Indian Journal of Geo Marine Sciences

Vol. 46 (10), October 2017, pp. 2054-2062

Genetic diversity of bioluminescent bacteria in diverse marine niches

CH. Ramesh* & R. Mohanraju
1
Department of Ocean Studies and Marine Biology, Pondicherry Central University,
Port Blair-744102, Andaman & Nicobar Islands, India.
2
Andaman and Nicobar Centre for Ocean Science and Technology,
ESSO-NIOT, Dollygunj, Port Blair-744103, Andaman and Nicobar Islands, India.
[E.mail : chrameshpu@gmail.com]

Received 29 September 2016 ; revised 28 November 2016

The biodiversity of marine luminous bacteria has been well defined from their typical habitats viz. light organ containing squids
and fishes. However, distribution and diversity of luminous bacteria among various non-luminous marine niches (non-typical
habitats) are not fully investigated yet. This report describes the genetic diversity of luminous bacteria among nonluminous
marine samples of different marine taxa collected from Andaman Islands. Twenty-one luminous bacterial isolates were obtained
from these samples and characterized using Restriction Fragment Length Polymorphism (RFLP) patterns by HhaI digestion of
PCR-amplified 16S rRNA gene products and 16s rRNA gene sequence analysis. Restriction patterns clearly discriminated genus
Vibrio and Photobacterium; and sequence analysis revealed the prevalence of harveyi clade members: Vibrio campbellii (9), V.
harveyi (3), V. rotiferianus (2), V. alginolyticus (2), V. owensii (2), and then Photobacterium damselae (2) and P. leiognathi (1).

[Keywords: Luminous bacteria, genetic diversity, RFLP, 16s rDNA analysis, Andaman Islands.]

