Journal of Genetic Engineering and Biotechnology (2014) 12, 27–35

Academy of Scientific Research & Technology and

National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology
www.elsevier.com/locate/jgeb

ARTICLE

RAPD-PCR and 16S rDNA phylogenetic

analysis of alkaline protease producing bacteria
isolated from soil of India: Identification and
detection of genetic variability
Sangeeta Saxena a, Jyoti Verma a,*
, Shikha b, Dinesh Raj Modi a

a
Department of Biotechnology, Babasaheb Bhimrao Ambedker University, Vidya Vihar, Lucknow 226025, U.P., India
b
Department of Environmental Sciences, Babasaheb Bhimrao Ambedker University, Vidya Vihar, Lucknow 226025, U.P., India

Received 21 November 2013; revised 7 February 2014; accepted 7 March 2014

Available online 6 April 2014

KEYWORDS Abstract 178 bacterial strains were isolated from the soil samples collected from different regions
Protease; of India out of which, 20 bacterial isolates were selected for alkaline protease production. The alka-
Bacteria; line protease production efficiency of organisms was monitored at regular intervals (24 h) upto
16S-rDNA; 7 days at 37 C, pH 10. The 16S rDNA sequencing and RAPD-PCR based technique were used
RAPD to identify the genetic variability among the 20 isolates of alkaline protease producing bacteria.
The phylogenetic analysis indicated that the isolates can be separated into two clusters which could
be further subdivided into five groups. Group 1 and 5 represented the family Bacillaceae, Groups 2
represented the Micrococcaceae family while Group 3 included the Arthrobacter bacterial group
(family Micrococcaceae) from different geographical locations, respectively. Group 4 was identified
as Pseudomonadaceae which was gram () bacteria. 21 different oligonucleotide primers were used
to amplify approximately 261 fragments from each DNA sample. The bands were scored on the
basis of their presence and absence and similarity between DNA samples was checked using
Jaccard’s coefficient. Isolates were distinguished into distinct groups based on RAPD profiles from
different geographical locations, morphological features and enzyme production efficiency. For
cluster analysis the dendrogram was constructed using the unweighted pair group method with

* Corresponding author. Tel.: +91 9415174692; fax: +91

5222441888.
E-mail addresses: dr_sangeeta_saxena@yahoo.com (S. Saxena),
jyoti.verma873@gmail.com (J. Verma), dr_shikha2003@yahoo.co.in
(Shikha), drmodilko@gmail.com (D. Raj Modi).
Peer review under responsibility of National Research Center, Egypt.

Production and hosting by Elsevier

1687-157X ª 2014 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
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28
arithmetic averages (UPGMA). The results indicated that 16S rDNA and RAPD-PCR are suitable
methods for rapid identification and differentiation of alkaline protease producing bacteria.
ª 2014 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research &
1. Introduction
Alkaline proteases are widely used biocatalytic enzyme and
well known for their emerging novel properties and their
exotic catalytic nature. Hence alkaline protease producing
organisms have attracted more attention of the scientific
community so as to enhance and ensure better utilization
of proteins [3,20]. These enzymes find diverse applications
in different industries such as detergent, food, feed, pharma-
ceutical, leather, silk and for recovery of silver from used X-
ray films [1,12,29]. Microorganisms represent an excellent
source of protease owing to their broad biochemical diversity
and susceptibility to genetic manipulation that are desirable
for various applications [11]. Alkaline proteases can be de-
fined as the proteases that are active in alkaline range of
pH [13,15]. Microbial proteases are among the most impor-
tant hydrolytic enzymes and have been studied extensively
since the advent of enzymology. Looking into the depth of
microbial diversity, there is always a chance of finding micro-
organism producing novel enzymes with better properties
and suitable for commercial exploitation. The multitude of
physiochemical diverse habitats has challenged nature to de-
velop equally numerous molecular adaptations in the micro-
bial world. The 16S rDNA method is sequence based
taxonomy and in the present scenario, molecular character-
ization based on RAPD fingerprinting is additionally used
to study the microbial diversity or variability and their eco-
logical distribution. RAPD is a very convenient and cost
effective method employed for bacterial identification and
variability estimation [17]. The PCR based method of gene
typing based on genomic polymorphism is a recent approach
which is widely used for the assessment of inter and intraspe-
cific genetic variation and uses a single short random oligo-
nucleotide primer [36]. In most cases of bacterial genetics,
RAPD assay generated the best DNA pattern for differenti-
ation of bacteria. A number of studies have reported success
in using RAPD assays to distinguish bacterial strains among
diverse species [28,4,9,10]. A study on genetic variability
among Arthrobacter strain by phylogenetic analysis of
RAPD-PCR fingerprint patterns has been reported by Saxe-
na and Singh (2013) [31].
2. Materials and methods
2.1. Sample collection
For the isolation of protease producing bacteria, soil sam-
ples were collected from agricultural fields of Central Soil
Salinity Research Institute, slaughter house, fish harbour
and dairy house etc. (Table 1). Soil samples were collected
in sterile polybags and stored at 4 C for further experi-
ments. Prior to collection of sample the pH of the soil
was monitored.
S. Saxena et al.
Technology.
2.2. Isolation and screening of alkaline protease producing
bacteria
The collected soil samples were serially diluted and 0.1 ml of
diluted sample was spread on plates containing alkaline agar
medium comprising of 1% glucose, 0.5% peptone, 0.5% yeast
extract, 0.1% KH2PO4, 0.02% MgSO4Æ7H2O, 1% Na2CO3
and 1.5% agar [14]. The colonies which appeared on plates
were purified by repeated streaking and stored on alkaline agar
slants at 4 C till further analysis. Further they were screened
by the method of Horikoshi [14].
2.2.1. Qualitative assay (Plate assay)
The isolated bacterial strains were screened for protease pro-
duction on alkaline agar medium comprising 10% skimmed
milk, 1% yeast extract, 1.5% Na2CO3 and 2% agar. Plates
containing alkaline agar medium were inoculated by bacterial
strains. The inoculated plates were incubated at 37 C for 24–
48 h [15] and observed for the formation of zone of hydrolysis.
The bacterial strains forming zone of skim milk hydrolysis
were purified and preserved on alkaline agar medium. The for-
mation of zone of hydrolysis by bacterial strains was consid-
ered as a positive indication of protease production by
bacterial strains [7].
2.2.2. Quantitative assay (Enzyme assay)
Bacterial strains screened positive for protease production in
plate test were grown in liquid media (alkaline broth) for pro-
tease assay. A well grown bacterial suspension was centrifuged
at 10,000·g for 10 min at 4 C. The crude enzyme activity was
assayed by the modified method of Tsuchida method [35] in
cell free culture supernatant using 1% Casein as a substrate
in 50 mM sodium phosphate buffer and Protein quantity was
determined by the Lowry method [23]. In this process, Bovine
serum albumin was used as standard. The crude enzyme activ-
ity was monitored at regular intervals (24 h) upto 7 days in
triplicate times.
2.3. Strain identification and characterization
2.3.1. Phenotypic identification of bacterial isolates
Promising bacterial strains were morphologically and bio-
chemically identified and characterized in laboratory.
2.3.2. Molecular Identification of bacterial isolates
Microscopic techniques were not found sufficient enough to re-
veal taxonomic details of the isolated strains. Therefore, the
modern molecular technique (r-DNA sequencing) was used
for identification of the isolated bacterial strains.
2.3.2.1. Isolation of genomic DNA and amplification by 16S-
rDNA. For their molecular characterization, Genomic DNA
was isolated by HimediaHiPurA Genomic DNA isolation
RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria 29

Table 1 Protease production level in bacterial strains isoalted from different geographical locations in India.
Strain code Collection of soil samples pH of soil samples Colony forming unit (CFU) Protease activity (U/ml)
Bact1 Allahabad (U.P.), India 9.86 2.7 · 107 10.70
Bact2 Lucknow (U.P.), India 9.61 4.7 · 106 23.21
Bact3 Panjab, India 9.00 2.0 · 107 2.75
Bact4 Lucknow (U.P.), India 8.78 1.9 · 107 3.40
Bact5 Lucknow (U.P.), India 9.00 2.3 · 106 4.51
Bact6 Delhi India 8.69 3.1 · 105 6.91
Bact7 Lucknow (U.P.), India 9.57 4.5 · 105 5.11
Bact8 Panjab, India 9.5 3.6 · 105 4.61
Bact9 Gujarat, India 9.5 2.7 · 107 4.33
Bact10 Orrisa , India 10 1.7 · 106 3.27
Bact11 Lucknow (U.P.), India 10.5 2.9 · 106 10.56
Bact12 Lucknow (U.P.), India 8.92 1.7 · 107 11.37
Bact13 Kanpur (U.P.) , India 9.68 2.1 · 107 9.40
Bact14 Lucknow (U.P.), India 9.12 3.0 · 107 10.17
Bact15 Delhi, India 8.45 3.7 · 106 9.69
Bact16 Gorakhpur (U.P.), India 8.90 2.8 · 107 7.42
Bact17 Lucknow (U.P.), India 9.05 1.9 · 107 4.81
Bact18 Lucknow (U.P.), India 10.05 2.6 · 107 8.15
Bact19 Lucknow (U.P.), India 10.50 4.1 · 106 16.08
Bact20 Lucknow (U.P.), India 9.78 3.2 · 105 4.05

kit (Himedia, India) and phenol:chloroform isolation method. tance matrix-based cluster algorithms viz. the unweighted pair
16S rDNA sequence was amplified from genomic DNA ob- group method with averages (UPGMA), neighbor-joining [30].
tained as above by PCR with the upstream primer: 50 -GAGA- The stability of trees obtained from the above cluster analyses
GTTTGATCCTGGCTGGCTCAG-30 and the downstream was assessed by using BOOTSTRAP program in sets of 1000
primer: 50 -AAGGAGGTGATCCAGCCGCA-30 , which gen- resamplings (MEGA 4).
erated a DNA fragment of approximately 1500 bp [6]. Ampli-
fication of DNA was carried out under the following 2.3.2.3. Sequence analysis. The purified PCR products were se-
conditions: denaturation at 94 C for 5 min followed by 30 cy- quenced from Amnion Biosciences Bangalore, India using Ge-
cles of 94 C for 30 s, 52 C for 30 s, 72 C for 1.5 min and final netic Analyzer (Applied Biosystems 3130 XL, Switzerland).
extension at 72 C for 10 min. Amplified PCR products of bac- The deduced sequence was subjected to BLAST algorithm
terial isolates were analyzed by electrophoresis with 1% aga- from the National Centre of Biotechnology, Bethesda, MD,
rose gel run at 7 V/cm (90 min). After electrophoresis the gel USA (http://www.ncbi.nlm.nih.gov) to retrieve for homolo-
was stained by ethidium bromide and then visualized and pho- gous sequences in GenBank and also subjected to Ribosomal
tographed under UV transilluminator. PCR product was puri- Database Project (RDP).
fied by PCR purification kit (Nucleospin, Germany).
2.4. Screening of alkaline protease producing bacterial isolates
2.3.2.2. Phylogenetic analysis of bacterial strain. The 16S of DNA
rDNA sequences of all bacterial isolates were aligned with ref-
erence sequences showing sequence homology from the NCBI The 20 accessions of bacterial samples were subjected to
database using the multiple sequence alignment program of RAPD screening with 21 random oligonucleotide primers (Ta-
MEGA 4.0 [33]. Phylogenetic trees were constructed by dis- ble 2). The primers from the Operon kit were used to produce
amplifiable products in the DNA from bacterial species to find
the RAPD markers producing bands.
Table 2 Details of 21 polymorphic randomly selected decam-
er primers. 2.4.1. Genetic variability estimation by RAPD fingerprinting
Primers Sequence Primers Sequence
The 20 alkaline protease producing bacterial isolates were sub-
jected to RAPD screening with 21 random oligonucleotide
OPA17 GACCGCTTGT OPX2 TTCCGCCACC primers (Operon kit, Germany) and were analyzed to obtain
OPN02 ACCAGGGGCA OPX7 GAGCGAGGCT
OPU13 GGCTGGTTCC OPX11 GGAGCCTCAG
OPU15 ACGGGCCAGT OPX12 TCGCCAGCCA
OPV10 CAAACGTGGG OPY1 GTGGCATCTC
OPV14 GGTCGATCTG OPY10 CAAACGTGGG
OPV20 AGCCGTGAAA OPY20 AGCCCTGGAA
OPW8 GACTGCCTCT OPZ1 TCTGTGCCAC
OPW11 CTGATGCGTG OPZ8 GGGTGGGTAA
OPW15 ACACCGGAAC OPZ10 CCGACAAACC
OPW20 TGTGGCAGCA Figure 1 16S rDNA-PCR amplification of alkaline protease
producing bacteria.
30 S. Saxena et al.

Table 3 Identification of bacterial strain on the basis of 16S rRNA gene sequence similarity.
Strain code Accession no. 99% Similarity/blast hit Bacterial name
Bact1 JX855288 JN033557.1 Bacillus flexus
Bact2 KC522120 JF274870.1 Citricoccus
Bact3 KC912707 HF750078.1 Bacillus flexus
Bact4 KC522121 JX007957.1 Micrococcus luteus
Bact5 KC522122 JN128828.1 Bacillus sp.
Bact6 KC522123 AB715339.1 Bacillus sp.
Bact7 KC522124 JX460830.1 Salinicoccus salitudinis
Bact8 KC522125 JX912420.1 Bacillus sp.
Bact9 KC522126 JN128828.1 Bacillus sp.
Bact10 KC522127 JN128828.1 Bacillus sp.
Bact11 KC522128 JQ809717.1 Arthrobacter sp.
Bact12 KC522129 HQ912420.1 Bacillus licheniformis
Bact13 KC522130 EF379937.1 Arthrobacter sp.
Bact14 KC522131 AB715339.1 Bacillus clausii
Bact15 KC912710 EF468655.1 Arthrobacter sp.
Bact16 KC522132 JQ347473.1 Uncultured bacterium
Bact17 KC522133 GQ093772.1 Uncultured bacterium
Bact18 KF528990 EF379937.1 Arthrobacter sp.
Bact19 KC581791 CP001878.2 Bacillus pseudofirmus
Bact20 KC912709 GQ340513.1 Bacillus licheniformis

94 C followed by 39 cycles at 94 C for 1 min, 32.5 C for

1 min, 72 C for 1 min and final extension for 10 min. The
amplified PCR products were separated by agarose gel electro-
phoresis in 1.2% run in 1· TAE buffer at 65 V for 3 h 30 min
till amplified fragments are separated and visualized under UV
transilluminator.

2.4.2. Analysis of amplicons (agarose gel electrophoresis)

The amplified PCR products were separated by electrophoresis
in 1.2% agarose gel run in 1· TAE buffer at 65 V for 3 h
30 min. till amplified fragments are separated. The gel was
photographed, stored and analyzed using a gel documentation
system with built in software.

2.4.3. