1.        Strains and culture conditions

1.1 The following organisms were used in the study:

1.2    Bacterial media

The marine agar 2216 will be prepared as directed by DIFCO/BBL. Nutrient agar w/3% NaCl will be prepared as per manufacturer’s instructions with the addition of 15g NaCl per liter. Nutrient agar with 1.5% NaCl will be prepared using 23g nutrient agar, 30g NaCl and 1 liter distilled water. The artificial sea water (ASW) will consist of 0.4 M NaCI, 0.1 M MgSO₄.7H₂0, 0.02 M KCI, and 0.02 M CaCl₂.2H₂O (Baumann, et al., 1971). The basal medium (BM) contained 50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.5), 190 mM NH₄C1, 0.33 mm K₂HPO₄.3H₂O, 0.1 mM FeSO₄ 7H₂O, and halfstrength ASW (Baumann, et al., 1971). Basal medium agar (BMA) was prepared by separately sterilizing and then mixing equal volumes of double-strength BM and 20 g of Oxoid lonagar per liter. Yeast extract broth (YEB) was made by supplementing BM with 5 g of Difco Yeast Extract per liter. Yeast extract agar (YEA) was prepared by solidifying YEB with 20 g of Difco Agar per liter. Wagatsuma medium consists of 0.3% yeast extract, 1% Bactopeptone (Difco), 7% NaCI, 0.5% K₂HPO₄, 1.5% agar, distilled water added to a final volume of 1 liter. After dissolving by heating (heat sterilization should be avoided), mannitol is added to a concentration of 1%, 0.1% crystal violet alcohol solution to 0.1 %, and human or rabbit defibrinated blood (or saline suspension of red blood cells) to 5 % (Miyamoto, et al., 1969). (Table 1).

Table 1.  Table showing the unknown organisms, type strains and controls for biochemical testing, 16SrRNA PCR, and RFLP

NR_027590

DQ868989

1.3        Gram staining, motility and cell structure

The growth medium and temperatures suggested in Table 5.  Table showing the unknown organisms, type strains and controls for biochemical testing, 16SrRNA PCR, and RFLP will be used to re-suspend the lyophilised stock cultures; produce samples for gram staining and also provide the subcultures for the biochemical tests. Each species will be inoculated from fresh specific agar slants into test tubes containing 5 ml of Yeast Extract Broth. After 4 to 6 hrs of growth and again at 18 to 24 hrs, the cultures will be examined by means of phase-contrast microscopy to determine cell shape and motility. The novel strains will be grown in 125-ml Erlenmeyer flasks containing 20 ml of Yeast Extract Broth. When the culture reaches a turbidity of 100 to 150 Klett units, the cells will be centrifuged and suspended in half-strength Artificial Sea Water containing 50 mM Trishydrochloride (pH 7.5) to a turbidity of 400 Klett units. Photomicrographs will be taken using a phase-contrast microscope (Baumann, et al., 1971).

1.4    Luminescence

Inoculate the test strains on marine agar and incubate at 25°C. After incubation for about 8 h and again at 16–20 h, take the plates into a room that can be totally darkened and set them on a bench top. Take in a flashlight, and turn off the overhead light. Observe the cultures for light. Light from the strong positive culture should be visible within a few seconds, but the weak positive culture may take several minutes. Continue to observe the test culture for luminescence and record as positive or negative after 5 and again at 10 minutes (Dworkin, et al., 2006).

1.5        Temperature

Growth at different temperatures will be determined by using YEB. Test tubes containing 5 ml of the medium will be inoculated and incubated with aeration at the appropriate temperature. Observations at 4°C were continued for 2 weeks. Observations at 25°C, 35°C and 40°C will be continued for 4 days (Baumann, et al., 1971).

1.6        Tests for sodium optimum

Each species listed in Table 5.  Table showing the unknown organisms, type strains and controls for biochemical testing, 16SrRNA PCR, and RFLPwould be grown on minimal media in the presence of 0%, 2%, 4%, 8% and 10% NaCl. BM and a similar medium differing only in the replacement of sodium by an equimolar amount of potassium. Cells from a fresh specific agar slants will be inoculated into 50-ml Erlenmeyer flasks containing 10 ml of the media and incubated with aeration for 48 hr. Turbidity will be measured using spectrophotometer. The tests will be performed in a medium containing 0.2% (v/v) glycerol as well as in a medium containing 0.2% (w/v) potassium lactate as the sole source of carbon and energy (Baumann, et al., 1971).

