African Journal of Microbiology Research Vol. 6(43), pp. 7111-7122, 13 November, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.1775 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Characterization and genotyping by pulsed-field gel electrophoresis (PFGE) of the first bioluminescent gigantis strains

Esra Ersoy Omeroglu* and Ismail Karaboz

Department of Biology, Basic and Industrial Microbiology Section, Faculty of Science, Ege University, 35100, Bornova-Izmir, Turkey.

Accepted 23 October, 2012

The genus Vibrio, which included 20 species in 1981, increased up to 63 in 2004 and in the last 10 years, 15 new species including Vibrio gigantis have been added. In this study, 20 bioluminescent were isolated and identified. The identification of the species whose various physiological and biochemical characteristics were identified was based on 16S rRNA gene region and it was determined that it belongs to the V. gigantis species. It was concluded that from the range of temperature and sodium chloride (NaCl) concentration needed, the phenotypic characteristics of the species of this genus used to distinguish them might vary. It was determined that their reproductive ability varied significantly especially in the media containing 0.5 and 9% of NaCl and at incubation temperatures of 4 and 30°C. These differences appeared in various enzymatic activities too. As a result of pulsed-field gel electrophoresis (PFGE) analysis, it was concluded that the of the V. gigantis strains were not completely identical, and had homology ranging from 70 to 94% of restriction fragment patterns homology. Therefore, this study is the first one indicating that V. gigantis strains have a bioluminescence feature and high rate of genomic polymorphism.

Key words: Vibrio gigantis, , 16S rRNA, pulsed-field gel electrophoresis (PFGE).

INTRODUCTION

Bioluminescence, known as the production and emission wide variety of ecological niche (fish light organs, of light by a living organism as a result of a chemical mammalian gut, nematode gut) and habitats (marine, reaction, is observed in many organisms, including fish, freshwater, terrestrial, and symbiotic relationship with the insects, medusae, dinoflagellates, fungi, bacteria and host) (Meighen, 1994). (Meighen, 1991; Peat and Adams, 2008). While When the marine sources of bioluminescent bacteria higher organisms such as insects and medusae can only are considered, sea water and sediments take the first produce intermittent flashes of light, the light steadily place (Ramesh et al., 1990; Reichelt and Baumann, produced by bioluminescent bacteria shows the 1973). Of course, bioluminescent bacteria are found not uniqueness of the light emitted by bacteria (Haygood, only in shallow coastal waters but also in the deep 1993). Bioluminescent bacteria mostly occur in marine pelagic zones of seas (Kita-Tsukamoto et al., 2006). environments but they are rare in freshwater, slightly They live not only in freely but also salty water and soil environments (Nealson, 1987). together with a variety of living and nonliving marine Bioluminescent bacteria are abundantly distributed in a organisms, and they colonize marine animals as saprophytes, commensals and parasites (Dunlap and Kita-Tsukamoto, 2006). When they freely live in seawater, their number is very low (0.01 to 40 cells/ml). *Corresponding author. E-mail: [email protected]. Tel: When they colonize the specialized organs of some 6 9 +902323112811. Fax: +902323881036. creatures, they reach very high numbers (10 -10 CFU/g) 7112 Afr. J. Microbiol. Res.

