Genetic Diversity of Bioluminescent Bacteria in Diverse Marine Niches

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

1Department of Ocean Studies and Marine Biology, Pondicherry Central University, Port Blair-744102, Andaman & Nicobar Islands, India. 2Andaman 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 and as milky seas7. They have also been isolated Bioluminescence is the phenomenon of emission from sediment, estuarine water8, sea water, of light that are regulated by various chemical thermocline layer water9, planktonic10, skins and reactions involved in diverse luminous organisms guts of finfish and shellfish6, artificial fibrous that spans from bacteria to fish1. The biological surfaces11, hatcheries12, surfaces of marine significance of bioluminescence is to render hydrozoans, bryozoans and polychaete worm13, interspecies signalling, alarming predators, luring human wounds, and several luminous squids and prey, and camouflaging in surrounding milieus2. fishes4. Recently uncultivable symbiont Among several luminous organisms, luminous bioluminescent bacteria associated with light bacteria are dominant in terms of abundance, organ of the fish Anomalops katoptron of the while other luminous organisms are more family Anomalopidae have been phylogenetically dominant in terms of biomass3. Luminous characterised as a new genus of luminescent bacteria are gram negative, facultative anaerobes, bacteria4. [except Shewanella hanedai and S. woodyi Remarkably luminous bacteria are found (aerobic)] known to inhabit several niches of to enter in aquaculture settings and cause larval different environments. Most of these light mortalities14. Phenomenons such as resistance to producing bacteria are of marine in origin, and antibiotics, expanding host adaptation15, quorum very few of them are terrestrial (Genus sensing16, and lateral transfer of lux genes among Photorhabdus) and freshwater (some strains of V. members of luminous and non-luminous bacteria cholerae) environments4. However, these have aroused global attention4. It is inferred that luminous bacteria show a strong lux genes changes in global environmental conditions might sequence identity5. drive these phenomenons involved in expanding Studies revealed the distribution of bacterial host range, lux gene transfer, and luminous bacteria in marine environment as regulation of luminescence emission17. associates with diverse bioluminescent and non- Recent studies are mainly focused on bioluminescent organisms, as free living, genetic characterization of luminous bacteria due saprophytic, commensals, symbiotic or parasitic6, to their impact on aquaculture. Furthermore, INDIAN J. MAR. SCI., VOL. 46, NO. 10, OCTOBER 2017 2055 importance of luminous bacteria in numerous Swab sampling technique was employed applications such as biosensors to detect several to obtain swab samples from surfaces of blue pollutants18, 19, and in detection of infectious starfish Linckia laevigata and sea anemone diseases using in vivo Bioluminescence Imaging Cryptodendrum sp. in the field after washing their studies has been investigated20, 21. Considering surfaces gently with sterile seawater25,26. These their significance, this study has been undertaken were then transferred into sterile test tubes. Other to understand their distribution in different samples such as seaweed Amphiroa anceps, marine niches of the Andaman Islands. seagrass Halophila ovata, sponge Rhabdastrella globostellata, polychaete Neries sp., and sea Materials and Methods squirt Clavinella moluccensis were handpicked Diverse marine samples of different taxa were using nitrile gloves and transferred to sterile collected by handpick method during receding plastic ziplock bags. All samples were tide from different stations following standard transported to laboratory within an hour and safety and aseptic techniques (Fig. 1 & Table 1). processed for isolation of luminous bacteria. Sediment sample was collected with a sterile The collected marine flora and faunal hand corer and transferred into a sterile plastic samples were gently rinsed with sterile seawater 22 tube . Coastal surface seawater sample was to remove debris and bounded epibiotic collected using a water sampler following the organisms. Using sterile cotton buds, swabs were 23 procedure as detailed by Dutka (1989) . Arabian obtained from surfaces of these samples. For red shrimp Aristeus alcocki was collected from sediment sample, sediment aliquot was prepared Junglighat fish landing centre. Live specimens of by diluting 1 gm of sediment in 9 ml of sterile both cuttlefish Sepia officinalis and leiognathid seawater (w/v)27. fish Leiognathus brevirostris were collected from All luminous bacterial strains were a fisherman. Zooplankton sample was collected cultured on Luminescent agar (LA)28, with using plankton net method as detailed by addition of 4% agar as suggested by Dunlap and 24 Agrawal and Gopal (2013) . The bottom part of Urbanczyk (2013) to prevent the growth of other stilt root of mangrove Rhizophora apiculata was non-luminescent bacterial contaminants. To cut with a sterile knife and transferred to a sterile freshly prepared LA plates 100µl of each sample container. Stone fish Synanceia verrucosa seawater (undiluted) and sediment aliquot was collected using a hand net with caution. 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 Fig. 1. Sampling locations in South Andaman. described by Nishiguchi et al. (2002)30. 2056 RAMESH & MOHANRAJU: GENETIC DIVERSITY OF BIOLUMINESCENT BACTERIA