1.7        Hemolysis

This test will be performed on a medium containing Trypticase Soy Agar (BBL), 2.5% NaCI, and 5% defibrinated blood (TSB) and on Wagatsuma's medium. The plates will be incubated at 29°C and examined at 24 and 48 hrs (Baumann, et al., 1971).

1.8        API 20E Test

This standardized test has been reported as an effective means of identifying These standardized test strips contain 20 microtubules. Each strip will be inoculated with a single isolated colony of the organism from a young culture (18 – 24hrs) suspended in 0.85% NaCl in accordance with manufacturer’s instruction. The strips will be inoculated in accordance with manufacturer’s instructions and incubated at 29°C - 30°C for 24hrs – 48hrs. The ability of the organism to utilise specific substrates and display fermentative/oxidative growth will be tested (Table 2).

Strains will be screened for their ability to utilize 150 carbon compounds as sole sources of carbon and energy. Using API 20E and API Strep tests.

Table 2. Substrates to be used to identify different aspects of the metabolism of the organisms.

1.9    Alternative tests

The following are a list of detailed biochemical tests to be used in the event that the API20E strips are inadequate for obtaining viable results for the Vibrio species.

1.9.1    Fermentation of carbohydrates

The ability of strains to ferment glucose will be determined by the use of two different fermentation media. The first (F-l) will be different from YEB by containing 100 mM Tris-hydrochloride (pH 7.5), 1 g of Oxoid lonagar per liter, and 10 g of filter-sterilized glucose per liter. 10 ml portions of the medium will be dispensed into test tubes and inoculated by means of a stab. A control medium lacking glucose will also be inoculated. About 5 ml of melted 10% (w/v) Oxoid lonagar, at 40°C, will be added to make an agar plug. The cultures will be examined for turbidity and gas production for a period of 6 days (Baumann, et al., 1971). The second fermentation medium (F-2) will differ from F-I by containing 1g of sodium thioglycolate per liter. The agar plug will be omitted. F-2 medium containing glucose as well as a control medium without glucose will be inoculated and examined for growth on the surface and throughout the medium at 24 and 48 hrs. At 48hrs, the pH of the culture is determined (Baumann, et al., 1971). The ability of strains to produce acid from D-ribose, D-gluconate, D-mannitol, and glycerol under anaerobic conditions will be tested on fermentation media (F-3) which will be different from BM by containing 25 mM Trishydrochloride (pH 7.5), 0.5 g of Difco Yeast Extract per liter, I g of Oxoid lonagar per liter, 2 ml of a 1.6% (w/v) alcoholic solution of Brom Cresol Purple per liter, and 10 g of the filter-sterilized carbohydrate per liter. Subsequent procedures are identical to those for F-1. The cultures will be examined for color change and gas production for a period of 6 days (Baumann, et al., 1971).

1.9.2    Production of 2, 3-butyleneglycol

Strains will be grown in test tubes containing 10 ml of YEB with 1% (w/v) D-glucose. After 72-hrs of incubation, the Voges- Proskauer (VP) test will be performed (Baumann, et al., 1971).

1.9.3   Nitrogen metabolism

            Strains will be tested for their ability to denitrify. The medium to be used would consist of YEB supplemented with 0.2% (v/v) glycerol, 3 g of NaNO₃ per liter, and 1 g of Oxoid lonagar per liter. Each strain is first grown in the denitrification medium under semianaerobic conditions. After 48 hrs of incubation, the cultures will be tested for nitrite by the use of the starch-iodine spot test method. A loopful of the semianaerobic culture will then be inoculated into a test tube containing homologous medium and sealed with an agar plug. Observations will continue for 8 days. Cultures producing gas will score positive for denitrification. Nitrogen fixation will be tested by inoculating cells from a fresh specific agar slant into BM from which NH₄Cl is omitted and which contains 0.2% (v/v) glycerol. Observations will continue for 6 days (Baumann, et al., 1971).