(Kita-Tsukamoto et al., 2006). Especially, certain disposable zip lock bags. Seawater complete (SWC) agar medium bioluminescent species have a symbiotic relationship was used for isolation, purification and storage of the isolates unique to the species, with various marine fish and (Suwanto and Suwanto, 2000). While the sediment samples were analyzed via the spread-plate method, the fish and samples squids, and inhabit their highly specialized light organs were dissected by aseptic techniques and swab samples obtained (Nealson and Hastings, 1992). Due to this bacterial from different places (intestinal content, gill, surface and intestinal bioluminescence, many marine fish species gain area of fishes and ink sac and mantle cavity of squid) of marine bioluminescent features (Peat and Adams, 2008). organisms were spread onto SWC agar. After incubation at 20C Until recently, three marine bioluminescent bacterial for 24 h, bioluminescent colonies were selected in a dark room and genera were known: Vibrio, , and sub-cultured on agar plates to obtain pure isolates (Ersoy Omeroglu et al., 2009). Shewanella. However, as a result of the study conducted by Urbanczyk et al. in 2007, especially the members of Vibrio fischeri were phylogenetically and phenotypically Phylogenetic analysis based on 16S rRNA gene sequence separated from the other genera of the Bioluminescent bacterial genomic for polymerase chain family and this group was classified into a new genus reaction (PCR) assays were isolated with the ZR Fungal/Bacterial called . In addition to the new regulations in DNA Kit (Zymo Research) according to the instructions of the systematics, new bioluminescent Vibrio species have manufacturer. To amplify 16S rRNA genes about 1500 bp, forward taken their place in since 2009. In studies primer (5-AGAGTTTGATCCTGGCTCAG-3) corresponding the 8- conducted by Yoshizawa et al. (2009, 2010), 2 new 27 position of E. coli sequence and reverse primer (5- species (Vibrio azureus, and Vibrio sagamiensis) were CTACGGCTACCTTGTTACGA-3), 1476-1495 were used. PCR added to the genus Vibrio which had 7 bioluminescent reactions were carried out in 50 µL mixtures containing 10X PCR buffer (Fermentas, 10X PCR, EP0402) , 1.5 mM MgCl2 (Fermentas species (, , Vibrio 3 mM, EP0402), 2 mM dNTP (Fermentas), 10 pmol (each) primer, mediterranee, Vibrio orientalis, Vibrio splendidus (biotype 1.5 U Taq polymerase (Fermentas 500U, EP0402) and 5 µL I), Vibrio vulnificus and Vibrio salmonicida) (Kita- genomic DNA template. The PCR conditions were as follows: initial Tsukamoto et al., 2006). In addition to new denaturation for 5 min at 94C, 40 cycles of denaturation for 1 min bioluminescent species, the Vibrio chagasii species that at 94C, annealing for 1 min at 56C, elongation for 1 min at 72C was initially considered not to have a bioluminescent and final elongation for 10 min at 72C (Mora et al., 1998). Then, feature is now included in bioluminescent species since it the bidirectional sequence analysis of purified amplicons (with Sephadex® G-50 kit) were conducted with ABI 3130xl genetic was found to have bioluminescent properties by studies analyzer (Applied Biosystems) using the same primers. performed later (Urbanczyk et al., 2008). After DNA sequencing analyses, identification of the As a result of the study by Roux et al. (2005), Vibrio bioluminescent strains was performed by comparing other rRNA gigantis was first isolated from the cultured oyster and in gene sequences in the public database. Partial 16S rRNA subsequent studies it was determined that V. gigantis sequences acquired were entered into GenBank database and was a new Vibrio species (Roux et al., 2005). In a study accession numbers for strains were obtained. To determine phylogenetic relationships on the basis of partial 16S rRNA carried out by Beleneva and Kukhlevskii in 2010, this sequences between bioluminescent strains isolated and identified species was isolated and identified, but so far it has not in this study, their phylogenetic dendrogram was constituted using been clarified whether this species has a bioluminescent Mega software version 4.0. feature. In this present study, the experiments and the results of these experiments on the isolation and Phenotypic characterization of bioluminescent strains identification of bioluminescent V. gigantis strains from a variety of sea water, sediment and marine life specimens With a view to determining physiological and biochemical properties collected from the Gulf of Izmir, determination of their of bioluminescent strains, the following features were examined: various biochemical and phenotypic characteristics, and Gram reaction, motility, poly-β-hydroxybutyrate (PHB) production, determination of their genetic relationships through growth abilities on thiosulfate citrate bile salts sucrose (TCBS) agar (Merck VM916963) (Liu et al., 2004), oxidation/fermentation of pulsed-filed gel electrophoresis technique are reported. glucose (Hamill et al., 2008), indole production, catalase (Taylor When the current information in the literature was and Achanzar, 1972) and oxidase activities, gas production from reviewed, it is seen that no V. gigantis species have been glucose, saccharose and lactose, gelatin liquefaction (Hendrie et identified as having the bioluminescent property; al., 1970), arginine dihydrolase, phenylalanine deaminase (Williams therefore, the data obtained in this study are of et al., 1971), sodium (0, 0.5, 1, 3, 5, 7, 9, 10 and 11% (w/v) NaCl) importance since they are the first documented ones. (Garnier et al., 2008) and temperature (4, 20, 27, 30, 40 and 50C) requirements (Garnier et al., 2008). At the same time, the hydrolyzing ability of starch (Mustafa and Kaur, 2009), skim milk (Chelossi et al., 2004), milk (Mustafa and Kaur, 2010), casein, MATERIALS AND METHODS tributyrine C4 (Mohankumar and Ranjitha, 2010), Tween-20 (Mustafa and Kaur, 2009) and Tween-80 (Soto-Rodriguez et al., Isolation of bioluminescent bacterial strains 2003) were also investigated. Except for salt requirement assessments, all the experiments To isolate bioluminescent bacteria, 3 sediment, 7 seawater and 10 were performed on appropriate medium supplemented with 2% fish samples were collected from different regions at discrete (w/v) NaCl. Haemolytic activities of bioluminescent strains were depths in the Gulf of Izmir (Figure 1). Sediment samples were screened on blood agar plates (Salubris) containing 5% (v/v) sheep collected with sterile 50 ml tubes and fish samples with sterile blood (Soto-Rodriguez et al., 2003). Salt and temperature Omeroglu and Karaboz 7113

