| INTRODUCTION | | | | PCR amplification of the NifH gene fragment (Burlage |
| Nitrogen fixation is the reduction of N2 (atmospheric | | | | et al., 1998). |
| nitrogen) to NH3 (ammonia).Free-living prokaryotes | | | | Nitrogenase Fe protein genes (NifH) were amplified |
| with the ability to fix atmospheric dinitrogen | | | | from Azotobacter sp derived genomic DNA, using |
| (diazotrophs) are ubiquitous in soil, but our knowledge | | | | the primer from OPERON diagnostic Ltd, USA. The |
| of their ecological importance and their diversity | | | | samples were amplified by PCR in a mixture |
| remains incomplete. Nitrogen fixation-related genes | | | | containing reaction buffer 5.0 µl, 10mM dNTP 1.0 µl, |
| have been highly conserved throughout evolution | | | | primer 1 (25 mer) 1.0 µl, primer 2 (24 mer) 1.0 µl, |
| even though they are widely distributed among | | | | template DNA 1.0 µl, enzyme Taq polymerase 0.5 |
| eubacteria and archaea. The great diversity of | | | | µl for 40 cycles (0.5 min at 94° C, 1 min at 54° |
| diazotrophs also extends to their physiological | | | | C and 0.5 min at 72° C) (Zehr et al., 1988). |
| characteristics, as N fixation is performed by | | | | One microlitre of DNA was used as template in PCR |
| chemotrophs and phototrophs and by autotrophs as | | | | study. Selected primers from A.vinelandi (M2568), |
| well as heterotrophs (Burgmann et al., 2003). | | | | Primer 1 5-GCIWTYTAYGGIAARGGIGG-3, |
| However, evidence has been accumulating which | | | | Primer 2 5-AAICCRCCRCAIACIACRTC-3respectively |
| documents the importance of bacterial N2 fixations in | | | | were used to amplify a 390-bp region of nif H gene. |
| many and diverse marine habitats. Brook’s et al | | | | Where I represents inosine, R represents A or G, W |
| provided the first strong evidence of in situ bacterial | | | | represents A or T and Y represents C or T. |
| N2 fixation occurring in estuarine sediments. Since | | | | RESULTS AND DISCUSSION |
| that time, the presence of heterotrophic N2 fixation | | | | Totally 120 samples were collected from Tondi |
| in the absence of light has been demonstrated for | | | | marine region of both water and sediments at the |
| the variety of marine and estuarine sediments | | | | intervals of approximately 30 days. The randomly |
| (Guerinot et al., 1985). The capacity for N2 fixation in | | | | collected samples after subjecting to further |
| natural populations of marine eubacteria is beginning | | | | processing on selective medium, the colony |
| to be evaluated using molecular and immunological | | | | morphology of Azotobacter strains were varying. |
| techniques. Most species are free-living with some | | | | The colonies were very clear, large, mucoid, watery |
| forming a major portion of the surface plankton; | | | | due drops. Pure culture of Azotobacter shows |
| others are symbiotic with teleost fishes and squid or | | | | rectangular or oval gram negative rods on |
| are pathogenic in fish and shellfish (Coyer et al., 1995). | | | | gram’s staining and positive results for catalase, |
| A number of free-living soil bacteria, e.g. bacteria of | | | | oxidase, starch hydrolysis, gelatin hydrolysis test |
| the genera Azotobacter (aerobic), Clostridium (strictly | | | | which are useful for identification of Azotobacter sp. |
| anaerobic), Klebsiella (optionally aerobic), and | | | | The DNA of the selected strains was isolated by |
| Rhodospirillum (anaerobic, photosynthetically active) | | | | lysozyme treatment and estimated OD at 260 nm, |
| belong to the nitrogen reducing species (Kennedy, | | | | the value ranges from 0.142 to 0.189. The estimated |
| 2004) | | | | value of the extracted DNA was ranging from 0.710 |
| Azotobacter sp is a large, obligately aerobic soil | | | | to 0.945. The presence and purity of DNA was |
| bacterium which has one of the highest respiratory | | | | checked by OD at 260nm/ OD at 280 nm, the value |
| rates known among living organisms and is able to | | | | ranges from 1.19 to 2.19. According to respective |
| grow on a wide variety of carbohydrates, alcohols | | | | values DNA was purified using the enzymes |
| and organic acids, in addition to be able to fix | | | | proteases and RNAase. The purified form of DNA |
| nitrogen. The biological nitrogen fixation reaction is | | | | was then subjected to agarose gel electrophoresis |