1.9.4    Extracellular enzymes

Strains will score positive for each of the extracellular enzymes only when the zone of reaction extended beyond the limits of growth. The production of gelatinase will be determined by inoculating onto YEA containing 50 g of gelatin per liter. After 48 hrs of incubation, the plates will be flooded with acidic mercuric chloride. Amylase production will be determined on YEA containing 2 g of starch per liter. After 48 hrs of incubation, the plates will be flooded with Lugol's Iodine solution. Lipase production will be determined on YEA supplemented with 0.01% (v/v) polyethylene sorbitan monooleate. The presence of alginase will be tested on a medium consisting of YEA overlaid with YEA containing 20 g of sodium alginate per liter. The presence of chitinase will be tested on a medium consisting of YEA overlaid with YEA containing 10 g of colloidal chitin per liter. Observations on the production of lipase, alginase, and chitinase will be continued for 6 days (Baumann, et al., 1971).

2.0       DNA extraction from cultured microorganisms

Genomic DNA will be extracted from the unknown marine Vibrio sp. and Vibrio ATCC type strains 7.1.1 for use in both multilocus gene analysis (MLSA) and direct genome restriction enzyme analysis (DGREA). DNA extraction will be carried out using the GenElute Bacterial Genomic DNA Kit (#NA2110-1KT, Sigma-Aldrich, St. Louis, MO). Overnight (16 hrs) broth cultures (1.5 ml) from unknown and reference organisms will be concentrated by centrifugation for two minutes at 12000 g. The supernatant will be removed and the pellet resuspended in 200 µl of lysozyme solution (45 mg/ml) and incubated for 30 min at 37^OC. RNA, nuclease and cell membrane degradation will be done by adding 20 µl of RNase for two minutes, followed by 20 µl of (20 mg/ml) Proteinase K solution and 200 µl of lysis solution C. The mixture will be vortexed for 15 seconds and then incubated for ten minutes at 55^OC. The DNA is then precipitated by adding 200 µl of 96% (v/v) ethanol to the lysate. After mixing for 10 seconds, the entire mixture will be added to the silica based binding column and centrifuged at 12,000 g for 1 minute. The binding column is then washed several times with GenElute washing solutions, followed by elution of the DNA using 200 µl GenElute elution solution.

The concentration and purity of the extracted DNA will be determined spectrophotometrically using the Nanodrop 2000c (Thermo Scientific, USA). The DNA if not used immediately will be stored at -20OC for long term usage.

2.1        Genomic fingerprinting

PFGE  protocol will adopt technique described by CDC (CDC, 2010; www.cdc.com).

Direct genome restriction enzyme analysis (DGREA)

2.1.1    Restriction Digest

Bacterial DNA will be extracted from single colonies cultured overnight on TSBA Agar using theSigma Wizard Genomic DNA extractionkit according to manufacturer’s instructions. Aliquots (10 ul) of genomic DNA from each strain listed in Table 7. Concentrations of vector plasmid and DNA amplicons for recombination step will be digested in a 50 ul restriction digestion mix containing 30 ul nuclease-free water, 5 ul 10x Fast Digest Buffer , 10 ul of genomic DNA and 5ul of HhaI, AatII, DpnI and EcoRI Fast Digest Restriction Enzyme separately for each strain selected. Digests will be incubated at 37^oC for 1 hour, except for EcoRI restriction digest which will be incubated for 30 minutes (Fast Digest – Fermentas INC, USA). Samples will then be incubated with Proteinase K at a final concentration of 0.2 mgm^-1 for 1 hour at 37^oC, to hydrolyze the restriction enzyme and the bovine serum albumen present in the buffer (Fuenzalida et al, 2006).