Figure 1. The sampling points of seawater, sediment, squid and fish samples in the Gulf of Izmir.

requirements of bacterial growth were determined in 96-well negative control (Sneath et al., 1986). microplates and a growth indicator (triphenyltetrazolium chloride, TTC, Sigma T8877) was used for the detection of salt concentrations and temperature in which microbial growth occurred. Emission spectra measurements For these assays, Nutrient Broth (Merck 1.05443) was used and supplemented with appropriate concentrations of NaCl. First, 180 Bacterial inoculum with turbidity equivalent to McFarland 1.0 of the µL of the medium was added to each well and then 20 µL of bioluminescent strains was prepared and 200 µL of these bacterial bacterial suspension with a turbidity equivalent to 1.0 McFarland suspensions were transferred into 3 microplate wells with opaque (Musa et al., 2008) was transferred into each well. Afterward, the speciality for each strain. The measurement was performed using microplates were incubated at 20C for 24 h and after incubation, Varioskan® Flash (Thermo Scientific) and the wavelength of cold 20 µL of sterile 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC; 0.1% light emitted by bioluminescent strains was determined. w/v) was added to each well (Ersoy Omeroglu et al., 2009). Following the incubation period, wells considered positive in terms of metabolic activity were evaluated. In this experiment, TTC Pulsed-field gel electrophoresis (PFGE) tetrazolium salt was used as an artificial electron acceptor in order to determine dehydrogenase activity in other words metabolically Bacterial suspensions with turbidity equivalent to McFarland 1.0 active bacteria, and a red insoluble formazan formed as the were prepared and pellets belonging to bioluminescent strains were reduction product of water-soluble colorless TTC (Griebe et al., obtained by centrifuging at 13.000 × g for 2 min at 4C. After 1997). discarding supernatants, the pellets were suspended in 200 µL cold Assimilation of 14 carbon sources (glucose, lactose, maltose, HST buffer (10 mM Tris-Cl, 50 mM EDTA and 20 mM NaCl). sucrose, mannitol, mannose, galactose, sodium citrate, sodium Following this step, agarose plugs were prepared by mixing an pyruvate, sodium lactate, sodium propionate, gluconic acid, acetate equal volume of bacterial suspensions with 2% low-melting agarose and cellobiose) and 5 nitrogen sources (glycine, arginine, proline, (0.2 g low-melting agarose in 9.5 ml HST buffer supplemented with serine and alanine) were detected on basal medium agar (BMA). 0.5ml 20% of SDS). Cells in the agarose plugs were lysed with lysis Carbon and nitrogen sources were added to BMA with final solution I containing 50 mM Tris-HCl, 50 mM concentration of 0.2 and 0.1% (w/v), respectively. Bacterial ethylenediaminetetraacetic acid (EDTA), 2.5 mg/ml lysosime and suspensions (10 µL) with a turbidity equivalent to McFarland 0.5 1.5 mg/ml proteinase K and lysis solution II containing 0.5 M EDTA, were spotted on agar media with carbon or nitrogen sources and 1% sarcosyl and 400 µg/ml proteinase K. also on the media including no carbon or nitrogen sources for The cells embedded into the plugs were transferred into 1.5 ml 7114 Afr. J. Microbiol. Res.

Table 1. Acquired and selected bioluminescent isolates and dates, sources, depths and coordinates of sampling places.

Isolation date Isolate Source Depth (m) Coordinate 30.03.2007 SW15 Seawater 0 - 15 382903N/ 264705E 30.03.2007 SWLiman Seawater 0 - 15 382722N/ 270965E 22.01.2008 S2W9 Seawater 0 - 15 383499N/ 263900E 22.01.2008 S2W42 Seawater 0 - 15 382458N/ 265688E 17.04.2008 S3W2 Seawater 0 - 15 384090N/ 263490E 17.04.2008 S3W28 Seawater 0 - 15 382350N/ 265500E 17.04.2008 S3W46 Seawater 0 - 15 382670N/ 270610E 30.03.2007 SeLu25 Sediment 0 - 15 382350N/ 263900E 22.01.2008 Se2Lu48 Sediment 0 - 15 382475N/ 265890E 17.04.2008 Se3Lu25 Sediment 0 - 15 382350N/ 263900E

30.03.2007 FU-9 Diplodus annularis (branchia) 67 between 383700 N-264220E and 30.03.2007 FU-10 Mullus barbatus (internal area) 67 383745 N-264330 E

