INTRODUCTION:

Actinomycetes are prokaryotic spore forming, gram positive bacteria that have DNA with high GC content (69 – 73%). Their filamentous nature, branching pattern and conidia formation are similar to those of fungi for the reason they are known as ray fungi. The spore size and spore characters are similar to bacteria. They are widely distributed in terrestrial soil, cultivable lands, marine soils, compost, fresh water basins and foodstuffs and beneficial effects should be manipulated according to our use. The name actinomycetes were derived from Greek akits ‘a ray’ and ‘mykes’ (fungus). Like fungi they form a mycelial new work of known as branching filamentous, conidia formation and are known as ‘ray fungi’. But like bacteria they have prokaryotic nuclei and are susceptible to antibacterial antibiotics. They are characterized by the formation of aerial and substrate mycelium.

Several antibiotics have been isolated from marine actinomycetes by many researchers. The antibiotics are entirely new and unique when compared to those from the terrestrial ones. Streptomyces, have been studied as potential procedures of metabolites with diverse chemical structures and biological activities. Tens of thousands of such compounds have been isolated and characterized many of which have been developed into drugs by treatment of wide range of human disease. Thus searching for novel actinomycetes constitutes on \essential component in natural product based drug discovery (Wang et al., 1991).

Phosphate Solubilizers:

Microbes which solubilize the bound phosphate and rock phosphate into simple phosphate are called phosphate solubilizers. They secrete organic acids such as formic acid, lactic acid, succinic acid, propionic acid, hydroxyl acid to solubilize the bound phosphate in the soil. The simple phosphates are readily available for the plants.The most essential function of phosphate in plants is in energy storage and transfer of adenosine di and tri phosphate(ADP and ATP) act as energy currency within the plants.

MATERIALS AND METHODS:

Sample collection:

The soil sample was collected from Manora at Thanjavur Dt., Tamil Nadu, India. Then it is brought to the laboratory in a sterile manner, which was then thoroughly mixed and dried.

Analysis of Physico-chemical characteristics of the soil:

Moisture content was estimated by finding the weight difference of known quantity of the soil before and after drying in a hot air oven at 60°C for 6 hours. Soil samples after removing the debris were suspended in distilled water (1:2 w/v) and allowed to settle down the sand particles. The pH of the suspension was read using pH meter (Systronics, India), to find out the soil pH.

Electrical conductivity of soil was determined in the filterate of the water extract using Conductivity bridge as described by Jackson (1973), cation exchange capacity (CEC) of the soil was determined by using 1N ammonium acetate solution as described by Jackson (1973).

Organic carbon content was determined by adopting chronic acid-wet digestion method as described by Walkley and Black (1934), available nitrogen was estimated by alkaline permanganate method as described by Subbiah Asija (1956) and available phorphorus by Braylk method as described by Bray and Kutz (1945). Available potassium was extracted from soil with neutral 1N ammonium acetate (1:5) and the potassium content in the extract was determined by using flame photometer (Standfold and English, 1949), Calcium (neutral 1 N NH₄ OAC extractable 1:5) was extracted with neutral 1N ammonium acetate and the available calcium in the extract was determined by Versenate method (Jackson, 19973). Available micronutrients such as Zn, Cu and Mn were determined in the diethylene triamine penta acetic extract of soil using Perklin-Elmer model 2280 Atomic absorption spectrophotometer (Lindsay and Norwell, 1978). Other nutrients such as magnesium, sodium and available iron were analysed following the method of Barnes (1959) and Muthuvel and Udayasoorian (1999).

Isolation of actinomycetes:

Isolation of actinomycetes was performed by plating technique using Starch Casein agar(Kuster and Williams.,1964) medium. The medium was prepared and sterilized at 121°C in 15Ibs pressure for 15 minutes. Then it was supplemented with Streptomycin (30µg) and griseoflavin (50 µg) to prevent the bacterial and fungal growth in the medium. The medium was poured into sterile petriplates. The collected soil Samples were diluted upto 10^-6and 0.1ml of the dilution sample were spreaded over the agar plates. The inoculated plates were incubated at 28±2°C for 7-10 days. After incubation actinomycetes colonies were observed on the medium and these colonies were used for the further investigation.

Purification of actinomycetes:

Streak plate method was used to purify the culture of actinomycetes. After inoculation plates were incubated at 28±2°C for 7-10 days. The starch Casein agar was used to purified the actinomycetes and stored at 4°C for further investigation.

