolise sulphur, such
as V. orientalis and V. coralliilyticus are exclusively found in the hypobranchial gland and also
utilize dimethylsulfoniopropionate, an organosulfur compound that produces dimethyl
sulphide and methanethiol as a breakdown product [95, 96]. Sulfitobacter mediterraneus was
detected in the foot and hypobranchial gland and is a sulfite-oxidizing bacteria  that may
catalyse the production of indoxyl sulfate. Thus, it is possible the various sulphur metabolizing
bacteria found in the hypobranchial gland play important roles in Tyrian purple precursor
A difference in the 16s rRNA bacterial profiles of male and female hypobranchial glands
was expected on the basis that previous chemical studies have suggested a difference in the
oxidation and reduction state of indole dye precursors in male and female hypobranchial glands.
Specifically, the female glands were found to contain higher amounts of reduced methanethiol
derivatised indoles, such as tyrindoleninone and tyriverdin, whereas males contained more
oxidised end-products 6-bromoisatin and 6,6’dibromoindirubin . This could imply the
presence of sulfur-reducing bacteria in the female hypobranchial glands, although we actually
found more evidence for known sulfur-reducing bacteria in the foot tissue (S3 Table) and none
were unique to the female hypobranchial gland. Nevertheless, the reducing environment of the
female gland could explain why the bacterial community structure was noticeably more
distinct from the foot communities than the male glands (Fig 5) and the tendency towards lower
phylogenetic complexity in the female compared to male hypobranchial glands (Table 1, Fig
3). However, consistent with a previous culture based study , there were no significant
difference in the bacterial communities isolated from male and female samples and no interaction
between tissue and gender. In both studies the lack of a significant gender effect could be
influenced by a consistent bacterial community structure within the foot tissues and low power to
detect a gender difference, specifically in the hypobranchial glands, due to relatively high
variability and low replication of male and female samples within this tissue type (e.g. Fig 5 PCO).
Consequently, future functional metagenomics studies aimed specifically towards examining
the sulphur metabolising bacteria in male and female hypobranchicial glands of Muricidae are
Overall, a larger number of bacterial taxa were found in the foot compared with the
hypobranchial gland of D. orbita, however, a higher abundance of Vibrio and some unique
microbial symbionts were observed in the hypobranchial gland. Some of the bacteria identified in the
hypobranchial gland are known to produce indole and bromoperoxidase or other enzymes
which may contribute to Tyrian purple precursor synthesis. Future studies will aim to culture
these microbial symbionts associated with the hypobranchial gland and further analysis will be
undertaken to identify genes that may be associated with Tyrian purple precursor production.
S1 Fig. Principal Coordinates Ordination (PCO) of bacterial genus associated with
hypobranchial gland (purple) and foot (orange) of female (F) and male (M) Dicathais orbita
after presence/ absence transformation.
S1 Table. Summary of statistical analyses for genus level using a reduced data set (F2H,
M1H, F3F, M2F and M3F) excluding samples with low number of reads (F1H, M2H and
F2F). Univariate PERMANOVA was performed on Euclidean distance similarity matrices for
genus level OTU richness and diversity, whereas multivariate PERMANOVA was performed
using Bray-Curtis similarity matrices for community composition based on the number of
S2 Table. Similarity of percentages (SIMPER) analysis showing the bacterial genus that
contribute the most to the similarity in A) hypobranchial gland (Average similarity: 45.47)
and B) the foot of Dicathais orbita (Average similarity: 60.14).
S3 Table. Dicathais orbita associated bacteria that have been previously shown to produce
indoles, brominated secondary metabolites or enzymes associated with their biosynthesis
or sulphur metabolizing bacteria.
We appreciate feedback on the draft manuscript from Bijayalakshmi Devi Nongmaithem,
Roselyn Regino, Jeanette Travis (SCU) and two anonymous reviewers.
Conceived and designed the experiments: KB AB AKN DW SW. Performed the experiments:
AKN AB. Analyzed the data: AKN AB KB. Contributed reagents/materials/analysis tools: KB
DW AB. Wrote the paper: AKN KB DW AB SW.
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