17.04.2008 E-10 Citharus linguatula (branchia) 42 - 44 17.04.2008 E-11 Arnoglossus laterna (internal area) 42 - 44 17.04.2008 E-14 Lepidotrigla cavillone (branchia) 42 - 44 17.04.2008 E-15 Diplodus annularis (surface) 42 - 44 between 383440 N-264610E and 17.04.2008 E-16 Boops boops (surface) 42 - 44 383345 N-264655 E 06.08.2008 H-2 Diplodus annularis (intestinal content) 42 - 44 06.08.2008 H-3 Merluccius merluccius (surface) 42 - 44 06.08.2008 H-16 Boops boops (branchia) 42 - 44

The numbers at the end of the sediment and seawater isolates are presenting the station no. The codes of E-H and FU isolates were isolated from between Uzunada-Gediz and Foca-Uzunada, respectively.

tubes in which 600 µL lysis solution I was added and incubated at RESULTS 37C for 1 h. After removing lysis solution I, lysis solution II was added to the 1.5 ml tubes and incubated at 55C for 2 h. First, the Bioluminescent isolates obtained in this study plugs were washed in ultra-pure water at 50C three times for 15 min each. As the last step of washing-out, the plugs were treated As shown in Figure 1, the samples were collected from with TE buffer (10 mM Tris-HCl, 0.1 mM EDTA) at the same different sources at different depths and 20 conditions and times. The agarose plugs containing DNA were cut by the ratio of ¼ and the plug section cut was placed into 100 µL bioluminescent isolates were isolated from these fresh enzyme buffer and was kept there for 10 min at 30C. For samples. Of the 20 isolates, 7 were obtained from sea treatment with restriction enzyme, 100 µL of buffer solutions were water, 3 from sediment samples and the remaining 10 prepared with 20 units of both SmaI (Fermentas 5X1200U) and the from different parts of different fish samples (Table 1). genomic DNA embedded in agarose plugs was digested with SmaI at 30C overnight (Durmaz et al., 2007). High molecular-weight restriction fragments were monitored by Identification based on 16S rRNA gene sequence electrophoresis using 1% (w/v) pulsed-field grade agarose (Bio- Rad) in the CHEF-Mapper PFGE system (Bio-Rad). The separation As a result of the 16S rRNA gene amplification, of restriction fragments was carried out in the following conditions: 6 2 amplicons with the length of approximately 1500 bp were V/cm via 120 angle of impact for 24 h at 14C with 5 to 25 s pulse obtained. As a result of the comparison of 16S rRNA times. A lambda ladder PFGE marker was used as the molecular size marker. After electrophoresis, to make restriction fragments gene sequences obtained from 20 bioluminescent strains apparent, the gel was stained with ethidium bromide (5 µg/ml) and with other gene sequences present in GenBank then destained with sterile deionized water (Haddock et al., 2010). database, it was determined that all the 20 strains and After the gel was photographed with PFGE displaying system of the LMG 227141 strain of V. gigantis shared 16S rRNA Versa DOC 4000MP (Bio-Rad), band profiles of bioluminescent gene homology by 98 - 99%. Partial 16S rRNA strains for both NotI and SmaI restriction enzymes were analyzed sequences obtained from the bioluminescent V. gigantis with the Bio-Profil-1D++ software (Vilber Lourmat) at 10% homology coefficient. Dendrograms belonging to PFGE band profiles were strains have been published in GenBank database with generated based on ‘unweighted pair group method with arithmetic the accession numbers given in the parentheses (SW15: mean (UPGMA)’ (Vilber Lourmat). JF412215, SWLiman: JF412216, S2W9: JF412219, Omeroglu and Karaboz 7115

S2W42 H-2

SWLiman SeLu25 Se3Lu25 53 E-15 E-11 H-3 51 S2W9 41 62 H-16 S346 SW15 E-16

94 S3W28

Vibrio gigantis LMG227141 Se2Lu48 FU-10 100 S3W2 E-14 62 61 E-10

90 FU-9 Vibrio orientalis ATCC 33934T 61 Vibrio harveyi ATCC 35084 69 Aliivibrio fischeri ATCC 7744T Aliivibrio logei ATCC 29985T Photobacterium kishitanii LMG 23892 Escherichia coli ATCC 25922

0.01

Figure 2. Phylogenetic dendrogram of V. gigantis strains performed using Mega 4,0 software. Phylogenetic tree was constituted using neighbor-joining algorithm based on 1424 bp sequences. Scale shows 1 base variance per 100 nucleotides (bootstrap analysis was carried out as 1000 repetitive). Numerals above branches indicate statistical bootstrap values (upwards of 40 are seen).

S2W42: JF412218, S3W2: JF412222, S3W28: JF412234). In order to determine the phylogenetic JF412221, S3W46: JF412220, SeLu25: JF412217, relationship between the V. gigantis strains obtained in Se2Lu48: JF412223, Se3Lu25: JF412224, FU-9: this study, the reference strain V. gigantis LMG 227141 JF412226, FU-10: JF412225, E-10: JF412230, E-11: and the external group Escherichia coli ATCC 25922, a JF412231, E-14: JF412227, E-15: JF412229, E-16: phylogenetic tree was formed with the neighbor-joining JF412228, H-2: JF412233, H-3: JF412232, H-16: algorithm (Figure 2). 7116 Afr. J. Microbiol. Res.