| catalyzed by a complex metalloenzyme called | | | | for the separation using 0.8% agarose gel. The |
| nitrogenase. Nitrogenase is composed of two | | | | agarose gel after electrophoresis was taken out and |
| separately purified proteins, both of which are | | | | placed on the UV transilluminator window / UV gel |
| extremely oxygen sensitive. The larger of the two | | | | documentation, it was hence concluded that there |
| proteins, designated the MoFe protein, has a | | | | were no substained difference between the banding |
| molecular mass of 230,000 Da. The MoFe protein is a | | | | pattern of the chromosomal DNA of different test |
| tetramer in its biologically active form and is | | | | isolates including marine and standard MTCC strains of |
| composed of two identical halves, each containing an | | | | Azotobacter sp on the agarose gel electrophoresis. |
| ?-subunit and a ?-subunit encoded by the NifD and | | | | The results of the PCR products were compared on |
| nifK genes, respectively. The smaller of the two | | | | 2% agarose gel electrophoresis. Selective NifH primer |
| proteins, designated the Fe protein, has a molecular | | | | from A.vinelandi (M2568), was used for the |
| mass of about 60,000 Da and is a dimer of identical | | | | amplification of the Azotobacter sp, the primers used |
| subunits encoded by the NifH gene. Besides the | | | | in my study was exactly matching the Azotobacter |
| structural genes of nitrogenase, there are a number | | | | genome. Free-living nitrogen-fixing prokaryotes |
| of nif-specific genes (20 identified to date) that | | | | (diazotrophs) are ubiquitous in soil and are |
| comprise the nif regulon (Lei et al., 1999). | | | | phylogenetically and physiologically highly diverse. |
| Methods for studying the community structure of | | | | Molecular methods based on universal PCR detection |
| N2-fixing bacteria in the rhizosphere usually require | | | | of the NifH marker gene have been successfully |
| enrichments of culturable bacteria, and functional | | | | applied to describe diazotroph populations in the |
| contributions are often assessed using the acetylene | | | | marine environment. However, the use of highly |
| reduction assay (ARA) of cultured isolates or soil | | | | degenerate primers and low-stringency amplification |
| cores. These methods provide incomplete information | | | | conditions render these methods prone to |
| because: i. only a small fraction of viable cells is | | | | amplification bias, while less degenerate primer sets |
| represented by culture-based methods, ii. N2-fixing | | | | will not amplify all NifH genes (Bürgmann et al., |
| bacteria that inhabit living root cells or tissues are not | | | | 2003). |
| identified, iii. There are technical problems associated | | | | REFERENCE |
| with the use of an indirect assay (ARA) for | | | | 1. BAGWELL, C.E., PICENO, Y.M., LUCAS, A.M. AND |
| nitrogenase activity, and iv. The relative functional in | | | | LOVELL, C.R., 1988. Physiological diversity of the |
| situ contributions of individuals in diverse populations | | | | rhizosphere diazotroph assemblages of selected salt |
| are not assessed. Molecular methodology facilitating | | | | marsh grasses. Appl. Environ. Microbiol., 64(11): |
| the characterization of the N2 -fixing bacterial | | | | 4276-4282 |
| community structure, when used with ARA and | | | | 2. BROOKS, R.H., P.L. Brezonik H.D. Putnam and H.A. |
| selective culturing methods, may provide a more | | | | Keirn , 1971 Nitrogen fixation in an estuarine |
| thorough analysis of N2-fixing bacterial population | | | | environments, the Waccasassa on the Florida Gulf |
| dynamics in the rhizosphere. (Lepo et al.,1983) | | | | coast, Limnol. Oceanogr 16 : 701-710. |
| MATERIALS AND METHOD | | | | 3. BÜRGMANN H, FRANCO WIDMER, WILLIAM |
| Sample collection and transport | | | | VON SIGLER, AND JOSEF ZEYER. New Molecular |
| Samples were collected from different locations of | | | | Screening Tools for Analysis of Free-Living |
| Tondi which comes under Rameshwaram marine | | | | Diazotrophs in soil. Environmental Microbiology, Vol.70, |
| region at the depth of 1–3 m. The samples were | | | | No1 p.240 - 247.2003. |
| collected in the sterile plastic bags (soil sample) and | | | | 4. COYER J A. T, PASINIT A M, SWIFTS H. N2 |
| water sampling bottles (water sample). The randomly | | | | fixation in marine heterotrophic bacteria: Dynamics of |
| collected samples were kept in an ice-cold box and | | | | environmental and molecular regulation. Department |
| transported safely to the lab for further analysis with | | | | of Molecular Genetics and Cell Biology, University of |
| in 12 hrs. | | | | Chicago,Contributed by Hewson Swift, December 6, |
| Processing and screening of Azotobacter sp from | | | | 1995 |
| marine soil and water samples (Kannan, 2002). | | | | 5. GUERINOT, M. L., AND PATRIQUIN D. G. 1981. The |
| The randomly collected samples were processed for | | | | association of N2-fixing bacteria with sea urchins. Mar |
| isolation of Azotobacter sp exclusively by using | | | | . Biol. Vol 62: 197 – 207 |
| various techniques like serial dilution followed by | | | | 6. LEI S, PULAKAT L, AND GAVINI N. Genetic |
| spread plating, pour plating on differential media like | | | | Analysis of nif Regulatory Genes by Utilizing the |
| Jensen’s Agar Medium (with 3.5% NaCl), | | | | Yeast Two-Hybrid System Detected Formation of a |
| Azotobacter Agar Medium (with 3.5% NaCl), | | | | nifL nif A Complex That Is Implicated in Regulated |
| Burk’s Medium (with 3.5% NaCl), Marine agar | | | | Expression of nif Genes. American Society for |
| medium. | | | | Microbiology 181(20): 6535–6539. |
| Characterization of Azotobacter sp (Bagwell et al., | | | | 7. KENNEDY C, RUDNICK P, MACDONALD M, |
| 1988) | | | | MELTON T.Genus Azotobacter in Garrity et al (eds) |
| Gram-staining characteristics and cell morphologies | | | | Bergey's Manual of Systematic Bacteriology. 2004. |
| were determined by standard methods (Gerhardt et | | | | 8. KELLEY, T.S., AND EISENSTARK, A. The genus |
| al., 1981). Motility was observed in wet mount using | | | | Azotobacter. Bull. Oklahoma Agr. Mech. Coll., 48, no. |
| phase contrast microscope. Preliminary physiological | | | | 16. 1990 |
| characterization such as catalase test, starch | | | | 9. LEPO J E, CHELIUS M K AND WEBER D E. |
| hydrolysis test, oxidase test, gelatin hydrolysis test | | | | Characterization of nitrogen-fixing bacterial |
| were also carried out. | | | | rhizosphere communities using restriction fragment |
| Extraction and purification of DNA (Kelly et al ., 1990) | | | | length polymorphisms of pcr amplified nifH. Center for |
| Azotobacter genomic DNA was isolated as previously | | | | Environmental Diagnostics and Bioremediation, |
| described (Robson et al., 1980). Pure culture of | | | | University of West Florida, Pensacola, Florida, 32514. |
| isolated and characterized marine Azotobacter Strains | | | | 1983. |
| (402,405,415,420,426,433,456,462,469,480,501 and | | | | 10. ROBSON Y. IGARASHI AND LANCE C. Nitrogen |
| 520) was selected based on their growth | | | | Fixation: The Mechanism of the Mo-Dependent |
| characteristic on selective media for the nucleic acid | | | | Nitrogenase Critical Reviews in Biochemistry and |
| extraction and purification. The reference (standard) | | | | Molecular Biology, 38:351–384 .1980. |
| cultures such as Azotobacter sp (2632), Azotobacter | | | | 11. ROBERTS, G. P., T. MACNEIL, D. MACNEIL, AND |
| chroococcum (2452), and Azotobacter lactinogens | | | | W. J. BRILL. Regulation and characterization of protein |
| (2633) procured from MTCC, Chandigarh, were also | | | | products coded by the Nif (nitrogen fixation) genes |
| used for the nucleic acid extraction and purification. | | | | of Klebsiella pneumoniae. J. Bacteriol. 136:267-279. |
| The purity of the DNA was checked | | | | 1978. |
| spectrophotometric method by using the formula OD | | | | 12. WILFINGER W.,MACKEY K. AND CHOMCZYNSKI |
| at 260 nm/ OD at 280 nm (Wilfinger et al., 1997). If | | | | P. Effect of Ph and ionic strength on the |
| the estimated value is 1.8 conforms the presence of | | | | spectrophotometric assessment of nucleic acid purity. |
| pure DNA. If the estimated value is lesser /greater | | | | Bio Techniques,22,474-481. 1997 |
| than 1.8 conforms the presence of DNA to protein / | | | | 13. ZEHR .J. P, MARK T. MELLON, AND SABINO ZANI. |
| RNA contamination, according to respective values | | | | New Nitrogen-Fixing Microorganisms Detected in |
| DNA was purified using the enzymes proteases and | | | | Oligotrophic Oceans by Amplification of Nitrogenase |
| RNAase. | | | | (NifH) Genes. |