Molecular analysis of 16s rRNA gene of an automated Sequencer (Applied Biosystems, these 21 strains was carried out according to the Foster City, USA) determined by Sanger method as described by Mohandas et al. (2012)31. dideoxynucleotide chain termination method. Polymerase Chain Reaction (PCR) amplification Phylogenetic analysis was performed using the of 16S rRNA gene was carried out with bacterial neighbour-joining method34, and the resultant 16S rDNA universal primer set, forward primer neighbour-joining tree topology was evaluated by 27F [5’-AGA GTT TGA TCC TGG CTC AG-3’] bootstrap analyses based on 1000 resamplings35. and reverse primer 1492R [5’-GGT TAC CTT Evolutionary distance matrices were generated GTT ACG ACT T-3’]32. PCR of the genomic according to Jukes and Cantor (1969)36. DNA isolates were conducted in a final volume Nucleotide sequence alignment and phylogenetic of 25μl. The reaction mixture contained 10x PCR tree construction were carried out using the buffer (Himedia, Mumbai), 10 mM DNTPs, 1 U MEGA 6 software37. All the 21 sequences were of Taq DNA polymerase, 10 µM of each forward checked for chimera detections using Black Box and reverse oligonucleotide primers and Chimera Check (B2C2) software38. Sequences approximately 20 ng of genomic DNA. PCR comparison with 16S rRNA gene sequence data’s amplification was performed using a GeneAmp available in GenBank-NCBI was performed using PCR system 2720-ThermoCycler, (Applied the Basic Local Alignment Search Tool Biosystem, Foster City, CA, USA). (BLAST)39. Subsequently sequences were The amplification profile consisted of an deposited in same data bank under assigned initial denaturation at 94 ºC for 3 min, followed accession numbers (Table 1). 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 Results extension step of 72 ºC for 7 min. The samples Results demonstrated the occurrence of luminous were held at 4 ºC until further analysis. Genus bacteria in all samples tested (Table 1). High level discrimination of these 21 strains was intense luminous bacteria of 21 strains were preliminarily confirmed using Restriction isolated and identified based on 16s rRNA gene Fragment Length Polymorphism (RFLP) analysis sequence analysis (Fig. 2). A total length of 1500 by HhaI Digestion of PCR-Amplified 16S rRNA base pairs of 16s rRNA genes of 21 strains were gene products according to the manufacture’s successfully amplified in PCR (Fig. 3). RFLP protocol (Imperial life sciences, Haryana). PCR analysis of PCR products of 16s rRNA gene products were visualised in 1.2% agarose gel run indicated an apparent discrepancy between the in TAE (Tris-acetate-EDTA) buffer and the gel two genera Photobacterium and Vibrio (Fig. 4). was stained with Ethidium Bromide and Further, 16S rRNA gene sequence analysis photographed with gel doc capture system. RFLP revealed grouping in two distinct clusters related products were resolved in 4% agarose gel33. to Vibrio and Photobacterium. Sequences of Prior to sequence, PCR products were these 21 strains showed 97 to 99% similarities purified with GeNoRime PCR Purification kit with sequence data of Vibrio and Photobacterium (Shrimpex, Chennai) and partially sequenced by 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 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 were found to represent merely harveyi clade 16s rRNA gene sequences was evaluated with top members as updated by Sawabe et al. (2013)40. three BLAST search hits obtained for each strain 16s rRNA gene sequence analysis of these strains and found unambiguous clustering in two distinct identified nine V. campbellii, three V. harveyi, groups as Photobacterium and Vibrio (Fig. 5). two V. rotiferianus, two V. alginolyticus, two V. Apparently strains LB2, APST1, and SQEG2 owensii, two Photobacterium damselae and one clustered with Photobacterium species, whereas P. leiognathi species (Fig. 6). all other strains clustered in a single group that