Evaluation of the phenotypic characteristics of PFGE profiles obtained with SmaI cut were analyzed bioluminescent V. gigantis strains with a 10% coefficient of homology and as a result of the analysis; the genome homology with the highest The evaluation of the physiological and biochemical percentage (94%) was exhibited by S2W9 and E-14 characteristics of strains revealed that all bioluminescent strains of V. gigantis. Analysis of Figure 3 reveals that strains shared the following characteristics: Gram two main clusters (A, B) are formed as a result of negative reaction; polar flagella motility; absence of poly- degradation of genomic DNA with SmaI, and that they p-hydroxybutyrate (PHB) accumulation; having exhibit 70% pattern homology. The cluster A is composed bioluminescent features; presence of indole production, of two sub-clusters (A1 and A2) exhibiting 75% pattern positive oxidase, catalase, capability of arginine homology among themselves. It was determined that dihydrolase activity and phenylalanine deaminase there were 12 different PFGE patterns within the cluster activity, gelatin, skim milk, milk casein, capability of A, and that pattern homologies varied between 75 and Tween-20 and Tween-80 hydrolysis, being fermentative 94% in this cluster. It was determined that there were in glucose-containing O/F medium, capability to different PFGE patterns within the cluster B, and that they reproduce at 20 and 27°C, inability to reproduce in the exhibited homology ranging between 75 and 88%. NB medium containing 0 and 11% NaCl, acid production Among all the V. gigantis strains, Se2Lu48 strain was as a result of glucose, sucrose and lactose fermentation determined to be the most distant strain with its 75% (Table 2). genome homology (Figure 3). As shown in Table 2, when their reproductive traits particularly in the TCBS medium are examined, all but V. gigantis SW15 strain formed yellow colonies. A similar DISCUSSION situation emerged in temperature requirements. It was determined that V. gigantis H-2 strain could not To the best of our knowledge, V. gigantis strains were reproduce at +4°C but all the other strains did. There was first isolated and identified in studies performed by Roux a significant difference between the temperature et al. (2005), and later by Beleneva and Kukhlevskii in requirements of the strains particularly at 30°C, and 50% 2010. However, both studies reported that these strains of the strains had the ability to reproduce. When the NaCl did not have bioluminescent features. In addition, amounts needed for growth and development were determination of genetic variations through the PFGE considered, a strain difference arose in the media technique has not been reported in any study so far. containing 0.5, 9 and 10% NaCl. There were also When the unique habitats where bioluminescent differences in amylase, lipase and hemolytic activities organisms have evolved are considered, it becomes and 55% of the V. gigantis strains were determined to be obvious that bioluminescence has many functions. Open β-hemolytic (Table 2). In addition, as shown in Table 3, it are a world with no place to hide and sunlight is was determined that all carbon and nitrogen sources filtered through the open waters and is reduced up to 10 were assimilated by all the strains except for the strain times almost every 75 m. All the visible light is lost below Se2Lu48 (Table 3). 1000 meters (Widder, 2010). In order to repair the damage occurring in their DNA, bioluminescent bacteria capable of surviving in this type of habitat use photo- Evaluation of emission spectra measurements reactivation mechanism which requires a specific wavelength of light, and in these environments where As a result of the measurements, it was determined that there is no light at all, the light necessary for photo- 20 bioluminescent V. gigantis strains emitted blue-green reactivation mechanism is produced within the cells light at a wavelength of 480 nm. (Czyz et al., 2000; Kozakiewicz et al., 2005). In addition, bioluminescence is estimated to have evolved at least 40 times independently of each other PFGE analysis carried out with the restriction based on a large number of light-producing chemical enzyme SmaI reactions despite the monophyletic origin (Haddock et al., 2010), which makes it important to discover PFGE analysis conducted using the restriction enzyme bioluminescent bacteria from new sources. Because it is SmaI resulted in 15 different PFGE fragment patterns. known that during this evolution process, while some Although the experiments were repeated 3 times, it was species that do not exhibit bioluminescent features may not possible to type 5 V. gigantis strains (S3W28, become bioluminescent, some other species exhibiting S3W46, M-15, H-2 and H-16) with the PFGE technique bioluminescent features may lose their bioluminescent using the SmaI enzyme. As a result of the analysis of 15 features as a result of their adaptation to the habitat they V. gigantis strains, it was revealed that genomic DNA live in. In this context, the results of our study revealed was cut by this enzyme and thus 15 different profiles that V. gigantis strains that have been reported not to occurred (Figure 3). exhibit bioluminescent features since 2005 when they Omeroglu and Karaboz 7117

Table 2. Biochemical and physiological characteristics of V. gigantis strains.