Table 1: Soil samples analytical report

S. No	Name of the parameter	Analytical values
1.	pH	7.56
2.	Electrical conductivity (dsm^-1)	0.26
3.	Organic carbon (%)	0.29
4.	Organic matter (%)	0.58
5.	Available Nitrogen (Kg/ac)	89.6
6.	Available Phosphorus (Kg/ac)	5.26
7.	Available Potassium (Kg/ac)	175
8.	Available Zin (ppm)	0.87
9.	Available Copper (ppm)	0.56
10.	Available Iron (ppm)	4.69
11.	Available Manganese (ppm)	2.45
12.	Cat ion exchange capacity (c. Mole. Proton^- /kg)	22.6
Exchangeable bases (c. Mole. Proton^- /kg)
13.	Calcium	12.4
14.	Magnesium	10.6
15	Sodium	1.69
16	Potassium	0.19

Characterization of actinomycetes:

Colony characterization:

Colony morphology was recorded with respect to colour aerial mycelium, size and nature of colony, reverse side and pigmentation.

Microscopic characterization:

Actinomycetes culture plates were inserted at an angle of 45°. The actinomycetes was slowly released at the intersection of medium to the cover slip. The plates were incubated at 28±2°C for 4-8 days. The cover slips were removed from the cultured medium and observed under the microscope. The photomicroscopy was taken using Nikon microscope. The morphological features of spores, sporangia, aerial and substrate mycelium was observed and recorded among the isolates, predominant organisms were selected for further studies.

Identification of actinomycetes:

The isolated actinomycetes were identified by using the gram’s staining, motility, and bio- chemical tests by using standard manuals.

Screening of phosphate solubilizing Actinomycetes:

The identified 10 Actinomycetes cultures were spot inoculated on Pikovskay’s medium (pH 6.8 – 7.8) containing 5 gm of tricalcium phosphate as sole phosphorus source for selectively screening the actinomycetes, which have the ability to release inorganic phosphate from tricalcium phosphate. The inoculated Pikovskay’s plates were incubated at 28 ±2°C for 4 to 5 days. After the completion of the incubation period, the plates were observed for the presence of clear zone around the colonies the transport zone around the microbial colonies indicate the extent of phosphate Solubilization (Sandara Rao and Sinha et al., 1963).

Table 2: Biochemical characterization of actinomycetes

S. No	Microorganisms	Test
S. No	Microorganisms	GS	I	MR	VP	Citrate	Catalase	Urease	Nitrate
1	Actinobispora yunnansis	+	-	+	-	+	+	+	-
2	Actinomadura citrea	+	+	+	-	+	-	+	+
3	Microtetrospora fastidiosa	+	+	+	+	+	-	+	+
4	Micromonospora echinospora	+	-	-	-	+	-	+	+
5	Saccharomonospora viridis	+	+	+	-	+	+	+	+
6	Saccharopolyspora hirsute	+	+	-	-	+	-	+	+
7	Streptomyces albus	+	+	-	-	+	-	+	+
8	Streptoverticillium album	+	-	-	-	+	+	-	-
9	Streptomyces albus	+	+	-	-	+	+	+	+
10	Streptomyces cyaneus	+	+	-	-	+	+	+	+

GS-Gram staining, I-Indole, MR-Methyl red, VP-Voges proskauer

(+) Positive; (-) Negative

Molecular characterization of Actinobispora ynnanensis

Isolation of chromosomal DNA (Wilson, 1990):

Isolates of Actinobispora ynnanensis were grown upto the late exponential phase in starch casein broth at 28 ±2°C, washed twice with Tris EDTA buffer or 10.3% sucrose prior to DNA preparation chromosomal DNA was isolated by resuspending 0.5 – 1.09 of cells with 15 ml lysis buffer (25mM Tris: 25mM EDTA, pH-8.0: 10-15mg lysozyme, 50µg/ml RNAse) and incubated for 30 – 80 min at 37°C followed by addition 500µl of 5M NaCl solution. The suspension was agitated on a vortex mixer until the cell suspension became translucent. Cells were lysed by the addition of 1.2ml of 10% SDS. They lysates of were incubated for 15 – 30 min at 65°C. After addition of 2.4 ml of 5M potassium acetate, the solution was mixed and left on ice for 20 min. The precipitate was removed by centrifugation for 30 min. at 6000 rpm and the volume of supernatant was adjusted to 8 ml. The DNA was recovered by precipitation was dissolved in 700 µl/g of 50mM Tris /10mM EDTA (pH 8.0). Any insoluble substances were spun off and the aqueous phase was transferred to a 1.5ml microfuge tube. Subsequently, 75µl 3M sodium acetate and 500µl isopropanol were added and the solution was centrifuged for 30 seconds to 2 min. The precipitate was washed with cold 70% ethanol, dried and dissolved in 100 µl TE (10mM tris/ 1mM EDTA pH 8.0).