2.1.2    Polyacrylamide Gel Electrophoresis (PAGE)

Restriction digest resolution will be carried out using 7.5 % Polyacrylamide Gel Electrophoresis. The gels will be  prepared using  7.13g Acrylamide, 0.37 g Bis-Acrylamide, 0.1 mL TEMED, 10.0 mL 10X Buffer stock, 82.4 ml water and 1.0 mL of 10% Ammonium persulfate. The two glass plates to be used for setting up the electrophoresis gel will be washed with warm detergent, and then rinsed with water followed by ethanol and allowed to dry. The plates will be intermittently treated with silicone to prevent the gel from sticking to them and tearing when it is removed. Immediately before the gel mixture is placed in the casting unit to be set, the Ammonium persulphate and TEMED solutions will be poured into the casting unit and mixed. After placing the complete mixture into the casting unit, a comb will be applied to the gel and it will be allowed to polymerize (Guilliat, 2002). Approximately 7ul of each digestion product will then be mixed with 2ul of loading dye buffer and placed into wells made on an 8x7x0.1cm gel (Fuenzalida et al, 2006). A 1X working solution of Tris-borate (TBE) electrophoresis buffer containing 89mM Tris (pH 7.6), 89mM boric acid and 2mM EDTA will be used to run the electrophoresis for 12 hours at 100 V (Guilliat, 2002).

2.1.3    Visualization and Analysis of Restriction Digest

Bands will be stained using silver nitrate, according to the methods of BMSL (2008) and visualized using Electrical Lite Power Source. Analysis of restriction fragments will carried out using GENtle (2003 V1.9.1) software (Magnus Manske, University of Cologne, Germany).

3.0   Multi locus sequence analysis (MLSA)

This method involves the analysis of several housekeeping genes (more than 5) and the establishing of relationships between taxa (Pascual, Macián, Arahal, Garay, & Pujalte, 2010). The MLSA utilizes PCR protocols followed by a phylogenetic analysis. Although it has been standard practise to differentiate species and subspecies by using the 16srRNA analysis the conservative nature of the gene means that the 16S rRNA genes of vibrios are not sufficiently polymorphic to ensure their reliable identification thus other genes and molecular markers, such as 23S rRNA, gapA , gyrB, hsp60, and recA, have been used to identify Vibrionaceae species (Ki, Zhang, Zhang, Huang, & Qian, 2009). The genes and PCR conditions for the MLSA used in this study are listed in Table 3.

Table 3. The genes and PCR protocols needed for the MLSA

3.1       PCR amplification of extracted DNA

The 16S rDNA gene has become the standard for phylogenetic classification (Brenner et al., 2005; Stackebrandt & Goebel, 1994). Its high level of gene conservation and negative selective pressure qualifies it as an ideal chronometer (Woese, 1987). Unfortunately, not all bacteria are able to be classified based solely on 16S rDNA analysis. Members within the gammaproteobacteria, especially vibrio have highly conserved 16S rDNA among intraspecies. This allows for little discriminatory power between Vibrio species even though DNA-DNA hybridization indicates the need for separate taxonomic denotations (Thompson et al., 2009). Additionally, Vibrio is one of the few organisms that have more than 5 copies of the 16S rDNA (Ki et al., 2009). This redundancy can lead to inadequate classification since each copy may undergo separate occurrences of recombination or point mutations. As a result of these factors, the use of 16S rDNA alone, cannot be used to determine phylogenetic lineage among Vibrio sp.. The use of multiple housekeeping genes for taxonomic identification was first proposed by Madien and Coworkers in 1998 and has been adopted as one of the most useful method to resolve between closely related Vibrio species (Taxonomy paper). The study of multiple conserved genes and concatenated gene sequences permits for the detection of detection of synonymous and nonsynonymous changes as well as recombinational events between strains (microevolution) (taxonomy paper).     

Seven different housekeeping/conserved genes (recA, pyrH, atpA, obj, tdh, rpoB and 16S rDNA) will be used to determine phylogenetic linage of the unknown Vibrio isolates. The sequences, expected size of gene product and percentage conserved homology of the genes are shown in Table 8. The genes and PCR protocols needed for the MLSA. Blank and collegeues discovered over 126 conserved genes within the gammaproteobacteria subfamily. Several of these conserved genes () have been previously used to determine phylogenetic lineage of vibrio. However, the listed seven are among the most commonly used that allows for detection of significant evolutionary disparities between species.