V. gigantis strains Characteristicss 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 V.g.* Gram reaction ------Flagellation + + + + + + + + + + + + + + + + + + + + + PHB accumulation + + + + + + + + + + + + + + + + + + + + ND Indole formation + + + + + + + + + + + + + + + + + + + + ND Luminescence + + + + + + + + + + + + + + + + + + + + - Oxidase + + + + + + + + + + + + + + + + + + + + + Catalase + + + + + + + + + + + + + + + + + + + + + Gelatin liquefaction + + + + + + + + + + + + + + + + + + + + + Arginine dihydrolase + + + + + + + + + + + + + + + + + + + + + Phenylalanine deaminase + + + + + + + + + + + + + + + + + + + + ND O/F F F F F F F F F F F F F F F F F F F F F ND Colony colour in TCBS G Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Growth at:

4C + + + + + + + + + + + + + + + + + - + + + 20C + + + + + + + + + + + + + + + + + + + + + 27C + + + + + + + + + + + + + + + + + + + + + 30C - - - + + + + - + + - + - + + - - + - - + 40C ------50C ------

Growth in NaCl at:

0% ------0.5% + - + - - - + - + - + + ------ND 1% + + + + + + + + + + + + + + + + + + + + ND 3% + + + + + + + + + + + + + + + + + + + + + 5% + + + + + + + + + + + + + + + + + + + + + 7% + + + + + + + + + + + + + + + + + + + + - 9% + - - - - + + + + + + + - + + + + + + + ND 10% + ------+ ------ND 11% ------ND

Fermentation to acid:

Glucose + + + + + + + + + + + + + + + + + + + + ND Saccharose + + + + + + + + + + + + + + + + + + + + ND Lactose + + + + + + + + + + + + + + + + + + + + ND Proteolytic activity on:

skim milk + + + + + + + + + + + + + + + + + + + + ND milk + + + + + + + + + + + + + + + + + + + + ND casein + + + + + + + + + + + + + + + + + + + + ND Production of:

amylase + + - + + + + - + + + + + + + + + + + + + lipase :

tributyrine C4 - + + + - + + - - - + + - - - - - + + + ND esterase:

Tween-20 + + + + + + + + + + + + + + + + + + + + ND 7118 Afr. J. Microbiol. Res.

Table 2. Continued.

Tween-80 + + + + + + + + + + + + + + + + + + + + + Haemolytic activity γ α γ β β β α β β α β α α β α β β β α β ND

F: fermentative; G: green; Y: yellow; 1: SW15; 2: SWLiman; 3: S2W9; 4: S2W42; 5: S3W2; 6: S3W28; 7: S3W46; 8: SeLu25; 9: Se2Lu48; 10: Se3Lu25; 11: FU-9; 12: FU-10; 13: E-10; 14: E-11; 15: E-14; 16: E-15; 17: E-16; 18: H-2; 19: H-3; 20: H-16; V.g.*: F. Le Roux et al., 2005 (V. gigantis); ND: not determined.

Table 3. Assimilation of carbon and nitrogen sources.

V. gigantis strains Assimilation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 V.g.* D-glucose + + + + + + + + + + + + + + + + + + + + + Lactose + + + + + + + + - + + + + + + + + + + + - Maltose + + + + + + + + + + + + + + + + + + + + ND Sucrose + + + + + + + + + + + + + + + + + + + + - D(-) Mannitole + + + + + + + + + + + + + + + + + + + + + D(+) Mannose + + + + + + + + + + + + + + + + + + + + +

sources D(+) Galactose + + + + + + + + + + + + + + + + + + + + + Tri-Sodium citrate + + + + + + + + + + + + + + + + + + + + ND Sodium pyruvate + + + + + + + + + + + + + + + + + + + + ND

Carbon Sodium lactate + + + + + + + + + + + + + + + + + + + + ND Sodium propionate + + + + + + + + + + + + + + + + + + + + ND Gluconic acid + + + + + + + + + + + + + + + + + + + + ND Acetate + + + + + + + + + + + + + + + + + + + + ND Cellobiose + + + + + + + + + + + + + + + + + + + + +

Glycine + + + + + + + + + + + + + + + + + + + + ND L-Arginine + + + + + + + + + + + + + + + + + + + + ND

sources L-Proline + + + + + + + + + + + + + + + + + + + + ND L-Serin + + + + + + + + + + + + + + + + + + + + ND

