The document describes the isolation and molecular characterization of Rhizobium bacteria from soil samples. Key steps included: 1. Isolating Rhizobium colonies from soil dilutions on nutrient agar plates. 2. Amplifying genes from the bacterial DNA using PCR with specific primers. 3. Analyzing the amplified genes using various bioinformatics tools like NCBI BLAST, codon usage analysis, and phylogenetic analysis to identify and characterize the bacteria.

1. Tusha Madan
M.Sc Biotech
IV th Sem
Jaipur National University

2.  Introduction
 Objectives
 Methods
 Result and Discussion
 Summary
 References

3.  Rhizobium is the most well- known species of a group of bacteria that act as
a primary symbiotic fixer of nitrogen.
 These bacteria can infect the roots of leguminous plants , leading to the
formation of lumps or nodules where the nitrogen fixation take place.
 In the soil the bacteria are free living and motile, feeding on the remains of
dead organism.
 Free living Rhizobium cannot fix nitrogen and they have a different shape
from the bacteria found in root nodules.
 All organism which reduce dinitrogen to ammonia do so with the aid of an
enzyme complex, nitrogenase.
 The nitrogenase enzymes are irreversibly inactivated by oxygen and the
process of nitrogen fixation use a large amount of energy.
 Biological nitrogen fixation is an important component of sustainable
agriculture, and the rhizobial inoculants have been applied frequently as
biofertilizers.

5.  Biotechnology is the use of living system and organism to develop or make
useful products or “any technological applications that use biological system,
living organism or derivatives thereof, to make or modify products or
processes for specific use.”
 A series of derived terms have been coined to identify several branches of
biotechnology for example.
 Blue Biotechnology: blue biotechnology is a term that has been used to
describe the marine and aquatic applications of biotechnology, but its use is
relatively rare.
 Green Biotechnology: green biotechnology is a biotechnology applied to
agricultural processes. An example would be the selection and domestication
of plants via micropropogation. Another example is the designing of
transgenic plants to grow under specific environments in the presence (or
absence) of chemicals.One hope is that green biotechnology might produce
more environmentally friendly solution than traditional industrial agriculture.

6.  Red Biotechnology: red biotechnology is applied to medical process some
example are the designing of organism to produce antibiotics and the
engineering of genetic cures through genetic manipulation.
 White Biotechnology: also known as industrial biotechnology applied to
industrial processes. An example is the designing of an organism to produce a
useful chemicals.

8.  Paulien Hogeweg coined the term ” Bioinformatics ” in 1970 to refer to study of
information processes in biotic system.
 Bioinformatics is a conceptualizing biology in terms of molecules (in the sense of
physical –chemistry) and then applying “ informatics “ techniques (derived from
disciplines such as applied maths, CS and statistics) to understand and organize
the information associated with these molecules, on a large scale.
 Complex machines are use to read biological data at a much faster rate than
before and used in decoding the code of life. Database and information system
are used to store and organize biological data. Analyzing biological data may
involve algorithms in artificial intelligence, soft computing, data mining, image
processing and stimulation
 Major research efforts in the field include sequence alignment gene finding,
genome assembly, drug design, drug discovery, protein structure alignment,
protein structure prediction and the modeling of evolution.
 In the field of genetics and genomics, it aids in sequencing and annotating
genomes and their observed mutations. It plays a role in the analysis of gene and
protein expression and regulation.

10.  Isolation of Rhizobium from soil sample
 Amplification of genes using reverse and forward primers
 Identification of molecular polymorphism in N2 fixation using
bioinformatics tools

11.  0.1g soil sample in 5ml distilled water.
 Prepare serial dilution upto 10 ¯1 to 10 ¯4
 Streak any two dilution plates that is 10¯2 and 10¯4
 Incubate the plates for 1 to 2 days and perform staining
 To isolate DNA from bacterial culture
 2ml sample (bacterial culture)
 10 min centrifuge in 1000 rpm
 Supernatant discarded and collect the pellet add 0.2ml cell suspension and mixed it
3-5 min
 Add 0.2ml cell lysis in a vial
 Add 0.2ml of salt solution (neutralize buffer) and again centrifuge in 1000 rpm for
10min supernatant is taken into a new vial

