Chapter 4 Molecular characterization of phosphate solubilizing bacterial isolates

Transcription

1 Chapter 4 Molecular characterization of phosphate solubilizing bacterial isolates

2 4.1 Introduction The potential advantage of phosphate solubilizers in plant growth promotion and prevention of environment pollution suggest that the research on phosphate solubilizing microorganisms needs to be accelerated greatly. Thus, the knowledge of genetic variation of phosphate solubilizing microorganisms is important. The abundance and diversity of microorganisms in a given environment i.e., soil is typically enormous. However, it is not possible to get complete information of the relative numbers and identities of the constituent organisms with any of the available biochemical and morphological means. The inability to completely categorize the microorganisms has hampered the efforts of microbiologists and it is recommended to follow the molecular tools for the biodiversity analysis. Molecular biology offers various techniques that can be applied for bacterial identification. Polymerase chain reaction (PCR) has become commonly used research methodology and has led to the development of several novel genetic assays based on selective amplification of DNA. For molecular characterization of bacteria, universal primers of 16S rrna were used to amplify DNA. Amplification by using primers of 16S rrna is routinely used for identification of unknown species. Generally, it is known that if universal DNA sequences are compared in closely-related bacteria, the difference between the DNA sequences would suggest the amount of change from a shared evolutionary ancestor. Thus, 16S rrna technique has proved to be useful because it is large with many domains, and has many positions that vary at different rates. In the present study, another PCR based approach, Randomly amplified polymorphic DNA (RAPD) is performed, as a means of comparing microbial communities using very small quantities of DNA. In particular, RAPD is used to compare a number of microbial communities and quantify their overall similarity (Williams et al., 1990). RAPD employs short primers of arbitrary sequences to amplify random portions of the sample DNA by PCR. Since each primer is short, it will anneal to many sites throughout the target DNA; a fragment is amplified whenever two of these primers anneal close enough and in the proper orientation with respect to one another. Individuals that have different sequences will have primers that anneal in different places and therefore produce a different spectrum of fragments from the PCR, a different DNA fingerprint. Because each primer generates relatively few (5 to 15) distinct bands when

3 separated on an agarose gel, several reactions must be run, using several different primers, and the results combined to obtain the desired number of markers. Pooled results can then be compared between samples and percent similarity computed. Using multiple primers also helps ensure that a sufficiently large region of the target DNA is scanned when an estimate of overall variance between samples is desired (Ogram and Feng, 1997). Typically, 10 to 15 primers (~100 bands) are required for statistical comparison of samples using RAPD markers (Xia et al., 199). Therefore, the combination of morphological, biochemical and molecular techniques is necessary to obtain a better understanding of the interaction between the microorganisms and their natural environment. Once this system is established, it may provide the basis for selection of parental material based on genetic diversity to help overcome some of the problems usually associated with microbial improvement. Moreover, development of molecular markers may be very useful for the management of germplasms and hence ex situ conservation. 4.2 Material and methods Forty-five different bacterial strains were isolated from different regions of Lucknow. The purified and identified isolates were stored in Nutrient agar medium at 4º C. The six unidentified bacteria PKU 5, PBS 4, PKN 3, PKS 4, PKS 3, and PCN 6 having efficient phosphate-solubilizing activity were considered for further molecular characterization Preparation of genomic DNA from bacterial culture The genomic DNA was isolated from the bacterial culture grown at 37 C for 24 h in Pikovskaya s broth by using a method of enzyme lysis (Sambrook et al., 1989). The cells were harvested from 1-5 ml of culture at 3000 rpm for 5 min, supernatant was discarded and pellet was resuspended in 0.5 ml of SET buffer (75 mm NaCl-0.435, 25 mm EDTA-0.930, 20 mm Tris in g/100 ml, ph 7.5) with lysozyme (1 mg/ml) and incubated for 1 h at 37 C. 1/10 th volume of 10 % SDS (50 µl) was added and incubated for 30 min at 37 C. Thereafter 5 M NaCl (150 µl) and 750 µl of chloroform: isoamyl alcohol (24:1) were added and incubated at room temperature for 30 min with frequent inversion. Samples were centrifuged at 10,000 rpm for 15 min and aqueous phase was transferred to a new tube. The DNA was precipitated by