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.

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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 and possibly due to the reciprocal genetic Distribution and biodiversity of luminous adaptation between host and bacteria45, or due to bacteria in the marine environment are well similarities and dissimilarities of genome known as associates with several bioluminescent contents that are found to play a crucial role in marine organisms4. Earlier studies showed that niche adaptations46. luminous bacteria were found to be attached to Globally the most widely encountered surfaces of different marine organisms6. luminous bacteria in marine environment are However, studies on prevalence of these Aliivibrio fischeri, Vibrio harveyi, bacteria on nonluminous marine samples Photobacterium leiognathi and P. phosphoreum. belonging to different taxa are not available. 16s However bacterial genera Vibrio are widespread rRNA gene sequence analysis in the present than genera Aliivibrio and Photobacterium are study confirmed the occurrence of luminous mostly attributed to symbiotic colonization with bacteria in diverse samples including seawater, various luminous organisms4. In the present sediment, zooplankton soup, Rhodophyta, study predominantly luminous Vibrio species Tracheophyta, Porifera, Cnideria, Polychaeta, have been encountered in all the samples, Arthropoda, Mollusca, Echinodermata, whereas Photobacterium species were observed Tunicata, and Pisces. Samples belonging to merely in luminous ponyfish L. brevirostris and Reptilia, Aves and Mammalia are yet to be in a nonluminous cuttlefish S. officinalis, and investigated for understanding the occurrence of genera Aliivibrio were not encountered. luminous bacteria (Fig. 5). Presence of Composition of luminous bacterial secondary nucleotide peaks in some sequence species has been reported to be dependent on chromatograms suggest that the sequence seasonal variations, light, depth, ocean dilution, ambiguity may be due to a PCR or sequencing pH, and nutrients8, 47. Study from South artifact, or presence of multiple copies of the rrn California had delineated that P. fischeri was operon as inferred by Dunlap and Ast (2005)41. found to occur throughout the year, whereas the In accordance to Thompson et al. dominance of V. harveyi was only in summer, (2004), species identification are to be and P. phosphoreum in winter48. In the considered when 16S rRNA sequence similarity Mediterranean Sea V. harveyi was found to is 97% or above42. 16s rRNA gene analysis occur throughout the year, and P. fischeri was demonstrated that diverse marine samples observed during winter49. In Gulf of Elat, P. examined in the present study are found to leiognathi was found throughout the year49, and harbour harveyi clade luminous bacterial also in India, V. harveyi occurred throughout the members. While, occurrence of P. damselae in year50. Occurrence of these bacterial groups luminous fish L. brevirostris, and P. leiognathi may be driven by key factors like temperature, in a nonluminous cuttlefish S. officinalis salinity variations in water column49, 50, and revealed the non-host-specific presence of depth10. luminous bacteria. This observation is in accord Distributions of luminous bacterial with the earlier study that identified more than species are found to be area-specific. Their one luminous bacterial species in a single colony numbers and species distribution squid43. Such incidence might depend on their depends on distribution of bioluminescent environmental distribution and physiology44, organisms such as squids which expel luminous

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