The proposed primers were obtained from published literature Table 9.  Taxonomic resolution of multi locus gene analysis of marine Vibrio.These primers were tested for specificity to the members within the Vibrio genus by utilizing the BLASTn search within the GenBank database at the National Center of Biotechnology Information (NCBI) (Zhang et al., 2000). Further analysis to check specificity was done using the ‘check probe’ facility of the Ribosomal Database Project (RDP) (http://rdp.cme.msu.edu/) (Cole et al., 2005; Cole et al., 2003). Mismatch with eukaryotic sequences were analyzed using the ‘probe check’ at Greengenes (http://greengenes.lbl.gov) (DeSantis et al., 2003; DeSantis et al., 2006). All forward primers were submitted as ‘target sequence’ and reverse primers were submitted as probes. The search was based on a 100% match, whereby all primer sequences had to align with the target sequence. Possibility of primer dimer formations were analyzed using the Quick primer test (sensitivity 3) function on FastPCR Ver. 5.3.119 (Kalendar et al., 2009), shows the list of primers that will be used, the samples that will be analyzed and the thermal settings for each PCR. All primers will be synthesized using the services of Invitrogen, USA.

3.2    Screening and extraction/purification of amplicons

PCR amplicons will be screened for correct nucleotide size using electrophoresis based on 1% UltraPure^TM Agarose (#15510-019, Invitrogen, Carlsbad, Ca, USA) gel stained with 9.4 µl (10 mg/ml) ethidium bromide (#E-1510, Sigma, St. Louis, MO). The gel will be run on an electrophoresis kit at 70 V for 90 minutes or 150 V for 20 minutes. Visualization of the DNA bands will then be carried out using UV-illumination at 240 nm wavelength. Amplicon size from samples that correlates to band size from the positive control, and produce no bands for negative controls will be excised and purified using Qiagen minielute gel extraction kit (#28604, Qiagen, Sweden).

Extraction is facilitated by first mixing the gel excised with twice its weight in QG solubilization buffer. The mixture is then heated at 50^oC for 10 minutes, 1× the gel weight of isopropanol added, and then 750 µl of the mixture is transferred into a mini-elute column and centrifuged for one minute at 10000 rpm. The effluent will then be discarded and the mini-elute column refilled with the DNA mixture and centrifuged until all the DNA is trapped in the filter. The DNA is then washed by adding 750 µl buffer PE to the columns. The mixture will then be centrifuged for one minute at 10000 rpm, the filtrate discarded; the tubes centrifuged for another one minute and the columns then transferred to new 1.5 ml eppendorf tubes. The DNA is then eluted from the column by adding 28 µl elution buffer, and centrifuging for one minute at 10000 rpm. The eluted DNA is then stored at 4^oC until cloning. 

3.3    Topota cloning

TOPOTA Cloning kit will be used to further screen the amplicons based on recombination of individual amplicons into vector plasmids and random cloning into competent E. coli cells. PCR amplification using Taq polymerase will be used to add 3’ sticky end poly A tail overhangs at the end of the amplicons. Reaction tubes will consist of 22 µl of the PCR product, 2.5 µl 10× PCR buffer, 1.0 µl dATP and 1.0 µl (1 U/µl) Taq polymerase. Tubes will be placed into the thermal cycler for 30 minutes at 72^OC. Recombination of the linear vector plasmid (pCR2.1-TOPO) with the amplicons will be carried out as shown in (Table 4). Different concentrations of DNA template will be used to determine concentration efficiency for transformation. Recombination is facilitated by incubating the tubes at room temperature (20^OC) for 30 minutes.

Table 4. Concentrations of vector plasmid and DNA amplicons for recombination step

To transform chemically competent E. coli cells with the vector plasmids, 3 µl of the transformed vector will be added to 50 µl of competent E. coli cells. The mixture is incubated on ice for 30 minutes, placed on a heating block for exactly 30 seconds and then placed back on ice. The cells are then allowed to repair by adding 250 µl of SOC medium to each tube and incubating on a shaking incubator at 37^OC for 1 hour at 200 rpm. After incubation, 100 µl and 150 µl of the transformed E. coli will then be streaked plated onto LB agar plates made selective with 50 µg/ml of ampicillin. The plates are then incubated for 24 hrs at 37^OC. Differentiation of transformed colonies are facilitated by the addition of 40 µl of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (xgal) (40 mg/ml) and 40 µl of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (100 mM) to each LB plate prior to inoculation. After incubation, six well-defined white colonies from each plate will be selected and replated onto selective LB agar. The selected colonies are then simultaneously inoculated into 2 ml of selective (kanamycin 0.04 mg/ml) yt medium and incubated at 37^OC for 16-20 hrs at 200 rpm.

