Genetic diversity of bioluminescent bacteria in

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[except Shewanella hanedai and S. woodyi. (aerobic)] known to inhabit several niches of different environments. Most of these light producing bacteria are of ...
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 : [email protected]] 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 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 nonbioluminescent organisms, as free living, saprophytic, commensals, symbiotic or parasitic6,

and as milky seas7. They have also been isolated 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,

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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.

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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.

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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

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).

Fig. 2. Varied intensities of luminescence of different colonies isolated.

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

GPS readings

Isolation source

Isolated strain

Burmanallah

Lat 11⁰33’52.24’’ N Long 92⁰44’01.51’’ E

Echinodermata: Blue starfish Linckia laevigata Cnidaria: Sea Anemone Cryptodendrum sp.

Chidiyatapu

Junglighat Kodiyaghat

Marina Park Sippighat

Lat 11⁰29’27.24’’ N Long 92⁰42’29.38’’ E

Lat 11⁰39’25.09’’ N Long 92⁰43’30.07’’ E Lat 11⁰31’47.16’’ N Long 92⁰43’25.97’’ E Lat 11⁰40’19.76’’ N Long 92⁰45’00.84’’ E Lat 11⁰36’13.23’’ N Long 92⁰41’10.26’’ E

Accession numbers of BLAST search top hit NR117424

Sequence identity in BLAST

Identified as species

SSBR3

GenBank sequence accession numbers KR811225

99%

Vibrio owensii

BSE1

KT354591

NR119050

99%

V. campbellii

Cnidaria: Sea Anemone Cryptodendrum sp. Cnidaria: Sea Anemone Cryptodendrum sp.

BSE4 BSE5

KT354589 KR811222

NR118091 NR119050

99% 99%

V. rotiferianus V. campbellii

Polychaeta: Ragworm Neries sp. Rhodophyta: Coralline red algae Amphiroa anceps Porifera: Sponge Rhabdastrella globostellata

BNE1 AMPHI2 SSPI1

KR811226 KR811219 KR811230

NR119050 NR122059 KC291496

99% 99% 98%

V. campbellii V. alginolyticus V. harveyi

Tunicata: Sea squirt Clavinella moluccensis

SQSU1

KR811221

NR119050

98%

V. campbellii

Sea Water Sea Water

BSECU1 BSECU3

KT354590 KR811224

NR119050 NR119050

99% 99%

V. campbellii V. campbellii

Sediment Tracheophyta: Seagrass Halophila ovata

VASE8 CHSE2

KR811218 KR811223

NR122050 NR117424

97% 99%

V. alginolyticus V. owensii

Tracheophyta: Mangrove Rhizophora mucronata

CHSE4

KR811231

JF412244

99%

V. harveyi

Zooplankton sample Arthropoda:Arabian red shrimp Aristeus alcocki Chordata: Stonefish Synanceia verrucosa Chordata: Stonefish S. verrucosa

JPL2 PEVI1 STF1 STF2

KR811220 KT354592 KR811228 KR811227

KP150442 NR119050 NR119050 NR119050

97% 99% 99% 99%

V. rotiferianus V. campbellii V. campbellii V. campbellii

Chordata: Stonefish S. verrucosa Mollusca: Cuttlefish Sepia officinalis

STF3 SQEG2

KR811229 KR911956

JQ948038 NR029253

98% 99%

Chordata: Shortnose ponyfish Leiognathus brevirostris

LB2

KR811216

HQ599852

97%

V. harveyi Photobacterium leiognathi P. damselae

Chordata: Shortnose ponyfish L. brevirostris

APST1

KR811217

NR113783

98%

P. damselae

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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 agarose gel. Arrow indicates the expected amplification 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. 4. Restriction patterns of PCR products of 16s rRNA genes of 21 luminous strains.

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.

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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,

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

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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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