4 adding one volume of isopropanol, mixed and kept for 30 min at -20 C. The DNA pellet was recovered after centrifugation at 12,000 rpm and washed with 100 µl of 70 % ethanol and again centrifuged. Ethanol was removed and the pellet was dried under vacuum in a speed vacuum system for 10 min and finally dissolved in 50 µl TE buffer (10:1) (ph 8.0). The extracted genomic DNA preparation along with the tracking dye (0.25 %) (Table 4.1) were electrophoresed on 1 % agarose gel with ethidium bromide (0.5 µg/ ml) at 80 volt for 30 min using 1X TAE buffer. 1X TAE was prepared by diluting from 50 X TAE, the composition of which is given in Table 4.2. Gel was monitored on Gel Documentation system (BIORAD). Table 4.1 Tracking dye Components Concentration Bromophenol blue 0.25 % Glycerol 15 % Distilled water Rest of volume Table 4.2 Tris Acetate-EDTA (TAE) Buffer Components Tris base Glacial acetic acid Contents (100 ml) (50X) 24.2 g 5.71 ml 0.5 M EDTA (ph 8.0) 10.0 ml Identification of bacterial isolates by 16S rrna PCR amplification The strain identity of isolates was confirmed by PCR using universal primers for rrna genes. Bacterial DNA samples were subjected to PCR amplification using 16S rrna-specific primers (Table 4.3). Each 25 l PCR mixture (Table 4.5) contained 1 l template DNA (100 ng 1.0 g), 2.5 l 1X PCR buffer, 1 l of deoxyribonucleoside triphosphate (dntp mix) (100 mm),

5 1.5 l MgCl 2 (25 mm), 1 l of forward and reverse primers (5 pmole) and 0.5 l Taq DNA polymerase (1 U/ml). The amplification cycle (Table 4.4) consisted of an initial denaturation step of 30 min at 94 C, followed by 35 cycles of 30 sec at 94 C (denaturation), 30 sec at 55 C (annealing) and 30 sec at 72 C (extension), with a final extension step for 5 min at 72 C (Table 4.4). For visualizing PCR products, 5 l of the amplified product was electrophoresed on 1 % agarose gel in 1X TAE buffer, stained with ethidium bromide (EtBr; 10 mg/ml) and analyzed by Gel Documentation system (BIORAD). Table S rrna-specific primers for bacterial isolates Universal primer Sequences Forward Primer (5 AGAGTTTGATCCTGGCTCAG 3 ) 16S rrna Reverse Primer (5 GTTACCTTGTTACGACTT 3 ) Table 4.4 Conditions for PCR reactions PCR steps Initial denaturation Denaturation Annealing Temperatures and time 94 C, 30 min 94 C, 30 sec 55 C, 30 sec Extension 72 C, 30 sec No. of cycles of denaturation, annealing, extension 35 Final Extension 72 C, 5 min

6 Table 4.5 Components of PCR Reagents Volume Concentration Reaction buffer, 10X 2.5 l 1X dntps mix 1.0 l 100 Mm MgCl l 25 mm Primer (forward) 1.0 l 5 pmol Primer (reverse) 1.0 l 5 pmol Taq DNA polymerase 0.5 l 1.0 U/ml Template DNA 2.0 l 100 ng 1.0 g Analysis of amplified products by agarose gel electrophoresis PCR amplified products obtained with different templates were electrophoresed on the agarose gels (1.0 to 2.0 %) by following the standard procedure as given by Sambrook et al., (1989). Electrophoresis procedure Agarose (1 % concentration) was prepared by dissolving the appropriate quantities of agarose in 1 X TAE buffer (ph 8.0) in a microwave oven or by keeping in boiling water bath. 0.5 µg/ ml Ethidium bromide stock was added directly to molten agarose solution before casting the gel. Working solution was prepared by diluting 20 ml of 50 X TAE (Table 4.2) to 1000 ml with distilled water. Molten agarose was cooled to 50 C, poured into respective moulds of mini-gel (50 ml) and midi-gel (100 ml) using appropriate combs (8 to 13 wells). The surface was leveled before pouring the gel. After complete solidifying of the gel, the comb was removed carefully and the gel plate was mounted on respective electrophoresis tanks. The respective electrophoresis tanks were filled with 1 X TAE electrophoresis buffer to cover the gel to a depth of about 1 mm. The DNA sample was mixed with 0.5 µl of tracking dye (1 X) and was loaded slowly into the slots of submarine gel using micropipette. Electrophoresis was carried out at 80 V for 1 h in mini-gel electrophoresis apparatus and 1.5 h in case of maxi-gel system till the tracking