Introduction and as milky seas7. They have also been isolated

Bioluminescence is the phenomenon of emission
of light that are regulated by various chemical
reactions involved in diverse luminous organisms
that spans from bacteria to fish1. The biological
significance of bioluminescence is to render
interspecies signalling, alarming predators, luring
prey, and camouflaging in surrounding milieus2.
Among several luminous organisms, luminous
bacteria are dominant in terms of abundance,
while other luminous organisms are more
dominant in terms of biomass3. Luminous
bacteria are gram negative, facultative anaerobes,
[except Shewanella hanedai and S. woodyi
(aerobic)] known to inhabit several niches of
different environments. Most of these light
producing bacteria are of marine in origin, and
very few of them are terrestrial (Genus
Photorhabdus) and freshwater (some strains of V.
cholerae) environments4. However, these
luminous bacteria show a strong lux genes
sequence identity5.
Studies revealed the distribution of
luminous bacteria in marine environment as
associates with diverse bioluminescent and non-
bioluminescent organisms, as free living,
saprophytic, commensals, symbiotic or parasitic6,
from sediment, estuarine water8, sea water,
thermocline layer water9, planktonic10, skins and
guts of finfish and shellfish6, artificial fibrous
surfaces11, hatcheries12, surfaces of marine
hydrozoans, bryozoans and polychaete worm13,
human wounds, and several luminous squids and
fishes4. Recently uncultivable symbiont
bioluminescent bacteria associated with light
organ of the fish Anomalops katoptron of the
family Anomalopidae have been phylogenetically
characterised as a new genus of luminescent
bacteria4.
Remarkably luminous bacteria are found
to enter in aquaculture settings and cause larval
mortalities14. Phenomenons such as resistance to
antibiotics, expanding host adaptation15, quorum
sensing16, and lateral transfer of lux genes among
members of luminous and non-luminous bacteria
have aroused global attention4. It is inferred that
changes in global environmental conditions might
drive these phenomenons involved in expanding
bacterial host range, lux gene transfer, and
regulation of luminescence emission17.
Recent studies are mainly focused on
genetic characterization of luminous bacteria due
to their impact on aquaculture. Furthermore,
 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017
importance of luminous bacteria in numerous
applications such as biosensors to detect several
pollutants18, 19, and in detection of infectious
diseases using in vivo Bioluminescence Imaging
studies has been investigated20, 21. Considering
their significance, this study has been undertaken
to understand their distribution in different
marine niches of the Andaman Islands.
Materials and Methods
Diverse marine samples of different taxa were
collected by handpick method during receding
tide from different stations following standard
safety and aseptic techniques (Fig. 1 & Table 1).
Sediment sample was collected with a sterile
hand corer and transferred into a sterile plastic
tube22. Coastal surface seawater sample was
collected using a water sampler following the
procedure as detailed by Dutka (1989)23. Arabian
red shrimp Aristeus alcocki was collected from
Junglighat fish landing centre. Live specimens of
both cuttlefish Sepia officinalis and leiognathid
fish Leiognathus brevirostris were collected from
a fisherman. Zooplankton sample was collected
using plankton net method as detailed by
Agrawal and Gopal (2013)24. The bottom part of
stilt root of mangrove Rhizophora apiculata was
cut with a sterile knife and transferred to a sterile
sample container. Stone fish Synanceia verrucosa
was collected using a hand net with caution.
Fig. 1. Sampling locations in South Andaman.
2055
Swab sampling technique was employed
to obtain swab samples from surfaces of blue
starfish Linckia laevigata and sea anemone
Cryptodendrum sp. in the field after washing their
surfaces gently with sterile seawater25,26. These
were then transferred into sterile test tubes. Other
samples such as seaweed Amphiroa anceps,
seagrass Halophila ovata, sponge Rhabdastrella
globostellata, polychaete Neries sp., and sea
squirt Clavinella moluccensis were handpicked
using nitrile gloves and transferred to sterile
plastic ziplock bags. All samples were
transported to laboratory within an hour and
processed for isolation of luminous bacteria.
The collected marine flora and faunal
samples were gently rinsed with sterile seawater
to remove debris and bounded epibiotic
organisms. Using sterile cotton buds, swabs were
obtained from surfaces of these samples. For
sediment sample, sediment aliquot was prepared
by diluting 1 gm of sediment in 9 ml of sterile
seawater (w/v)27.
All luminous bacterial strains were
cultured on Luminescent agar (LA)28, with
addition of 4% agar as suggested by Dunlap and
Urbanczyk (2013) to prevent the growth of other
non-luminescent bacterial contaminants. To
freshly prepared LA plates 100µl of each
seawater (undiluted) and sediment aliquot
samples were spread evenly by using L- glass
spreader following the spread plate method27.
Swab samples were also spread evenly on to
surface of freshly prepared LA media plates.
These plates were incubated at 32⁰C for 24 hours
and luminous colony counts were made following
incubation.
After incubation, the petri plates were
examined in a dark room after straining the eyes
for about 10 to 15 minutes for luminous bacterial
colonies. High intense luminous colonies were
identified visually and picked up with sterile
tooth picks by adjusting red light29, and streaked
onto fresh plates. This was repeated twice or
thrice to obtain pure isolated single colonies.
Intense luminous pure isolates obtained
were assigned with strain codes and stored as
agar slants on LA and as agar stabs in Marine
agar maintained at 4⁰C; and 10% glycerol stocks
were maintained at -20⁰C. Further subcultures
were prepared from these stock cultures for
further studies.
Genomic DNA isolation from the
cultures grown in Marine broth at 35⁰C overnight
was performed by following CTAB method as
described by Nishiguchi et al. (2002)30.
2056 RAMESH & MOHANRAJU: GENETIC DIVERSITY OF BIOLUMINESCENT BACTERIA
Molecular analysis of 16s rRNA gene of
these 21 strains was carried out according to the
method as described by Mohandas et al. (2012)31.
Polymerase Chain Reaction (PCR) amplification
of 16S rRNA gene was carried out with bacterial
16S rDNA universal primer set, forward primer
27F [5’-AGA GTT TGA TCC TGG CTC AG-3’]
and reverse primer 1492R [5’-GGT TAC CTT
GTT ACG ACT T-3’]32. PCR of the genomic
DNA isolates were conducted in a final volume
of 25μl. The reaction mixture contained 10x PCR
buffer (Himedia, Mumbai), 10 mM DNTPs, 1 U
of Taq DNA polymerase, 10 µM of each forward
and reverse oligonucleotide primers and
approximately 20 ng of genomic DNA. PCR
amplification was performed using a GeneAmp
PCR system 2720-ThermoCycler, (Applied
Biosystem, Foster City, CA, USA).
The amplification profile consisted of an
initial denaturation at 94 ºC for 3 min, followed
by 35 cycles at 94 ºC for 1 min, 55 ºC for 1 min
and 72 ºC for 1 min. This was followed by a final
extension step of 72 ºC for 7 min. The samples
were held at 4 ºC until further analysis. Genus
level discrimination of these 21 strains was
preliminarily confirmed using Restriction
Fragment Length Polymorphism (RFLP) analysis
by HhaI Digestion of PCR-Amplified 16S rRNA
gene products according to the manufacture’s
protocol (Imperial life sciences, Haryana). PCR
products were visualised in 1.2% agarose gel run
in TAE (Tris-acetate-EDTA) buffer and the gel
was stained with Ethidium Bromide and
photographed with gel doc capture system. RFLP
products were resolved in 4% agarose gel33.
Prior to sequence, PCR products were
purified with GeNoRime PCR Purification kit
(Shrimpex, Chennai) and partially sequenced by
Fig. 2. Varied intensities of luminescence of different colonies isolated.
an automated Sequencer (Applied Biosystems,
Foster City, USA) determined by Sanger
dideoxynucleotide chain termination method.
Phylogenetic analysis was performed using the
neighbour-joining method34, and the resultant
neighbour-joining tree topology was evaluated by
bootstrap analyses based on 1000 resamplings35.
Evolutionary distance matrices were generated
according to Jukes and Cantor (1969)36.
Nucleotide sequence alignment and phylogenetic
tree construction were carried out using the
MEGA 6 software37. All the 21 sequences were
checked for chimera detections using Black Box
Chimera Check (B2C2) software38. Sequences
comparison with 16S rRNA gene sequence data’s
available in GenBank-NCBI was performed using
the Basic Local Alignment Search Tool
(BLAST)39. Subsequently sequences were
deposited in same data bank under assigned
accession numbers (Table 1).
Results
Results demonstrated the occurrence of luminous
bacteria in all samples tested (Table 1). High
intense luminous bacteria of 21 strains were
isolated and identified based on 16s rRNA gene
sequence analysis (Fig. 2). A total length of 1500
base pairs of 16s rRNA genes of 21 strains were
successfully amplified in PCR (Fig. 3). RFLP
analysis of PCR products of 16s rRNA gene
indicated an apparent discrepancy between the
two genera Photobacterium and Vibrio (Fig. 4).
Further, 16S rRNA gene sequence analysis
revealed grouping in two distinct clusters related
to Vibrio and Photobacterium. Sequences of
these 21 strains showed 97 to 99% similarities
with sequence data of Vibrio and Photobacterium
species available in GenBank (Table 1).
 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017 2057

