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 Vibrio 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 bacteria 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 genomes of the bioluminescence V. gigantis strains were not completely identical, and had genome 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, bioluminescent bacteria, 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). squids (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 marine habitats 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 squid 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 20C Until recently, three marine bioluminescent bacterial for 24 h, bioluminescent colonies were selected in a dark room and genera were known: Vibrio, Photobacterium, 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 Vibrionaceae Bioluminescent bacterial genomic DNAs for polymerase chain family and this group was classified into a new genus reaction (PCR) assays were isolated with the ZR Fungal/Bacterial called Aliivibrio. 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 taxonomy 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 cholerae, Vibrio harveyi, 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 94C, 40 cycles of denaturation for 1 min bioluminescent species, the Vibrio chagasii species that at 94C, annealing for 1 min at 56C, elongation for 1 min at 72C was initially considered not to have a bioluminescent and final elongation for 10 min at 72C (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 50C) 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