12.  Add 100% ethanol and again centrifuge for 15 min
 Then 70% ethanol and repeat the above step
 Air dry the vial for some time
 20ul T.E buffer is added and collect the DNA bands
A basic PCR set up requires several components and reagents. These components
include:
• DNA template that contains the DNA region (Target) to be amplified.
• Two primers that are complementary to the 3’ ends of each of the sense and
anti-sense strand of the DNA target.
• Taq polymerase with an optimum temperature of around 70.C
• Deoxyribonucleotide triphosphate (dNTPs), the building blocks from which
the DNA polymerases synthesizes a new DNA strand.
• Buffer solution, providing a suitable chemical environment for optimum
activity and stability of DNA polymerase.
o All the components were kept on ice.
oPCR tube was label and kept it on ice.
o 10ul of isolated genomic DNA was taken in to PCR tube.
o PCR ‘Master Mix’ was prepared adding the following components into PCR
tube

13.  10ul 5’ Genomic primer (reverse primer)
 10ul 3’ Genomic primer(forward primer)
 20ul Taq reaction buffer
 20ul dNTPs
 20ul Taq polymerase
 10ul PCR grade water
o ‘Master Mix’ was thoroughly mixed in PCR tube.
o Thermo cycler was programmed
 1 cycle of 96°C for 2 minutes
 33 cycles 94°C for 1 min, 55°C for 1 min, 68°C for 2 min
 1 cycle 68°C for 7 minutes
 1 cycle of 4°C forever
 Place the tube in the thermo cycler.
o Use 50ul of your amplified DNA were used for visualization

14.  After visualization different types of bioinformatics tools have been used for
further study which are discussed below
 NCBI: The National Centre of Biotechnology Information is a part of
United States National Library of Medicine (NLM), a branch of the
National Institute of Health. The NCBI is located in Bethesda Maryland and
was founded in 1988 through legislation sponsored by Senature Claude
pepper. Here a few steps to perform NCBI
 Step 1: Database
 Step 2: Sequence
 The seq. can be in GCG, FASTA, EMBL, PHYLP, NBRF, and GeneBank
 Seq. file upload
 Seq. type
 Indicates if the seq. is protein or DNA/RNA
 Step 3: Parameters

16.  BLAST: In bioinformatics BLAST is for Basic Local Alignment Search Tool
is an algorithm for comparing primary biological seq. information such as the
amino acid seq. of different proteins or the nucleotides of DNA seq. A
BLAST search enables a researcher to compare a query seq. with a library or
databases of seq. and identify library seq. that resembles the query seq. above
a certain thresholds. For example
 Protein Database query
 Step 1: select your database
 Step 2: enter your input seq.
 Step 3: set your parameters
 BLAST
 Step 4: submit your job

18.  Codon usage: Refers to differences in the frequency of occurrence
of synonymous codons in coding DNA. A Codon is a series of
three nucleotide (triplets) that encodes specific amino acid residue in
a polypeptide chain or for the termination of translation stop codons. There are
64 different codons (61 codons encoding for amino acids plus 3 stop codons)
but only 20 different translated amino acids. The abundance in the number of
codons allows many amino acids to be encoded by more than one Codon.
Because of such redundancy it is said that the genetic code is degenerate.
Different organisms often show particular preferences for one of the several
codons that encode the same amino acid- that is; a greater frequency of one will
be found than expected by chance. How such preferences arise is a much
debated area of molecular evolution
 Open Reading Frame: In molecular genetics an ORF is a part of reading frame
that contains no stop codons. The transcription termination pause site is located
after the ORF, beyond the translation stop Codon, because if transcription were
to cease before the stop Codon, an incomplete protein would be made during
translation.
 The ORF finder is a graphical analysis tool which finds all Open Reading
Frames of a selectable minimum size in a user’s seq. or in a seq. already in
database

20.  CpG Islands: CpG Islands are regions with a high frequency of CpG site,
through objectives definitions for CpG islands are limited. The usual formal
definition of CpG islands is a region with at least 200 bp, and a CG percentage
that is greater than 50% and with an observed – to- expected CpG ratio that is
greater than 60%. The “observed- to- expected CpG ratio” is calculated by
formula (num of CpG/ Num of C* Num of G * Total no. of nucleotide in the
seq.
 Restriction Enzyme Maps: Restriction enzyme map is a map of known
restriction sites within the sequence of DNA. Restriction mapping require the
use of restriction enzyme. In molecular biology, restriction maps are used as
reference to engineer plasmid or other relatively short piece of DNA, and
sometimes for larger genomic DNA
 DNA Stats: DNA returns the numbers of occurrence of each residue in the
sequence you enter. Percentage totals are also given for each residue, and for
certain group of residue, allowing you to quickly compare the results obtained
for different sequence

21.  Codon plot: Codon plot accepts a DNA sequence and generates a graphical
plot consisting of a horizontal bar for each Codon. The length of the bar is
proportional to the frequency of the Codon in the Codon frequency table you
enter.
 Reverse complement: Reverse complement sequence converts a DNA
sequence into its reverse, complement or reverse-complement counterpart.
The entire IUPAC DNA alphabet is supported, and the case of each input
sequence character is maintained.

22.  Colonies of Rhizobium were obtained on nutrient agar medium after
inoculation at 24°C from 2 days. The colonies were having sticky
appearance showing the production of mucus through at lower levels.
Analysis of colony morphology indicated round colonies, white colonies
till after 3-4 days of growth and turning yellowish in color after 4 days.
DNA bands are collected from Gel Electrophoresis and perform their
structural and functional analysis.

24.  Genes are identified and perform their phylogenetic analysis through different
bioinformatics tools which are as follow:
FASTA seq. of Gene 1
Gene name
 nodN dehydrates nodulation protein [Rhizobium etli CFN 42]
Fasta sequence
Rhizobium etli CFN 42 chromosome, complete genome
NCBI Reference Sequence: NC_007761.1
>gi|86355669:3804959-3805447 Rhizobium etli CFN 42 chromosome, complete
genome
ATGGTCACGGAAATTTCACTCTCCGACGTGCGGGGATTGATCGGCA
TGGAAACGGGTCTTTCGGATTGGATCACCGTTGACCAGACAATGAT
CGACGCCTTTGCGGGGGCGACCGACGACCATCAGTTCATTCATGTC
GATCCCGAGCGTGCGGCAGCCGAAAGCCCCTTCGGCGGCACCATC
GCCCATGGTTTCCTGACGCTGTCCCTATTGTCGGCGATGAACTACAA
TTGCCTGCCGAAAGTGCGCGAGCAGACAATGGGCATCAACTACGGT
TTCGACCGCGTCCGCTTCATGACGCCGGTCAGGAGCGGCGCCCGCG
TGCGCGGCCGCTTCCTGCTCTGCGACGCCCGCTTCCGCGGCGGCGG
CATGCTGATGACCACCTATGACGTGACGATCGAAATCGAAAACGAG
AAGAAGCCGGCGCTGATGGCAAAATGGATCACCATCATCCAATTTG
ATCCGAAGGACCGCCCAGAAGACGCGTAG

26. FASTA seq. Gene 2
• nodN nodulation protein N [Rhizobium tropici CIAT 899]
Fasta Sequence
Rhizobium tropici CIAT 899, complete genome
NCBI Reference Sequence: NC_020059.1
>gi|440224888:3088373-3088861 Rhizobium tropici CIAT 899, complete
genome
ATGCCTGGCGAAATTTCGCTTGTTGAGATCATGAAGCTTGCGGGA
ACGGAAATCGGCGTTTCCGATTGGATCACCGTCGACCAGACGATG
ATCGACACCTTTGCCGATGCGACGCTCGATCACCAATTCATCCAC
GTCGATCCCGAGCGCGCCAAAGCGGAAACCCCCTATGGCGGCAC
CATCGCCCATGGCTTCCTGACACTCTCGCTGCTCTCGGCGATGAA
CTATAGCGCATTGCCGAAGATCCGCGAACAGACCATGGGCATCAA
CTACGGCTTCGAGAAGATCCGCTTCATGTCGCCGGTCAAGTGCGG
AGCGCGGGTGCGAGGACGTTTCACACTCGCCGAAACGCGGCTGC
GCGGCGCCAATATGCTGATGCTGACCTATGACGTGACAGTCGAGA
TCGAAAACGAACGCAAACCGGCGCTAACCGCGACATGGACGACC
ATATCGCAGTTCGACCCGAAGGATCGGCCCGAAGAGGGTTAA

34.  >gi|86355669:3804959-3805447 Rhizobium etli CFN 42 chromosome,
complete genome reverse complement
CTACGCGTCTTCTGGGCGGTCCTTCGGATCAAATTGGATGATGGTGA
TCCATTTTGCCATCAGCGCCGGCTTCTTCTCGTTTTCGATTTCGATCG
TCACGTCATAGGTGGTCATCAGCATGCCGCCGCCGCGGAAGCGGGC
GTCGCAGAGCAGGAAGCGGCCGCGCACGCGGGCGCCGCTCCTGAC
CGGCGTCATGAAGCGGACGCGGTCGAAACCGTAGTTGATGCCCATT
GTCTGCTCGCGCACTTTCGGCAGGCAATTGTAGTTCATCGCCGACAA
TAGGGACAGCGTCAGGAAACCATGGGCGATGGTGCCGCCGAAGGG
GCTTTCGGCTGCCGCACGCTCGGGATCGACATGAATGAACTGATGGT
CGTCGGTCGCCCCCGCAAAGGCGTCGATCATTGTCTGGTCAACGGT
GATCCAATCCGAAAGACCCGTTTCCATGCCGATCAATCCCCGCACGT
CGGAGAGTGAAATTTCCGTGACCAT

35.  >gi|440224888:3088373-3088861 Rhizobium tropici CIAT 899,
complete genome reverse complement
TTAACCCTCTTCGGGCCGATCCTTCGGGTCGAACTGCGATATGGT
CGTCCATGTCGCGGTTAGCGCCGGTTTGCGTTCGTTTTCGATCTCG
ACTGTCACGTCATAGGTCAGCATCAGCATATTGGCGCCGCGCAGC
CGCGTTTCGGCGAGTGTGAAACGTCCTCGCACCCGCGCTCCGCA
CTTGACCGGCGACATGAAGCGGATCTTCTCGAAGCCGTAGTTGAT
GCCCATGGTCTGTTCGCGGATCTTCGGCAATGCGCTATAGTTCATC
GCCGAGAGCAGCGAGAGTGTCAGGAAGCCATGGGCGATGGTGCC
GCCATAGGGGGTTTCCGCTTTGGCGCGCTCGGGATCGACGTGGAT
GAATTGGTGATCGAGCGTCGCATCGGCAAAGGTGTCGATCATCGT
CTGGTCGACGGTGATCCAATCGGAAACGCCGATTTCCGTTCCCGC
AAGCTTCATGATCTCAACAAGCGAAATTTCGCCAGGCAT

36.  Results for 489 residue sequence "gi|86355669:3804959-3805447 Rhizobium etli CFN 42
chromosome, complete
genome" starting "ATGGTCACGG"
>ORF number 1 in reading frame 1 on the reverse strand extends from base 1 to base 282.
CTACGCGTCTTCTGGGCGGTCCTTCGGATCAAATTGGATGATGGTGATCCATTTTGCCATCAGCGCCGG
CTTCTTCTCGTTTTCGATTTCGATCGTCACGTCATAGGTGGTCATCAGCATGCCGCCGCCGCGGAAGCG
GGCGTCGCAGAGCAGGAAGCGGCCGCGCACGCGGGCGCCGCTCCTGACCGGCGTCATGAAGCGGAC
GCGGTCGAAACCGTAGTTGATGCCCATTGTCTGCTCGCGCACTTTCGGCAGGCAATTGTAGTTCATCG
CCGACAATAG
>Translation of ORF number 1 in reading frame 1 on the reverse strand.
LRVFWAVLRIKLDDGDPFCHQRRLLLVFDFDRHVIGGHQHAAAAEAGVAEQEAAAHAGAA
PDRRHEADAVETVVDAHCLLAHFRQAIVVHRRQ*
>ORF number 2 in reading frame 1 on the reverse strand extends from base 283 to base 474.
GGACAGCGTCAGGAAACCATGGGCGATGGTGCCGCCGAAGGGGCTTTCGGCTGCCGCACGCTCGGGATC
GACATGAATGAACTGATGGTCGTCGGTCGCCCCCGCAAAGGCGTCGATCATTGTCTGGTCAACGGTG
ATCCAATCCGAAAGACCCGTTTCCATGCCGATCAATCCCCGCACGTCGGAGAGTGA
>Translation of ORF number 2 in reading frame 1 on the reverse strand.
GQRQETMGDGAAEGAFGCRTLGIDMNELMVVGRPRKGVDHCLVNGDPIRKTRFHADQSPHVGE