Table 1. Details of isolation sources, sequence similarities and nucleotide sequence accession numbers of 21 luminous bacterial strains.

Samples GPS readings Isolation source Isolated GenBank Accession Sequence Identified as
collected strain sequence numbers of identity in species
stations accession BLAST search BLAST
numbers top hit
Burmanallah Lat 11⁰33’52.24’’ N Echinodermata: Blue starfish Linckia laevigata SSBR3 KR811225 NR117424 99% Vibrio owensii
Long 92⁰44’01.51’’ E Cnidaria: Sea Anemone Cryptodendrum sp. BSE1 KT354591 NR119050 99% V. campbellii
Cnidaria: Sea Anemone Cryptodendrum sp. BSE4 KT354589 NR118091 99% V. rotiferianus
Cnidaria: Sea Anemone Cryptodendrum sp. BSE5 KR811222 NR119050 99% V. campbellii
Polychaeta: Ragworm Neries sp. BNE1 KR811226 NR119050 99% V. campbellii
Rhodophyta: Coralline red algae Amphiroa anceps AMPHI2 KR811219 NR122059 99% V. alginolyticus
Chidiyatapu Lat 11⁰29’27.24’’ N Porifera: Sponge Rhabdastrella globostellata SSPI1 KR811230 KC291496 98% V. harveyi
Long 92⁰42’29.38’’ E
Tunicata: Sea squirt Clavinella moluccensis SQSU1 KR811221 NR119050 98% V. campbellii

Sea Water BSECU1 KT354590 NR119050 99% V. campbellii

Sea Water BSECU3 KR811224 NR119050 99% V. campbellii
Sediment VASE8 KR811218 NR122050 97% V. alginolyticus
Tracheophyta: Seagrass Halophila ovata CHSE2 KR811223 NR117424 99% V. owensii
Tracheophyta: Mangrove Rhizophora mucronata CHSE4 KR811231 JF412244 99% V. harveyi
Junglighat Lat 11⁰39’25.09’’ N Zooplankton sample JPL2 KR811220 KP150442 97% V. rotiferianus
Long 92⁰43’30.07’’ E Arthropoda:Arabian red shrimp Aristeus alcocki PEVI1 KT354592 NR119050 99% V. campbellii
Kodiyaghat Lat 11⁰31’47.16’’ N Chordata: Stonefish Synanceia verrucosa STF1 KR811228 NR119050 99% V. campbellii
Long 92⁰43’25.97’’ E Chordata: Stonefish S. verrucosa STF2 KR811227 NR119050 99% V. campbellii
Chordata: Stonefish S. verrucosa STF3 KR811229 JQ948038 98% V. harveyi
Marina Park Lat 11⁰40’19.76’’ N Mollusca: Cuttlefish Sepia officinalis SQEG2 KR911956 NR029253 99% Photobacterium
Long 92⁰45’00.84’’ E leiognathi
Sippighat Lat 11⁰36’13.23’’ N Chordata: Shortnose ponyfish Leiognathus brevirostris LB2 KR811216 HQ599852 97% P. damselae
Long 92⁰41’10.26’’ E Chordata: Shortnose ponyfish L. brevirostris APST1 KR811217 NR113783 98% P. damselae
2058 RAMESH & MOHANRAJU: GENETIC DIVERSITY OF BIOLUMINESCENT BACTERIA

Phylogenetic analysis of these strains based on
16s rRNA gene sequences was evaluated with top
three BLAST search hits obtained for each strain
and found unambiguous clustering in two distinct
groups as Photobacterium and Vibrio (Fig. 5).
Apparently strains LB2, APST1, and SQEG2
clustered with Photobacterium species, whereas
all other strains clustered in a single group that
were found to represent merely harveyi clade
members as updated by Sawabe et al. (2013)40.
16s rRNA gene sequence analysis of these strains
identified nine V. campbellii, three V. harveyi,
two V. rotiferianus, two V. alginolyticus, two V.
owensii, two Photobacterium damselae and one
P. leiognathi species (Fig. 6).

Fig. 3. Amplified products of 16s rRNA gene in 1.2% TAE Fig. 4. Restriction patterns of PCR products of 16s rRNA
agarose gel. Arrow indicates the expected amplification genes of 21 luminous strains.
products size approximately 1500 bp. Lanes: 1 to 21 refers to
respective strain code depicted in Table 1. Lane M, size
marker (1 kb ladder).

Fig. 5. Phylogenetic tree constructed based on the neighbour-joining tree resulting from analysis of the 16s rRNA genes of 21
luminous strains along with top three hits obtained for each strain from NCBI-BLAST analysis. Blue dots denote the strains
obtained in this study.
 INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017
Fig. 6. Luminous bacterial species identified from various marine niches studied in this study. Star indicates marine niches yet to
be studied.
Discussion
Distribution and biodiversity of luminous
bacteria in the marine environment are well
known as associates with several bioluminescent
marine organisms4. Earlier studies showed that
luminous bacteria were found to be attached to
surfaces of different marine organisms6.
However, studies on prevalence of these
bacteria on nonluminous marine samples
belonging to different taxa are not available. 16s
rRNA gene sequence analysis in the present
study confirmed the occurrence of luminous
bacteria in diverse samples including seawater,
sediment, zooplankton soup, Rhodophyta,
Tracheophyta, Porifera, Cnideria, Polychaeta,
Arthropoda, Mollusca, Echinodermata,
Tunicata, and Pisces. Samples belonging to
Reptilia, Aves and Mammalia are yet to be
investigated for understanding the occurrence of
luminous bacteria (Fig. 5). Presence of
secondary nucleotide peaks in some sequence
chromatograms suggest that the sequence
ambiguity may be due to a PCR or sequencing
artifact, or presence of multiple copies of the rrn
operon as inferred by Dunlap and Ast (2005)41.
In accordance to Thompson et al.
(2004), species identification are to be
considered when 16S rRNA sequence similarity
is 97% or above42. 16s rRNA gene analysis
demonstrated that diverse marine samples
examined in the present study are found to
harbour harveyi clade luminous bacterial
members. While, occurrence of P. damselae in
luminous fish L. brevirostris, and P. leiognathi
in a nonluminous cuttlefish S. officinalis
revealed the non-host-specific presence of
luminous bacteria. This observation is in accord
with the earlier study that identified more than
one luminous bacterial species in a single
squid43. Such incidence might depend on their
environmental distribution and physiology44,
2059
and possibly due to the reciprocal genetic
adaptation between host and bacteria45, or due to
similarities and dissimilarities of genome
contents that are found to play a crucial role in
niche adaptations46.
Globally the most widely encountered
luminous bacteria in marine environment are
Aliivibrio fischeri, Vibrio harveyi,
Photobacterium leiognathi and P. phosphoreum.
However bacterial genera Vibrio are widespread
than genera Aliivibrio and Photobacterium are
mostly attributed to symbiotic colonization with
various luminous organisms4. In the present
study predominantly luminous Vibrio species
have been encountered in all the samples,
whereas Photobacterium species were observed
merely in luminous ponyfish L. brevirostris and
in a nonluminous cuttlefish S. officinalis, and
genera Aliivibrio were not encountered.
Composition of luminous bacterial
species has been reported to be dependent on
seasonal variations, light, depth, ocean dilution,
pH, and nutrients8, 47. Study from South
California had delineated that P. fischeri was
found to occur throughout the year, whereas the
dominance of V. harveyi was only in summer,
and P. phosphoreum in winter48. In the
Mediterranean Sea V. harveyi was found to
occur throughout the year, and P. fischeri was
observed during winter49. In Gulf of Elat, P.
leiognathi was found throughout the year49, and
also in India, V. harveyi occurred throughout the
year50. Occurrence of these bacterial groups
may be driven by key factors like temperature,
salinity variations in water column49, 50, and
depth10.
Distributions of luminous bacterial
species are found to be area-specific. Their
colony numbers and species distribution
depends on distribution of bioluminescent
organisms such as squids which expel luminous
2060 RAMESH & MOHANRAJU: GENETIC DIVERSITY OF BIOLUMINESCENT BACTERIA
bacteria into the surrounding environment51.
Gentile et al. (2009) observed copious
distribution of P. phosphoreum and P. kishitanii
clades in seawater sample from Tyrrhenian
Sea52. Quantitative estimation of luminous
bacteria by De Luca (2006) showed the
dominance of P. phosphoreum (87%), and
sporadic occurrence of Vibrio and Shewanella
species in the Strait of Sicily and Ionian Sea53.
Recently dominance of S. hanedai was found in
samples of seawater, sediment, squid, and cuttle
fish collected from Karaikal coast, Bay of
Bengal54. Gallardo et al. (2004) reported the
occurrence of Vibrio (20%) and Photobacterium
(2%) groups in the marine algae. Significantly
in this study V. campbellii, V. harveyi, and V.
rotiferianus were found in most of the samples.
Results of this study demonstrate the presence
of presaging seven luminous bacterial species P.
damselae, P. leiognathi, V. alginolyticus, V.
campbellii, V. harveyi, V. owensii, and V.
rotiferianus in Andaman waters.
Acknowledgements
The corresponding author thanks the
Department of Science and Technology for
providing the INSPIRE fellowship
DST/IF120230.
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