PCR will be used to screen the selected colonies for the presence of the vector insert. A dot of each colony is selected using a sterile pipette tip and added to reaction tubes containing 12.5 µl of 2× iProof HF Master Mix, 10 µM of M13F/M13R and 11.5 µl of sterile nuclease free MQ water. The PCR thermal cycles consist of 1 cycle at 98^oC for 5 min; 30 Cycles at 98^oC for 1 min, 60^oC for 1 min, 72^oC for 2:00 min; 1 cycle at 72^oC for 7 min and hold at 4^oC. Screening of amplicons will be carried out using gel electrophoresis as previously described 7.8.2. Amplicons that correspond to the requisite base size indicate colonies that contained successfully recombined plasmids. DNA is then extracted for sequencing, from the corresponding positive colonies grown in the yt broth.

Cells are first pelleted by centrifuging the culture tubes at 8000 rpm for 3 minutes. The plasmid DNA is then extracted using the QIAprep Spin miniprep kit (#27104, Qiagen, USA). Plasmid extraction is carried out by first mixing the pellet with 250 µl of RNAase (Buffer P1), followed by 250 µl of lysis buffer P2 and then 350 µl of neutralization buffer N3. The solution is mixed thoroughly after each addition, by inverting the tube 4-6 times. The mixture is then centrifuged at 13000 rpm for 10 minutes and the supernatant poured into a QIAprep spin column. The columns are centrifuged at 13000 rpm for 60 seconds and the filtrate discarded. The spin column is then washed with 0.5 ml of PB wash buffer (binding buffer) centrifuged for 60 seconds, the filtrate discarded and 0.75 ml of washing buffer PE added. The mixture is centrifuged twice for another 60 seconds at 13000 rpm and the filtrate discarded each time. Elution of the DNA will then be carried out by adding 50 µl of Buffer EB to the spin column followed by centrifugation at 13000 rpm for one minute. The DNA rich filtrate is then stored at 4^OC until use. Sequencing of the plasmid insert will be carried out using the services of MWG Biotech Germany.

3.4    Phylogenetic analysis

Sequenced DNA will be aligned and checked for primers, chimeras and vector sequences using the editing software VecScreen (Altschul et al., 1997) from the NCBI, and RDP web-based chimera detection (Cole et al., 2003). The edited sequences will then be compared to the NCBI database using the BLASTn program (Zhang et al., 2000).  A multiple sequence alignment of the sequences and their closest type strain relatives will be produced using MUSCLE v.3.6 (Edgar, 2004) from the European Molecular Biology Laboratory: European Bioinformatics Institute (EMBL-EBI) (EMBL-EBI, 2009). Additional Vibrio type strains will be added to the alignment so as to visualize the position of each major clad within the genus. These type strains will include, V. harveyi CECT 525T, V. rotiferianus LPD 1-1-11, V. campbellii CECT 532T, V. parahaemolyticus CECT 511T and V. alginolyticus CECT 521T. The tree will be rooted using Photobacterium Damselae CDC 23-81.  Manual alignment and editing will be done using BIOEDIT v.7.0.9.0 sequence alignment editor (Hall, 1999). Phylogenetic trees will then be produced based on Jukes-Cantor distance correction tree model (Jukes & Cantor, 1969) in conjunction with the Neighbor-Joining algorithm (Saitou & Nei, 1987), using the MEGA v.4.0.1 software (Tamura et al., 2007). Confidence in the tree will be verified by using a bootstrap value of 1000 (Felsenstein, 1985). Separate phylogenetic trees based on three to seven concatenated genes will be created to improve and validate the resolution and tree topology for individual gene evolution. MLSA based taxonomic resolution based on individual genes used in this study is shown in Table 5.

Table 5.  Taxonomic resolution of multi locus gene analysis of marine Vibrio.