Molecular Study On Azotobacter Nif H Gene By Pcr

Introduction				1.0 µl, template DNA 1.0 µl, enzyme Taq
Nitrogen fixation is the reduction of N2				polymerase 1.0 µl for 35 cycles ( 1 min at 94°
(atmospheric nitrogen) to NH3 (ammonia). Free				C,1 min at 54° C and 1 min at 72° C) (Zehr et
living prokaryotes with the ability to fix				al., 1988).
atmospheric dinitrogen (diazotrophs) are ubiquitous				Results and Discussion
in soil. But our knowledge of their ecological				Totally 100 samples were collected in marine
importance and their diversity remains incomplete.				region of both water and sediments in the
In natural ecosystems, biological N2 fixation is				intervals of approximately 20 days .Out of 70
most important source of N. The capacity for				marine water samples collected, all the 70
nitrogen fixation is widespread among bacteria and				samples were showing the presence of
archaea. The estimated contribution of free-living				Azotobacter, but only 23 marine sediments out of
N-fixing prokaryotes to the N input of soil ranges				30 were showing the presence of Azotobacter.
from 0-60 kg/ha /year (Burgmann et al., 2003).				These samples were processed through the
Dinitrogen (N2)-fixing microorganisms (diazotrophs)				commonly used procedures such as selective
play important roles in ocean biogeochemistry and				media. Gram’s staining, Phase contrast
plankton productivity (Church et al., 2005).				observation for motility, starch hydrolysis test and
Nitrogen fixation can be an important source of				Catalase test for identification of free- living
nitrogen for biological productivity in the marine				diazotrophic organism i.e Azotobacter from the
environment. Biological nitrogen fixation is				above samples, and that can be processed, result
catalyzed by the enzyme nitrogenase, which is				shows that Azotobacter sp are motile, gram
possessed by diverse microorganisms				negative, catalase and starch hydrolysis positve.
representing virtually all phylogenetic groups.				The colony morphology of Azotobacter strains
Interest in nitrogen fixation in the sea has usually				were varying during the isolation in the selective
been focused on rates of nitrogen fixation, but				media. The colonies were very clear, large,
information on the types of species present with				mucoid, watery due drops like initially i.e. from the
the capability for nitrogen fixation can be				marine source. The mother culture was sub
important for predicting nitrogen fixation rates in				cultured in the same media; the colony
situ (Zehr et al., 1998).				morphology differs slightly i.e. small, and circular,
Nitrogenase catalyzes the reduction of nitrogen				convex in nature. All the isolated Azotobacter
gas to ammonium in an ATP-and reductant				strains were numbered for the easy identification
dependent reaction. It is one of the best				and convenience. From these isolates, well defined
characterized metalloenzyme and is an excellent				pure culture of Azotobacter strains (1, 16, 27, 82,
model for elucidating metalloprotein assembly.				101, 103, 108, 115, 125, and 132) were selected
Nitrogenase is composed of two oxygen-labile				for the nucleic acid analysis.
metallo protein; dinitrogenase and dinitrogenase				The reference (standard) cultures such as
reductase. Dinitrogenase is a 240-KDa				Azotobacter beijerinckii (123), A. chroococcum
alpha2-beta2 tetramer of the nifD and nifK gene				(446), and A. vinelandii (124) procured from
products. Dinitrogenase reductase is a 60-KDa				MTCC, (Chandigarh, India) were also used along
alpha2 dimer of the nifH gene products that				with marine isolates for the nucleic acid extraction
contains a single 4Fe-4S center coordinated				and purification. The DNA of the selected strains
between the two subunits (Rubio et al., 2005).				was isolated and estimated OD at 260 nm, the
Understanding how fixed N regulates nitrogenase				value ranges from 0.141 to 0.177. The estimated
availability is necessary for devising strategies to				value of the extracted DNA was ranging from
increase the amount of ammonium synthesized				0.70 to 0.88.The purity of the DNA was analyzed
by nitrogen fixing bacteria with the potential to be				by spectrophotometric method using OD at 260
used in agriculture (Kennedy et al., 2004).				and OD at 280 nm. The presence and purity of
Molecular tools for detection and characterization				DNA was checked by OD at 260nm/ OD at 280
of the nitrogenase (Nif) genes and immunoassays				nm, the value ranges from 1.13 to 2.21. If the
for nitrogenase protein can provide new				estimated value is 1.8 conforms the presence of
information on the factors regulating the				pure DNA. If the estimated value is lesser
distribution and activity of diverse nitrogen fixing				greater than 1.8 conforms the presence of DNA
organisms in the marine environment. Amplification				to protein / RNA contamination, according to
and characterization of NifH sequences has made				respective values DNA was purified using the
it possible to identify the type(s) of organism				enzymes proteases and RNase. The purified form
responsible for nitrogen fixation, such as in				of the DNA was separated by the agarose gel
aggregates of the cyanobacterium and				electrophoresis for the comparison the banding
Trichodesmium. Differences in nitrogen fixation				pattern between the randomly selected marine
patterns have been linked to genetic differences				samples and the standard strains. There is no
between Trichodesmium strains. Further				substantial difference between the banding
development of these approaches will provide				patterns of the chromosomal DNA on the gel as
new and powerful ways to link the genetic				shown in the plates. This result confirms that
potential for nitrogen fixation to nitrogen fixation				molecular weight of chromosomal DNA in all
rates in the ocean (Zehr et al., 1998)				strains is similar.
Nitrogenase gene (NifH) sequences amplified				One µl of DNA was used as template in PCR.
directly from oceanic waters showed that the				selected primers,
open ocean contains more diverse diazotrophic
microbial populations and more diverse habitats				Primer2:5-CGGATCCGDNGCCATCATYTCNCC-3
for nitrogen fixers than previously observed by				procured from OPERON diagnostics LTD, USA,
classical microbiological techniques (Zehr et al.,				respectively was used to amplify a 324-bp region
1998). Understanding how fixed N regulates				between sequence positions 336 and 660. All N2
nitrogenase availability is necessary for devising				fixers carry a NifH gene, which encodes the Fe
strategies to increase the amount of ammonium				protein of the nitrogenase (Poly et al., 2001). The
synthesized by nitrogen fixing bacteria with the				results of the PCR products were compared on
potential to be used in agriculture (Kennedy et al.,				2% agarose gel electrophoresis. Selective NifH
2004).				primer from Anabaena sp strain PCC7120 was
The commercial history of biofertilizer began with				used for the amplification of the Azotobacter sp,
the launch of "Nitrogin" by Nobbe and Hiltner; a				the primers used in my study was exactly
laboratory culture of rhizobia in 1895, followed by				matching the Azotobacter genome. Free-living
the discovery of Azotobacter and then the blue				nitrogen-fixing prokaryotes (diazotrophs) are
green algae and a host of other microorganisms.				ubiquitous in soil and are phylogenetically and
Azotobacter is used as a biofertilizer in the				physiologically highly diverse. Molecular methods
cultivation of most crops. Azotobacter is an				based on universal PCR detection of the NifH
obligate aerobic diazotrophic soil-dwelling organism				marker gene have been successfully applied to
with a wide variety of metabolic capabilities, which				describe diazotroph populations in the environment.
include the ability to fix atmospheric nitrogen by				However, the use of highly degenerate primers
converting it to ammonia. Azotobacter naturally,				and low-stringency amplification conditions render
fixes atmospheric nitrogen in the rhizosphere.				these methods prone to amplification bias, while
There are different strains of Azotobacter each				less degenerate primer sets will not amplify all
has varied chemical, biological and other				NifH genes (Bürgmann et al., 2003).
characters. However, some strains have higher				References
nitrogen fixing ability than others (Burgmann et al.,				Bagwell, C.E., Piceno, Y.M., Lucas, A.M. and Lovell,
2003). Besides, nitrogen fixation, Azotobacter also				C.R., 1988. Physiological diversity of the
produces, Thiamin, Riboflavin, indol acetic acid and				rhizosphere diazotroph assemblages of selected
gibberellins. When Azotobacter is applied to seeds,				salt marsh grasses. Appl. Environ. Microbiol., 64(11):
seed germination is improved to a considerable				4276-4282.
extent, so also it controls plant diseases due to				Bali, A., Gonzalo Blanco, Susan Hill, and Christina
above substances produced by Azotobacter				Kennedy 1992. Execretion of Ammonium by a
(Kader et al., 2002.)				NifL Mutant of Azotobacter vinelandii Fixing
This NifH gene has been largely studied by				Nitrogen. Appl. Environ. Microbiol., 58(5 ): 1711-1718.
culture-independent approaches. These				Bürgmann H, Franco Widmer, William Von Sigler,
approaches provide a more complete picture of				and Josef Zeyer. New Molecular Screening Tools
the diazotrophic community than culture-based				for Analysis of Free-Living Diazotrophs in soil.
approaches. Various techniques, such as PCR				Environmental Microbiology, Vol.70, No1 p.240 -
cloning, denaturing gradient gel electrophoresis,				247.1999.
PCR-restriction fragment length polymorphism				Bürgmann H, Manuel Pesaro, Franco Widmer
(RFLP), and fluorescently labeled terminal				and Josef Zeyer strategy for optimizing quality
(FLT)-RFLP, have been used to analyze the				and quantity of DNA extracted from soil.
composition of NifH gene pools in various				Bacteriological Reviews, vol.36, No.2 p.295-341.
environments. These studies found that the NifH				2003.
gene is present in diverse environments: forest				Church M J, Cindy M. Short, Bethany D. Jenkins,
soil, the rhizosphere of native wetland species,				David M. Karl, and Jonathan P. Zehr, Temporal
such as Spartina, or of crop species, such as rice,				Patterns of Nitrogenase Gene (NifH) Expression in
aquatic or polar cyanobacteria, and the bacteria				the Oligotrophic North Pacific Ocean’ Ocean
found in termite guts. All these studies described a				Sciences Department, Environmental Microbiology,
large number of unknown sequences which				Vol 134, No.1 p.155-193, 1999.
correspond to diverse unidentified diazotrophs.				Wood,N.R.Krieg,andG.B.phillips.1981.Manual of
Some NifH genes are characteristic of an				method for generalbacteriology.American
ecological niche (Shaffer et al., 2000) evoked the				SOCIETY FOR microbiology, Washington, D.C.
possible relationship between the habitats of soil				Joerger, R.D., Jacobson, M.R., Premakumar, R.,
nitrogen-fixing bacteria and the structure of NifH				Wolfinger, E.W. and Bishop, P.E., 1989. Nucleotide
gene pools (Poly et al., 2001).				sequence and mutational analysis of the structural
Nitrogen fixation in A. vinelandii is complicated by				genes (anfHDGK) for the second alternative
the presence of three biochemically and				nitrogenase from Azotobacter vinelandii. J.
genetically distant nitrogenase enzymes, each of				Bacteriol., 171(2): 1075-1086.
which is synthesized under different conditions of				Kader.M.A, Mian.M.H, Hoque.M.S. Effect or
metal supply. The regulation of conventional				Azotobacter inoculant on the yield and nitrogen
molybdenum nitrogenase, whose subunits are				uptake by wheat. Department of soil science,
encoded by the Nif-HDK genes and which is similar				Bangladesh Agricultural University, Mymensigh,
to the enzyme purified from number of other				Bangladesh. Vol 2(4) p 251-261,2002.
nitrogen-fixing organisms. (Sabra et al., 2000). The				Kelly, M.J.S., Poole, R.K., Yates, M.G. and Kennedy,
Nif-HDK genes are located in a large cluster of nif				C., 1990. Cloning and mutagenesis of genes
genes, which includes, in order,				encoding the cytochrome bd terminal oxidase
NifHDKTYENXUSVWZMF (Bali et al., 1992).				complex in Azotobacter vinelandii: mutants
Molecular methods based on universal PCR				deficient in the cytochrome d complex are unable
detection of nifH marker genes have been				to fix nitrogen in air. J. Bacteriol., 172(10):
successfully applied to describe diazotroph				6010-6019.
population in the environment (Burgmann et al.,				Kennedy.C, R.K.Poole, M.G.Yates, and M.J.S.Kelly.
2003).				Cloning and Mutagenesis of Genes Encoding the
Materials and Methods				Cytochrome bd Terminal Oxidase Complex in
Sample collection				Azotobacter vinelandii: Mutants Deficient in the
Samples were collected in different locations of				Cytochrome d Complex are unable To Fix
Rameshwaram marine region (Gulf of Mannar) at				Nitrogen in Air.Journal of Bacteriology, Vol.172,No.10
the depth of 1–5 m. The randomly collected				p.6010-6019 . 1990
samples in the sterile plastic bags (soil sample) and				Maniatis,T.,E.F.Fritsch,and J.Sambrook.1982.
water sampling bottles (water sample) bottles				Molecular cloning: a laboratory manual. Cold spring
were kept in an ice-cold box and transported				Harbour Laboratory,Cold Spring Harbor,N.Y
safely to the lab for further analysis with in 12				Mary. L. G and rita r. colwell, Enumeration ,isolation
hrs. The sample with media tubes were packed				and characterization of N2 fixing bacteria from
and transported safely to the laboratory.				sea water . Department of microbiology,
Isolation of Azotobacter from water and				University of Maryland. Vol 50 no .2. 1985
sediment samples (Mary et al., 1985)				Poly .F, Lionel Ranjard, Sylvie Nazaret, François
Different selective media were used for the				Gourbière, and Lucile Jocteur Monrozier..
isolation of Azotobacter sp from marine source				Comparison of NifH Gene Pools in Soils and Soil
as described previously. Azotobacter strains used				Microenvironments with Contrasting Properties in
for this study were maintained and cultured in				Applied and Environmental Microbiology Vol. 67 p.
Burk medium as previously described (Joerger et				2255-2262. 2001
al., 1988). As the isolates are of marine origin, the				dley, and J.R.Postgate. 1984.Genomic size and
media were prepared by the 3.5% sodium				complexity in Azotobacter chroococcum.J.Gen
chloride (NaCl). Media used for the isolation of				Microbial.130:1603-1612.
nitrogen fixing organism (Azotobacter) from				Rubio .L M, singer.S.W, and Ludden P.W. Purification
marine sources were Jensen’s agar medium,				and Characterization of NafY from Azotobacter
Azotobacter agar medium, Burk’s Medium				vinelandii. Department of plant and
and marine agar medium.				microbiology,college of natural resources,
Culture characteristics (Bagwell et al., 1988)				University of California, California. 2005
Gram-staining characteristics and cell morphologies				Sabra, W., Zeng, A.P., Lunsdorf, H. and Deckwer,
were determined by standard methods(Gerhardt				W.D., 2000. Effect of oxygen on formation and
et al., 1981). Motility was observed in wet mount				structure of Azotobaceter vinelandii alginate and
using phase contrast microscope. Preliminary				its role in protecting nitrogenase. Appl. Environ.
physiological characterization such as catalase test,				Microbiol., 66(9): 4037-4044.
starch hydrolysis test were also carried out.				Shaffer, B. T., F. Widmer, L. A. Porteous, and R. J.
Extraction and purification of DNA (Kelly et al .,				Seidler. Temporal and spatial distribution of the
1990)				NifH gene of N2-fixing bacteria in forests and
Azotobacter genomic DNA was isolated as				clear-cut in western Oregon. Microb. Ecol. 39
previously described (Robson et al., 1980). Linear				P.12-21. 2000.
DNA fragments were analyzed by electrophoresis				Wilfinger W.,Mackey K. and Chomczynski P.1997
in agarose gel in TEB buffer (Maniatis et al., 1982).				Effect of Ph and ionic strength on the
The purity of the DNA was checked				spectrophotometric assessment of nucleic acid
spectrophotometric method by using the formula				purity. Bio Techniques,22,474-481.
OD at 260 nm/ OD at 280 nm (Wilfinger et al.,				Zehr .J. P ,Mark T. Mellon, and Sabino Zani. New
1997).				Nitrogen-Fixing Microorganisms Detected in
PCR amplification of the NifH gene fragment				Oligotrophic Oceans by Amplification of
Nitrogenase Fe protein genes (NifH) were amplified				Nitrogenase (NifH) Genes. 1998.
from Azotobacter sp derived genomic DNA, using				Zehr .J. P. Sarah Braun, Yibu Chen and Mark Mellon
the primer from OPERON diagnostic Ltd, USA.				Nitrogen fixation in the marine environment:
The samples were amplified by PCR in a mixture				relating genetic potential to nitrogenase activity.
containing reaction buffer 5.0 µl, 10mM dNTP 1.0				Department of Biology, Rensselaer Polytechnic
µl, primer 1 (25 mer) 1.0 µl, primer 2 (24 mer)				Institute, Troy, NY 12180-3590, USA . 1999.

Lab Focus

Molecular Study On Azotobacter Nif H Gene By Pcr

Articles