37.  Results for 489 residue sequence "gi|440224888:3088373-3088861 Rhizobium tropici
CIAT 899, complete genome"
starting "ATGCCTGGCG"
>ORF number 1 in reading frame 1 on the reverse strand extends from base 64 to base
489.
CGCCGGTTTGCGTTCGTTTTCGATCTCGACTGTCACGTCATAGGTCAGCATC
AGCATATTGGCGCCGCGCAGCCGCGTTTCGGCGAGTGTGAAACGTCCTCGC
ACCCGCGCTCCGCACTTGACCGGCGACATGAAGCGGATCTTCTCGAAGCC
GTAGTTGATGCCCATGGTCTGTTCGCGGATCTTCGGCAATGCGCTATAGTTC
ATCGCCGAGAGCAGCGAGAGTGTCAGGAAGCCATGGGCGATGGTGCCGCC
ATAGGGGGTTTCCGCTTTGGCGCGCTCGGGATCGACGTGGATGAATTGGTG
ATCGAGCGTCGCATCGGCAAAGGTGTCGATCATCGTCTGGTCGACGGTGAT
CCAATCGGAAACGCCGATTTCCGTTCCCGCAAGCTTCATGATCTCAACAAG
CGAAATTTCGCCAGGCAT
>Translation of ORF number 1 in reading frame 1 on the reverse strand.
RRFAFVFDLDCHVIGQHQHIGAAQPRFGECETSSHPRSALDRRHEADLLEAVV
DAHGLFADLRQCAIVHRREQRECQEAMGDGAAIGGFRFGALGIDVDELVIER
RIGKGVDHRLVDGDPIGNADFRSRKLHDLNKRNFARH

41.  Microorganism are capable of transforming atmospheric nitrogen into fixed nitrogen,
inorganic compounds usable by plants. The symbiotic nitrogen –fixing bacteria invade
the root hairs of host plants, where they multiply and stimulate formation of root
nodules, enlargements of plants cells and bacteria in intimate association. Within the
nodules the bacteria converts free nitrogen to nitrates, which the host plants utilize for
its development. Biological nitrogen fixation is brought about bath by free- living soil
microorganism with higher plants. Biological nitrogen fixation is the process of
capturing atmospheric nitrogen by biological process.
 Rhizobium etli is a nitrogen-fixing soil bacterium that is able to form a root
nodule symbiosis with leguminous plants, specially Phaseolus vulgaris (common
bean), one of the most important crop in Mexico and Latin, America. In R.etli CFN42
and other nitrogen- fixing symbiotic bacteria, NiFA- RPoN is a mater regulator of
nitrogen fixation genes and NIFA plays a central role in ensuring expression of the
nitrogen fixation apparatus during symbiosis.
 Polymerase Chain Reaction has enabled great advances in the study of taxonomy
used genetic diversity of Rhizobia, because this tech permits the sequencing and
comparison of defined fragments nucleotide seq.
 In the present work, we used a genomic approach to detect and measure a variation
and to analyze the contribution of recombination to the genomic diversification of
R.etli strains. In R.etli we found a great amount of variation when any draft genome
was compared to the complete genome of CFN42.

42.  Book References
 Aneja KR 2003. Experiments in Microbiology plant pathology and
biotechnology. 4th edition, New Age International publisher, New Delhi: India,
241p
 Atlas RM and Bartha R. 1981. Microbial Ecology Fundamentals and
applications. Addission Wesley Publishing Company Inc.., Philippines, 560p
• Humoon Rashidi, Lukask. Buehler 2005. Application in Biological Science and
Medicine .2nd edition, CRC press/ Taylor and Francis group, 335p
• Stephen A. Krawetz , David D. Womble 2003. Introduction to Bioinformatics. 1st
edition , Human press publisher, 760p
 Web References
Galter Health Science Library
https:// www.galter.northwestern.edu
Web resources for C. elegans studies
https:// blast -ncbi.nlm.nih.gov
DNA sequencing technology
http:// the tools of DNA Sequencing Technology