PCR amplification of 16S rDNA (Weisburg et al., 1991):

A mixture of sterile deionized water 49 ml upstream primer (100 pmols) 10µl (5^/ AGAGTTGATCCTGGCTCAG 3’). Down stream primer (100pmds) 10µl (5’ – AGGGCTACTACCTTGTTACGACTT-3’) 10X PCR buffer 25mM, Mgcl₂ 8µl, dNTP mix 6 µl, Streptomyces template DNA (50 nq), 5 µl and Taq DNA polymerase (30/ µl), 2 µl in a 0.5 ml micro centrifuge tube was taken. The total 100 µl mixture in a tube was gently span for 10 sec and allowed to settle the contents. The samples were kept in eppendrof PCR Thermal cycler. The amplification was carried out in the following manner of 35 cycles, denaturation for 60 sec at 92°C, primer annealing for 60 seconds at 54°C and polymerization for 90 sec at 72°C. Finally the tubes were ensured complete polymerization at 72°C for 15 min. 10µl of PCR products with 2 µl of loading dye was mixed and loaded on a 1.2% agarose gel and analysed electrophoretically at 60 volts for 45 min. The gel was observed on UV transilluminator and compared with 1kb DNA ladder.

16S rDNA sequencing:

The PCR products were purified using micron PCR centrigual filter device (Millipore crop. Bedford Hass) and sequenced using the facility at macrogene (Seoul, Korea).

Nucleotide sequence accession:

The 16s rDNA sequence for the Actinobispora ynnanensis have been deposited in Gene Bank http://www.nebi.nlm.nih.90v/gene bank

The reference sequences required for comparison were down loaded from the EMBL database using the site http://www.nebi.nlm.nih.90v/gene bank. All the sequence were aligned using the multiple sequence alignment program CLUSTAL X . The aligned sequences were then checked for gaps manually and arranged in a block of 250bp in each row and saved as an format in software MEGA v 2.1- the pairwise evolutionary distances were computed using the Kimura(1980). To obtain the confidence values the original data set was resampled 1,000 times using Boot strap. The program of PHYLOGENY and subjected to bootstrap analysis. The bootstrap analysis. The bootstrap data set was used directly for constructing the phylogenetic tree using the MEGA program or used for calculating the multiple distance matrix obtained was then used to construct phylogenetic trees using Neighbour-joining method of Saitou and Nei (1987). All these analysis were performed using the MEGA v2-1 (Kumar et al., 2007).

16S rDNA Secondary structure prediction of Actinobispora ynnanensis:

The secondary structure of (Actinobispora ynnanensis) was predicted using the bioinformatics tools available online www.genebee.msu.sul services/rna2 reduced- html.

Restriction site analysis in 16S rDNA of Actinobispora ynnanensis:

The restriction sites in 16S rDNA of Actinobispora ynnanensis were analysed using NEB culture programme version 2.0 tools in online www.neb.com/NEB cutles 2/index.php.

Table 3: Phosphate solubilization of actinomycetes

S. No	Name of the organisms	Zone of clearance (mm in diameter)
1.	Actinobispora yunnanensis.	32
2.	Actinomodura citrea	--
3	Microtetrospora fastidiosa	_
4.	Micromonospora echinospora.	18
5.	Sacchromonospora viridis	15
6.	Saccharopolyspora hirsuta	12
7.	Streptomyces albus	-
8.	Streptoverticillium album	12
9.	Streptomyces cyaneus	-
10	Thermonospora mesophila	12.5

RESULTS AND DISCUSSION:

Physico-chemical analysis of the soil sample:

Physically, the texture of the soil sample was sandy loam. The physico-chemical parameters such as pH (7.56), Electrical conductivity (0.26 dsm^-1), organic carbon (0.29%), organic matter (0.58%), Available nitrogen (89.6 kg/ac), available phosphorus (5.26 kg/ac), available potassium (175 kg/ac), available zinc (0.87ppm), available copper (0.56 ppm), available iron (4.69 ppm), available manganese (2.45 ppm), cation exchange capacity (22.6 C. mole proton ⁺ kg), calcium (12.4), magnesium (10.6), sodium (1.69), potassium, (0.19) were recorded from the soil sample of coastal area Manora, Thanjavur (Dt).

Isolation of Actinomycetes from marine soil sample:

By direct plating 10 species of actinomycetes were isolated from marine soil sample. The isolated actinomycetes were Actinobispora yunnanensis, Actinomodura citrea, Microtetrospora astidiosa, Micromonospora echinospora, Sacchromonospora viridis, Saccharopolyspora hirsute, Streptomyces albus, Streptoverticillium album, Streptomyces cyaneus, Thermonospora mesophila.