7 dye reached bottom of the gel. After completion of electrophoresis, the gels were taken, out of the chamber and examined under UV transilluminator and photograph taken in gel documentation system (BIORAD) Sequencing of bacterial isolates DNA of the six most promising bacterial isolates were isolated and amplified with 16S rrna gene-specific primers and send to Amnion Biosciences Pvt. Ltd., 112, 16 th cross, Doddannna, Industrial area, Ishwaneedam Post, Bengaluru for sequencing Analysis of DNA sequences A nucleotide Blast search ( was performed for the sequenced DNA. Sequences were aligned using ClustalX version 1.81 and phylogenetic tree were prepared and analyzed Random amplification of polymorphic DNA The RAPD primers are decamer (10 nucleotide length) DNA fragments used for PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals, although not necessarily in a reproducible way. Random decameric primers were obtained from Operon Technologies, Alameda, USA (OPA primers; Table 4.6) and were used for amplification of bacterial genomic DNA for analysis of genetic diversity. The RAPD analysis was carried out through PCR amplification of total DNA. Amplification reactions were performed in a total volume of 25 µl containing 2.5 µl 10X PCR-buffer, 2.5 µl 2 mm dntp mix, 3 µl oligonucleotide primer (15 ng), 5 µl template DNA (20 ng) and 0.3 µl Taq DNA polymerase (1 U/ml). The final volume was made up to 25 µl using sterile distilled water. The amplification reaction was performed for 45 cycles, each cycle comprising of 3 min at 94ºC (denaturation), 1 min at 37ºC (annealing) and 2 min at 72ºC (extension), with a final extension at 72 C for 10 min. The amplified products were electrophoresed on 1.2 % agarose gel in 1 X TAE buffer, ph 8.0 along with DNA EcoRI/Hind III double digest as molecular weight marker and analyzed using gel documentation system (BIORAD).

8 Table 4.6 Random primers sequence and their annealing temperature Random primer Primer sequences Annealing temperature OPA 1 CAGGCCCTTC 34 C OPA 2 TGCCGAGCTG 34 C OPA 3 AGTCAGCCAC 32 C OPA 4 AATCGGGCTG 32 C OPA 5 AGGGGTCTTG 32 C OPA 6 GGTCCCTGAC 32 C OPA 7 GAAACGGGTG 32 C OPA 8 GTGACGTAGG 32 C OPA 9 GGGTAACGCC 34 C OPA 10 GTGATCGCAG 32 C OPA11 CAATCGCCGT 32 C OPA12 TCGGCGATAG 32 C OPA 13 CAGCACCCAC 34 C OPA 14 TCTGTGCTG 32 C OPA 15 TTCCGAACCC 32 C OPA 16 AGCCAGCGAA 32 C OPA 17 GACCGCTTGT 32 C OPA 18 AGGTGACCGT 32 C OPA 19 CAAACGTCGG 32 C OPA 20 GTTGCGATCC 32 C Analysis of RAPD profiles The amplification profiles for all the primers were compared with each other and the bands of DNA fragments were scored as present (1) or absent (0) generating the 0, 1 matrices. The genetic similarity was estimated by computing Jaccard s co-efficient using Numerical Taxonomy

9 System, Version 2.0 for Windows XP, Vista, & Win7 (NTSYS pc-2.0) software programme (Nei and Li, 1979). The clustering was done and dendrogram were drawn by following Unweighted Pair-Group Method using Arithmetic mean" (UPGMA) routine using the above programme. Other parameters computed were, Percent polymorphism = Total of polymorphic bands * 100 Total number of bands Differentiation power = No. of unique RAPD phenotypes * 100 Total number of phenotypes 4.3 Results and discussion Since morphological and biochemical characterization did not give complete information about bacteria, molecular characterization was done. Bacteria having most efficient phosphate solubilization efficiency were sequenced and characterized Genomic DNA isolation Genomic DNA of 21 bacterial isolates tested positive by plate assay (Chapter 2) were isolated. The extracted DNA was analyzed on agarose gel electrophoresis. A good quality of intact DNA having high molecular weight was obtained and results are presented in Fig M PKN 1 PKU 5 PKS 6 PKU 4 PKS 5 PKN 2

10 M PIN 4 PKU 3 PIN 3 PKN 3 PCN 3 PKS 4 PUS 4 Marker PKU 1 PKN 4 PKS 3 PIU 1 PKU 2 Figure 4.1 Genomic DNA of bacterial isolates. Lane M; DNA size Marker, Lane 2-20 (21 bacterial isolates) Identification of bacterial DNA by 16S rrna PCR amplification The genomic DNA of 21 bacterial isolates was amplified by 16S rrna was run on agarose gel (1 %). The isolates indicated the positive signal in the form of discrete and distinct 1.2 Kbp bands on gel. The PCR products were purified by ethanol precipitation and collected (Fig. 4.2).

11 M PKN 1 PKU 5 PKS 6 PKU 4 PKS 5 PKN 2 PBS 4 PIN 4 PKU 3 PIN M PKN 3 PCN 3 PKS 4 PUS 4 PIN 2 PKU 1 PKN 4 PKS 3 PIU 1 PKU 2 PCN Figure 4.2 PCR amplification of genomic DNA using 16S rrna primers, Lane M DNA size Marker; Lane 2-20 (21 bacterial isolates) Sequencing of bacterial isolates and BLAST results Genomic DNA of six most promising unidentified bacterial isolates were isolated and amplified with 16S rrna universal primers and the amplified product were sent to Amnion Biotech Company for sequencing. The Blast search performed against GenBank ( revealed a large number of similar 16S rrna gene sequences. The blast results of most promising bacterial isolates showed % similarity

12 between available GenBank entries. The sequencing results of most promising bacterial isolates PKU 5, PBS 4, PKN 3, PKS 3, PKS 4 and PCN 6 are shown in Table 4.7 and their DNA sequence alignment (BLAST analysis) results are as follows. Isolate no. PKU 5: Pseudomonas fluorescence (JX ) GTGCTTGCACCTCTTGAGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGG TAGTGGGGGATAACGCTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGA AAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGT TGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATC AGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAA TATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCT TCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTTACCTAATACGTAATTGT TTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAAT ACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTT CGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGTC GAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAG ATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGA GGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAA ACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTA AGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGG GCCCGCACAAGCGGTGGAGCATGTGGTTT Isolate no. PBS 4: Pantoea eucalypti (KC ) AAACGGTGGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCC TCTCACTATCGGATGAACCCAGATGGGATTAGCTAGTAGGCGGGGTAATGGCCCAC CTAGGCGACAATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGA CACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAA GCCTGATGCACCCATGCCGCGTGTATGAAAAAAGCCTTCGGGTTGTAAAGTACTTTC AGCGGGGAGGAAGGCGGTGAGGTTAATAACCTTACCGATTGACGTTACCCGCAGAA AAAACACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTT AATCGGAATTACTGGGCGTAAAGCGCACGCACGCGGTCTGTTAAGTCACATGTGAA ATCCCCGGGCTTAACCTGGGAACTGCATTTGAAACTGGCAGGCTTGAGTCTTGTAGA GGGGGGTATAATTCCACGTGTATCGGTGAAATGCGTATAGATCTGGAGGAATACCC GTGGCGAAAGCGGCCCCCTGGACAAAGAC Isolate no. PKN 3: Bacillus thuringiensis (NR ) TGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGG TGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGG CTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCT GTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGTAACGGCTCACCAA GGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACAC

13 GGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTC TGACGGAGCAACCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTA GGGAAGAACAAGTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCC ACGGCTAACTACGGAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAAT TATTGGGCGTAAGCCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAA CCGTGGAGGTCATGGAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCA TGTGTAGCGGTGAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTT CTGGTCTGTAATGACACTGAGGCGCGA Isolate no. PKS 4: Bacillus cereus (KC ) ACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACC GGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTA TGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAAC GATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGG AGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGA AGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCA CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGA ATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACG GCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGT GGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGA AGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACA GGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAG Isolate no. PKS 3: Staphylococcus succinus (NR ) TGCAAGTCGAGCGAACGGATAAGGAGCTTGCTCCTTTGAAGTTAGCGGCGGACGGG TGAGTAACACGTGGGTAACCTACCTATAAGACTGGAATAACTTCGGGAAACCGGAG CTAATGCCGGATAACATATAGAACCGCATTTCTATAGTGAAAGATGGTTTTGCTATC ACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGG CGACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGG TCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTG ACGGAGCACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTATTAGG GAAGAACAAATCGTAAGTAACTGTGCGCATCTTGACGGTACCTAATCAGAAAGCCA CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGA ATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACG GCTCAACCGTGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAAAGT GGAATTCCATGTGTAGCGGTGAAATGGCAGAGATATGGAGGAACACCAGTGGCGAA GGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAG GATTAGATACCCTGGTAGTCCAC Isolate no. PCN 6: Pseudomonas fragi (JX )

14 ACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTT GCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAA GGCTACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACAC GGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCC TGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGT TGGGAGGAAGGGCATTAACCTAATACGTTGGTGTCTTGACGTTACCGACAGAATAA GCACCGGCTAACTCTGTGCCAGCACCCGCGGTAATACAGAGGGTGCAAGCGTTAAT CGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGAATGTGAAATCC CCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAAAGTATGGTAGAGGG TAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTG GCGAAGGCGACTACCTGG The sequencing results of bacterial isolates are given in Table 4.7. Table 4.7 Identification of six most promising bacterial isolates based on their sequencing results and BLAST analysis No. of strains Bacterial strains Sequenced results 1 PKU 5 2 PBS 4 3 PKN 3 4 PKS 4 Pseudomonas fluorescence (JX ) Pseudomonas fragi (JX ) Bacillus thuringiensis (NR ) Bacillus cereus (KC ) % Similarity 98% 99 % 99 % 95 % 5 PKS 3 Staphylococcus succinus (NR ) 100 % 6 PCN 6 Pantoea eucalypti (KC ) 99 % Six amplified products using 16S rrna primers were given for sequencing and identified as Pseudomonas fragi, Staphylococcus succinus, Bacillus cereus, Bacillus thuringiensis, Pantoea eucalypti and Pseudomonas fluorescence Amplification of DNA from the six most promising bacterial isolates using random primers

15 The results of the present study suggest that RAPD is a useful technique for studying variation among microbial communities. RAPD analysis was done to find out the molecular diversity amongst the different bacterial isolates. The total genomic DNA of all the strains were isolated from the six most promising strains having high phosphate solubilization efficiencies which includes PKU 5, PCN 6, PKS 3, PKS 4, PKN 3 and PBS 4 and used as template for RAPD analysis with 20 different decamer primers obtained from Operon Technologies (OPA primers). Out of 20 OPA primers, 12 were found as positive primers. Bands obtained on the agarose gel with 12 positive OPA primers were documented through Gel documentation system (Fig. 4.3, Fig. 4.5, Fig. 4.7, Fig. 4.9, Fig. 4.11, Fig. 4.13, Fig. 4.15, Fig. 4.17, Fig. 4.19, Fig. 4.21, Fig. 4.23, Fig ). The amplification profiles of all the primers were compared with each other and bands on the gels were scored for presence or absence of band. The results presented here suggest that, with careful standardization of reagents and amplification conditions, the impact of variation among individual PCR reactions on overall community profiles is negligible. The RAPD patterns of each isolate was evaluated, assigning character state 1 to indicate the presence of band in the gel and 0 for its absence in the gel. Thus, a data matrix was created which was used to calculate the Jaccard s similarity coefficient for each pairwise comparison using SIMQUAL programme. Jaccard s coefficient were clustered to generate dendrogram (Fig 4.4, Fig. 4.6, Fig. 4.8, Fig. 4.10, Fig. 4.12, Fig. 4.14, Fig. 4.16, Fig. 4.18, Fig. 4.20, Fig. 4.22, Fig. 4.24) using the SHAN clustering programme, selecting UPGMA algorithm with NTSYSpc, version 2.0 (Table 4.8).

16 M Figure 4.3 Agarose gel showing random amplified segments by using OPA 3 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.4 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 3 primer. M

17 Figure 4.5 Agarose gel showing random amplified segments by using OPA 7 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.6 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 7 primer. M

18 Figure 4.7 Agarose gel showing random amplified segments by using OPA 9 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.8 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 9 primer M

19 Figure 4.9 Agarose gel showing random amplified segments by using OPA 11 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.10 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 11 primer.

20 M Figure 4.11 Agarose gel showing random amplified segments by using OPA 13 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.12 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 13 primer.

21 M Figure 4.13 Agarose gel showing random amplified segments by using OPA 14 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.14 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 14 primer.

22 M Figure 4.15 Agarose gel showing random amplified segments by using OPA 15 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.16 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 15 primer.

23 M Figure 4.17 Agarose gel showing random amplified segments by using OPA 16 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.18 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 16 primer.

24 M Figure 4.19 Agarose gel showing random amplified segments by using OPA 17 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.20 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 17 primer.