Nitrogen DL-Alanine + + + + + + + + + + + + + + + + + + + + ND

1: SW15; 2: SWLiman; 3: S2W9; 4: S2W42; 5: S3W2; 6: S3W28; 7: S3W46; 8: SeLu25; 9: Se2Lu48; 10: Se3Lu25; 11: FU-9; 12: FU-10; 13: E-10; 14: E-11; 15: E-14; 16: E-15; 17: E-16; 18: H-2; 19: H-3; 20: H-16; V.g.*: F. Le Roux et al., 2005 (V. gigantis); ND: not determined.

were first isolated and identified (Beleneva and ability to produce indole, oxidase and catalase; they Kukhlevskii, 2010; Roux et al., 2005), include cannot reproduce in the media including 0 and 11% NaCl bioluminescent strains too. Since our data is the first one at 40 and 50° C; they have the ability to produce acids as on this issue, it is of importance because it was a result of glucose, sucrose and lactose fermentation; determined that the strains isolated from different sources they have the ability to produce extracellular protease (in and stations within the scope of our study have the medium with all the three sources); and they have bioluminescent features and that they belong to V. gelatinase and esterase enzymes; they exhibit phenyl gigantis. alanine deaminase and arginine dihydrolase activities Within the scope of our study, 20 bioluminescent V. and they use the glucose, the only carbon source in the gigantis strains (7 from sea water, 3 from sediment medium, through fermentation. In this context, these samples and 10 from different parts of sea fish samples features can be said to be the common characteristics of such as their surfaces, gills, intestinal contents and all bioluminescent V. gigantis strains. In addition, as a internal organs) were collected. The following were result of isolation and identification from different parts of determined as the common features of these Boops boops and Diplodus annularis collected from bioluminescent strains: they have Gram-negative cells different sampling points at different times, it was with the bent-rod morphology; they have the ability to concluded that V. gigantis is the only bioluminescent move with at least one polar ; they can produce species colonizing these marine fishes in the present PHB while not producing endospores; they have the study. Omeroglu and Karaboz 7119

Figure 3. Dendrogram showing the genetic relationships among bioluminescent V. gigantis strains based on PFGE analysis of genomic restriction fragments formed by SmaI. PFGE profiles were analysed with 10% homology coefficient and UPGMA clustering. The names of the clusters and subclusters and V. gigantis strains are indicated on right and left.

Of these common features, one has not been reported 1979). It is known that strains showing bioluminescent in the literature yet. In the previous studies including the feature when they are first isolated lose this property due first isolation study so far, it has been stated that V. to dark mutants. In addition, horizontal gene transfer gigantis strains do not exhibit bioluminescent features (HGT) which plays an important role in bacterial evolution (Beleneva and Kukhlevskii, 2010; Roux et al., 2005). and which frequently occurs in bacteria in nature can However, the results of our study revealed that these contribute to the acquisition of lux genes (Urbanczyk et strains can exhibit bioluminescent features, which is of al., 2008). In this context, it is thought that Photorhabdus importance since these are the first data on this topic. It (a terrestrial species) and Shewanella (which has only was determined that the 20 strains identified belong to V. two bioluminescent species) may have acquired lux gigantis and that the light they emit has blue-green genes from a member of Vibrionaceae by HGT (Dunlap properties according to its wavelength. In various studies, and Kita-Tsukamoto, 2006; Forst et al., 1997; Kasai et the presence and causes of similar situations have been al., 2007). In this respect, it may be suggested that reported. In the data on bacterial bioluminescence, the different strains of the same species may experience existence of dark variants is known (Kenyan and changes in their bioluminescent features depending on Hastings, 1961; Nealson, 1972; Nealson and Hastings, the habitat in which they evolve. A similar change 7120 Afr. J. Microbiol. Res.

occurred in V. chagasii species too. When it was first organisms. The reason why rRNA genes are preferred as isolated, it was reported not to be bioluminescent optimal tools for these purposes is due to their ribosomal (Thompson et al., 2003), but in their study conducted in protection characteristics. In bacteria, the three rRNA 2008, Urbanczyk et al. identified it as a bioluminescent genes are organized into a gene cluster, which is species and this was reported in the literature. Previously expressed as a single that may be present in characterized V. chagasii strains were determined not to multiple copies in the genome (Pei et al., 2010). In many have luxA and luxB, but the existence of strains bearing bacteria, the number of 16S rRNA gene copies is more these genes have been identified by HGT (Urbanczyk et than 1 and 15 at most. It is thought that different copies al., 2008). do not differentiate but evolve in harmony, which is In addition to these common features, it has been achieved by a mechanism called gene protection (Harth determined that variations may occur in physiological and et al., 2007). Therefore, the 16S rRNA is widely used as biochemical characteristics of bioluminescent V. gigantis a marker for the taxonomic classification of prokaryotic strains. Consistent with our study, in several other studies organisms (Pei et al., 2010), and it is considered that conducted on V. gigantis, it has been revealed that there there should be at least 97% 16S rRNA similarity for the may be differences in their various characteristics due to identification of species (Thompson et al., 2004). strain differences (Roux et al., 2005). It has been Genetic proximity of strains belonging to the same determined that especially with NaCl concentration, some species can be determined with various ribotyping extracellular enzymatic activities and temperature techniques. However, for the determination of these requirements in which V. gigantis species can reproduce relationships, it is suggested to use PFGE, which is can results in variations between V. gigantis strains. The accepted as the gold standard method and thus in this results obtained indicated that of the V. gigantis strains, study, PFGE was preferred (Seifert and Gerner-Smidt, 70% did not have reproductive ability in a medium 1995). This is because DNA damage can be prevented containing 0.5% NaCl, 25% in a medium containing 9% and thus more significant results can be achieved NaCl and 95% in a medium containing 10% NaCl. A through this technique in which the entire genome is similar situation occurred in the temperature analyzed and there is no DNA isolation phase although it requirements, and it was determined that 5% of the requires a lot more experience, time and expense strains were not capable of reproduction at 4°C and 50% compared to other methods. The decision about the of them at 30°C. It was also determined that 90% of the restriction enzyme to be used in this stage should be strains exhibited amylase activity while 55% of them made carefully. The careful examination of studies degraded hemoglobin in the blood completely. Except for particularly performed on Vibrio and bioluminescent one strain, similar results were obtained for all the strains species reveals that NotI enzyme is preferred more in terms of reproduction characteristics in the TCBS agar (Goering, 2010). Suwanta et al. (1998) used 5 different medium, and carbon and nitrogen sources they restriction enzymes (ApaI, EagI, NotI, SmaI and SpeI) for assimilated. the investigation of genetic diversity and genotyping of The phenotypic identification of genera and species of Vibrio isolates and they achieved their DNA. NotI was Vibrionaceae is problematic and open to debate. The used to determine the genome size and chromosomal main problem leading to this problem is that phenotypic position of Photobacterium kishitanii, a new traits exhibit high variability. Their reproductive ability in bioluminescent species introduced into the literature in different salt concentrations and temperatures, and the 2007 (Ast et al., 2007). In our study, it was found that carbon sources used are among the phenotypic tests SmaI enzyme could be used for the investigation of the and, at the end of our study, variations were determined genetic diversity of bioluminescent V. gigantis strains with between these properties of the strains. Therefore, these PFGE technique. distinguishing features lead to misinterpretations of the At the end of the PFGE analysis of the entire genome, identification of Vibrionaceae isolates (Thompson et al., although the PFGE experiments were repeated 3 times, 2004). In a study conducted by Montes et al. (2003), the PFGE patterns of S3W46, S3W28, M-15, H-2 and H-16 species previously identified as V. mediterranee, Vibrio strains of V. gigantis were not obtained. Therefore, it was anguillarum and Vibrio ordalii with phenotypic concluded that typing of these species cannot be fulfilled characterization were then identified as Vibrio with PFGE technique. Pseudomonas aeruginosa scophthalmi, Vibrio splendidus-V. lentus and V. (Romling et al., 1994), Clostridium difficile (Kristjansson scophthalmi, respectively, with molecular et al., 1994), and V. cholerae (Kam et al., 2003) are the characterization. Therefore in this study, phenotypic examples of strains that cannot be typed with the PFGE characteristics were used only for the determination of technique. Such problems arise as a result of methylation the characteristics of bioluminescent isolates not for the or nuclease activity (Goering, 2010). Upon the completion identification of species. For species identification, the of PFGE analysis of all the genomes, it was determined 16S rRNA gene region was preferred because rRNA that the 15 bioluminescent V. gigantis strains formed 15 genes are widely used for the prediction of evolutionary genotypes and were divided into 2 main clusters and that histories and taxonomic identification of individual the genome homology between these 2 clusters was Omeroglu and Karaboz 7121

70%. Examination of the sources of the strains forming ACKNOWLEDGEMENTS the cluster B showed that they were V. gigantis strains (E-10, FU-10 and Se2Lu48), each of which was collected The authors would like to extend their thanks to the Izmir from different stations and samples, and unlike other Institute of Technology, Biotechnology and strains. Evaluation of the similarities in terms of their Bioengineering Research and Application Center for physiological and biochemical characteristics showed molecular assays. This work was supported by BAP that, of the 20 strains, V. gigantis SW15 exhibited the Project (2007 FEN 024), Ege University. most different properties and that whereas all the strains were identical in the assimilation of carbon and nitrogen REFERENCES sources, only Se2Lu48 strain appeared to be different from the others. As a result of the analysis of all the Ast JC, Cleenwerck I, Engelbeen K, Urbanczyk H, Thompson FL, De Vos P, Dunlap PV (2007). 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