Screening of phosphate solubilizing Actinomycetes:

Detection of the phosphate solubilizing ability of microorganisms have been possible using plate-screening methods (Pikovskaya, 1948). Phosphate solubilizing microorganisms produce clearing zones around the microbial colonies in the Pikovskaya’s medium, a specific medium for the isolation of phosphate solubilizers.

The production of clearance zones is due to the microbes ability to solubilize the insoluble mineral phosphates such as tricalcium phosphate or hydroxyapatite present in the medium. Among the screened 10 species of actinomycetes, Saccharomonospora hirsute and Streptomyces, cyaneus, Actinomadura citrea, Microtetrospora fastidiosa have no phosphate solubilizing activity.

The maximum zone of clearance was found with the Actinobispora yunnanensis (32 mm) compared to that of the Micromonospsora echinospora (18 mm), Sacchraromonospora viridis Saccharopolyspora hirsute, (12mm), Thermonospora mesophila (12.5mm) and streptoverticillium album,(12mm). These results coincide with that of Ayyakkannu and Chandramohan, 1970a, who reported about the occurrence and distribution of phosphate solubilizing micro organisms in the natural marine environment. They also stated that, among the different microbes, bacteria are the potential candidates for dissolving the insoluble organic and inorganic phosphorus compounds.

Molecular characterization of Actinobispora yunnanensis:

The molecular charecterization of Actinobispora yunnanensis was evaluated by PCR amplification of 16S r DNA gene .the genomic DNA and amplified produts were separated in agarose gel.The 16S r DNA genes of Actinobispora yunnanensis from the marine soil was partially sequenced using 16 S r DNA sequence primer(3’TGC CAG CGG CGG TAA TAA 5’- forward primer and 5’ CCG CCG ACG ACG TCT TTA 3’ reverse primer).The sequence of Actinobispora yunnanensis 16 S r DNA was deposited in NCBI to get the accession number. The sequence comparisons with sequences in the EMBL data base, the phylogenetic analysis (neighbour joining tree) revealed that the sequence of the marine isolate is Actinobispora yunnanensis similar (98%) to the existing uncultured Actinobispora yunnanensis ncd90h09c1 and it has a lesser percentage of similarity with and Actinobispora yunnanensis sp.J31 strain.

The secondary structure of 16S r DNA of Actinobispora yunnanensis showed 33 loops, 33 stems and 24 hairpin loops(Fig- 4).The restriction sites found in B.epidermidis was shown in figure 1 to 3.Totally 155 restriction enzyme sites were observed. The GC and AT content of Actinobispora yunnanensis was found to be 56% and 44% respectively, using NEB cutter programmeV2.0 in WWW.neb.com/neb cutter 2/ index.php.

REFERENCES:

1. Goldstein, A.H., 1995. Recent progress in understanding the molecular genetics and biochemistry of calcium phosphate Solubilization by Gram Negative bacteria. Biol. Agric. Hort., 12: 18-26.

2. Kathiresan, K. and Duraiswamy, A., 2006. ENVIS News letter, Department of Zoology, University of Madras., 4: 3-5.

3. Kim, K.Y., Jordan, D., Krishnan, H.B., 1997. Rahnella aqualitis a bacterium isolated from soybean rhizosphere can solublize hydroxyapatite. FEMS Microbiol. Lett., 153: 273-277

4. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111 – 120.

5. O’ Donnel,A.G., Embley,T.M. and Good fellow, M., 1993. Future of bacterial systematics.In: Handbook of new bacterial systematics, London, academic press,pp:513-524.

6. Sci. 2:137-143 Seidman.J.G. and Struhi, K., eds. Chapter 2-4, Greene publishing and Wiley inter science, New Yark.

7. Sundara Rao, W.V.B. and Sinha, M.K., 1963. Phosphate dissolving organisms in the soil and rhizosphere. Ind. J. Agric. Sci., 33: 272-278.

8. Wang, Y., Zhang, Z.S., Ruar, T.S., Wang, Y.M. and Ali, S.M., 1999. Investigation of actinomycetes diversity in the tropical rainforests of Singapore. J. Ind. Microbiol. Biotechnol. 23: 178-187.

9. Weishburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J., 1991. 16S rDNA amplification for phylogenetic study. J. Bacteriol. : 173: 697-703

10. Wilson, K., 1990. Preparation of genomic DNA from bacteria. In current protocol in Smith J.A.,

11. Yokota,T.,1997.The structure, biosynthesis and function of brassinosteroids.Trends. plant.

Received on 11.10.2011 Accepted on 20.11.2011

Asian J. Pharm. Tech. 1(4): Oct. - Dec. 2011; Page 119-122