25 M Figure 4.21 Agarose gel showing random amplified segments by using OPA 18 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.22 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 18 primer.

26 M 6 Figure 4.23 Figure 4.5 Agarose gel showing random amplified segments by using OPA 19 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.24 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 19 primer.

27 M Figure 4.25 Agarose gel showing random amplified segments by using OPA 20 primer. Lane M-Marker, Lane 1: Isolate No. PKU 5; Pseudomonas fluorescence, Lane 2: Isolate No. PCN 6; Pseudomonas fragi, Lane 3: Isolate No. PKS 3; Staphylococcus succinus, Lane 4: Isolate No PKS 4; Bacillus cereus, Lane 5: Isolate No. PKN 3; Bacillus thuringiensis, Lane 6: Isolate No. PBS 4; Pantoea eucalypti Figure 4.26 Combined UPGMA dendrogram showing relationship among 6 bacterial isolates using OPA 20.

28 Table 4.8 Polymorphic ratio of bacterial strains with different OPA primers Primers Sequence detail No. of amplified bands No. of polymorphic bands Polymorphic ratio (%) OPA 03 AGTCAGCCAC OPA 07 GAAACGGGTG OPA 09 GGGTAACGCC OPA 11 CAATCGCCGT OPA 13 CAGCACCCAC OPA 14 TCTGTGCTGG OPA 15 TTCCGAACCC OPA 16 AGCCAGCGAA OPA 17 GACCGCTTGT OPA 18 AGGTGACCGT OPA 19 CAAACGTCGG OPA 20 GTTGCGATCC Average Total The RAPD analysis of all the identified six isolates was done by using different OPA primers, which helped in finding the genetic diversity amongst known bacterial isolates. The amplification profiles of all the primers were compared with each other by bands on the agarose gels. All bacterial isolates exhibited different genetic pattern. All the amplified products with the random primers had shown polymorphic and distinguishable banding patterns indicating the genetic diversity.. The genetic similarity was estimated by computing Jaccard s co-efficient using NTSYS pc-2.0 software programme. The clustering was done and dendrogram were drawn

29 by following UPGMA routine. The dendrogram indicated the existence of a great genetic diversity between known bacterial isolates. Analysis of the group or the clustering indicated significant connections between the taxonomically related isolates by establishing similarity and difference among all the six identified PSBs using different OPA primers. On the basis of highest banding pattern (OPA 11), one cluster included the individuals from P. fluorescence to P. eucalypti and the other cluster included the individual S. succinus, B. cereus and B. thuringiensis. The cluster formed by the individuals, P. fluorescence to P. eucalypti was divided in two subgroups, one contained individuals, P. fluorescence and P. fragi, and the other included P. eucalypti while the other cluster contained B. cereus and B. thuringiensis and on the other side was S. succinus. The results obtained after the dendrogram analysis indicated the fact that the genetic similarity of the individuals belonging to the analyzed families is variable, depending on the geographic origin. Among all, highest 10 reproducible and scorable polymorphic bands were generated with primers OPA 11. Similarity range was found 0.1 to 0.5 among all the bacterial isolates and the highest similarity coefficient was found between B. cereus and B. thuringiensis. Thus OPA 11, OPA 3 and OPA 7 has given a large genetic diversity while less number of bands were generated with primers OPA 15 and OPA 20, thus, showing very less genetic diversity among the known bacteria. 4.4 Conclusion In the present study, bacterial isolates were analyzed by isolation of genomic DNA, amplification of bacterial DNA by using 16S rrna universal primers. Six amplified products of bacteria having high efficiency for phosphate solubilization were given for sequencing and identified as Pseudomonas fragi, Staphylococcus succinus, Bacillus cereus, Bacillus thuringiensis, Pantoea eucalypti and Pseudomonas fluorescence. To check the reproducible polymorphism among bacterial isolates, RAPD analysis was done using different OPA primers. All amplified products in RAPD analysis had shown polymorphic and discriminated banding patterns on agarose gel that indicated the genetic diversity. It revealed the polymorphism and specific identification of bacterial isolates. Total 96 reproducible DNA fragments were scored for generation of dendrogram showing a wide range of diversity within the bacterial isolates with % genetic similarities whereas 126 polymorphic bands were scored and the average polymorphic ratio was 73.36%. The investigation confirmed

30 the higher level of genetic variability among studied PSBs, pointing out the dissimilarity between these isolates obtained from different regions of Lucknow.