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INTRODUCTION

Nitrogen fixation is the reduction of N2 (atmospheric nitrogen) to NH3 (ammonia).Free-living prokaryotes with the ability to fix atmospheric dinitrogen (diazotrophs) are ubiquitous in soil, but our knowledge of their ecological importance and their diversity remains incomplete. Nitrogen fixation-related genes have been highly conserved throughout evolution even though they are widely distributed among eubacteria and archaea. The great diversity of diazotrophs also extends to their physiological characteristics, as N fixation is performed by chemotrophs and phototrophs and by autotrophs as well as heterotrophs (Burgmann et al., 2003).

However, evidence has been accumulating which documents the importance of bacterial N2 fixations in many and diverse marine habitats. Brook’s et al provided the first strong evidence of in situ bacterial N2 fixation occurring in estuarine sediments. Since that time, the presence of heterotrophic N2 fixation in the absence of light has been demonstrated for the variety of marine and estuarine sediments (Guerinot et al., 1985). The capacity for N2 fixation in natural populations of marine eubacteria is beginning to be evaluated using molecular and immunological techniques. Most species are free-living with some forming a major portion of the surface plankton; others are symbiotic with teleost fishes and squid or are pathogenic in fish and shellfish (Coyer et al., 1995). A number of free-living soil bacteria, e.g. bacteria of the genera Azotobacter (aerobic), Clostridium (strictly anaerobic), Klebsiella (optionally aerobic), and Rhodospirillum (anaerobic, photosynthetically active) belong to the nitrogen reducing species (Kennedy, 2004)

Azotobacter sp is a large, obligately aerobic soil bacterium which has one of the highest respiratory rates known among living organisms and is able to grow on a wide variety of carbohydrates, alcohols and organic acids, in addition to be able to fix nitrogen. The biological nitrogen fixation reaction is catalyzed by a complex metalloenzyme called nitrogenase. Nitrogenase is composed of two separately purified proteins, both of which are extremely oxygen sensitive. The larger of the two proteins, designated the MoFe protein, has a molecular mass of 230,000 Da. The MoFe protein is a tetramer in its biologically active form and is composed of two identical halves, each containing an ?-subunit and a ?-subunit encoded by the NifD and nifK genes, respectively. The smaller of the two proteins, designated the Fe protein, has a molecular mass of about 60,000 Da and is a dimer of identical subunits encoded by the NifH gene. Besides the structural genes of nitrogenase, there are a number of nif-specific genes (20 identified to date) that comprise the nif regulon (Lei et al., 1999).

Methods for studying the community structure of N2-fixing bacteria in the rhizosphere usually require enrichments of culturable bacteria, and functional contributions are often assessed using the acetylene reduction assay (ARA) of cultured isolates or soil cores. These methods provide incomplete information because: i. only a small fraction of viable cells is represented by culture-based methods, ii. N2-fixing bacteria that inhabit living root cells or tissues are not identified, iii. There are technical problems associated with the use of an indirect assay (ARA) for nitrogenase activity, and iv. The relative functional in situ contributions of individuals in diverse populations are not assessed. Molecular methodology facilitating the characterization of the N2 -fixing bacterial community structure, when used with ARA and selective culturing methods, may provide a more thorough analysis of N2-fixing bacterial population dynamics in the rhizosphere. (Lepo et al.,1983)

MATERIALS AND METHOD

Sample collection and transport

Samples were collected from different locations of Tondi which comes under Rameshwaram marine region at the depth of 1–3 m. The samples were collected in the sterile plastic bags (soil sample) and water sampling bottles (water sample). The randomly collected samples were kept in an ice-cold box and transported safely to the lab for further analysis with in 12 hrs.

Processing and screening of Azotobacter sp from marine soil and water samples (Kannan, 2002).

The randomly collected samples were processed for isolation of Azotobacter sp exclusively by using various techniques like serial dilution followed by spread plating, pour plating on differential media like Jensen’s Agar Medium (with 3.5% NaCl), Azotobacter Agar Medium (with 3.5% NaCl), Burk’s Medium (with 3.5% NaCl), Marine agar medium.

Characterization of Azotobacter sp (Bagwell et al., 1988)

Gram-staining characteristics and cell morphologies were determined by standard methods (Gerhardt et al., 1981). Motility was observed in wet mount using phase contrast microscope. Preliminary physiological characterization such as catalase test, starch hydrolysis test, oxidase test, gelatin hydrolysis test were also carried out.

Extraction and purification of DNA (Kelly et al ., 1990)

Azotobacter genomic DNA was isolated as previously described (Robson et al., 1980). Pure culture of isolated and characterized marine Azotobacter Strains (402,405,415,420,426,433,456,462,469,480,501 and 520) was selected based on their growth characteristic on selective media for the nucleic acid extraction and purification. The reference (standard) cultures such as Azotobacter sp (2632), Azotobacter chroococcum (2452), and Azotobacter lactinogens (2633) procured from MTCC, Chandigarh, were also used for the nucleic acid extraction and purification. The purity of the DNA was checked spectrophotometric method by using the formula OD at 260 nm/ OD at 280 nm (Wilfinger et al., 1997). If the estimated value is 1.8 conforms the presence of pure DNA. If the estimated value is lesser /greater than 1.8 conforms the presence of DNA to protein / RNA contamination, according to respective values DNA was purified using the enzymes proteases and RNAase.

PCR amplification of the NifH gene fragment (Burlage et al., 1998).

Nitrogenase Fe protein genes (NifH) were amplified from Azotobacter sp derived genomic DNA, using the primer from OPERON diagnostic Ltd, USA. The samples were amplified by PCR in a mixture containing reaction buffer 5.0 µl, 10mM dNTP 1.0 µl, primer 1 (25 mer) 1.0 µl, primer 2 (24 mer) 1.0 µl, template DNA 1.0 µl, enzyme Taq polymerase 0.5 µl for 40 cycles (0.5 min at 94° C, 1 min at 54° C and 0.5 min at 72° C) (Zehr et al., 1988).

One microlitre of DNA was used as template in PCR study. Selected primers from A.vinelandi (M2568),

Primer 1 5-GCIWTYTAYGGIAARGGIGG-3,

Primer 2 5-AAICCRCCRCAIACIACRTC-3

respectively were used to amplify a 390-bp region of nif H gene. Where I represents inosine, R represents A or G, W represents A or T and Y represents C or T.

RESULTS AND DISCUSSION

Totally 120 samples were collected from Tondi marine region of both water and sediments at the intervals of approximately 30 days. The randomly collected samples after subjecting to further processing on selective medium, the colony morphology of Azotobacter strains were varying. The colonies were very clear, large, mucoid, watery due drops. Pure culture of Azotobacter shows rectangular or oval gram negative rods on gram’s staining and positive results for catalase, oxidase, starch hydrolysis, gelatin hydrolysis test which are useful for identification of Azotobacter sp.

The DNA of the selected strains was isolated by lysozyme treatment and estimated OD at 260 nm, the value ranges from 0.142 to 0.189. The estimated value of the extracted DNA was ranging from 0.710 to 0.945. The presence and purity of DNA was checked by OD at 260nm/ OD at 280 nm, the value ranges from 1.19 to 2.19. According to respective values DNA was purified using the enzymes proteases and RNAase. The purified form of DNA was then subjected to agarose gel electrophoresis for the separation using 0.8% agarose gel. The agarose gel after electrophoresis was taken out and placed on the UV transilluminator window / UV gel documentation, it was hence concluded that there were no substained difference between the banding pattern of the chromosomal DNA of different test isolates including marine and standard MTCC strains of Azotobacter sp on the agarose gel electrophoresis.

The results of the PCR products were compared on 2% agarose gel electrophoresis. Selective NifH primer from A.vinelandi (M2568), was used for the amplification of the Azotobacter sp, the primers used in my study was exactly matching the Azotobacter genome. Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the NifH marker gene have been successfully applied to describe diazotroph populations in the marine environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all NifH genes (Bürgmann et al., 2003).

REFERENCE

1. BAGWELL, C.E., PICENO, Y.M., LUCAS, A.M. AND LOVELL, C.R., 1988. Physiological diversity of the rhizosphere diazotroph assemblages of selected salt marsh grasses. Appl. Environ. Microbiol., 64(11): 4276-4282

2. BROOKS, R.H., P.L. Brezonik H.D. Putnam and H.A. Keirn , 1971 Nitrogen fixation in an estuarine environments, the Waccasassa on the Florida Gulf coast, Limnol. Oceanogr 16 : 701-710.

3. BÜRGMANN H, FRANCO WIDMER, WILLIAM VON SIGLER, AND JOSEF ZEYER. New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in soil. Environmental Microbiology, Vol.70, No1 p.240 – 247.2003.

4. COYER J A. T, PASINIT A M, SWIFTS H. N2 fixation in marine heterotrophic bacteria: Dynamics of environmental and molecular regulation. Department of Molecular Genetics and Cell Biology, University of Chicago,Contributed by Hewson Swift, December 6, 1995

5. GUERINOT, M. L., AND PATRIQUIN D. G. 1981. The association of N2-fixing bacteria with sea urchins. Mar . Biol. Vol 62: 197 – 207

6. LEI S, PULAKAT L, AND GAVINI N. Genetic Analysis of nif Regulatory Genes by Utilizing the Yeast Two-Hybrid System Detected Formation of a nifL nif A Complex That Is Implicated in Regulated Expression of nif Genes. American Society for Microbiology 181(20): 6535–6539.

7. KENNEDY C, RUDNICK P, MACDONALD M, MELTON T.Genus Azotobacter in Garrity et al (eds) Bergey’s Manual of Systematic Bacteriology. 2004.

8. KELLEY, T.S., AND EISENSTARK, A. The genus Azotobacter. Bull. Oklahoma Agr. Mech. Coll., 48, no. 16. 1990

9. LEPO J E, CHELIUS M K AND WEBER D E. Characterization of nitrogen-fixing bacterial rhizosphere communities using restriction fragment length polymorphisms of pcr amplified nifH. Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, Florida, 32514. 1983.

10. ROBSON Y. IGARASHI AND LANCE C. Nitrogen Fixation: The Mechanism of the Mo-Dependent Nitrogenase Critical Reviews in Biochemistry and Molecular Biology, 38:351–384 .1980.

11. ROBERTS, G. P., T. MACNEIL, D. MACNEIL, AND W. J. BRILL. Regulation and characterization of protein products coded by the Nif (nitrogen fixation) genes of Klebsiella pneumoniae. J. Bacteriol. 136:267-279. 1978.

12. WILFINGER W.,MACKEY K. AND CHOMCZYNSKI P. Effect of Ph and ionic strength on the spectrophotometric assessment of nucleic acid purity. Bio Techniques,22,474-481. 1997

13. ZEHR .J. P, MARK T. MELLON, AND SABINO ZANI. New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (NifH) Genes. 1998

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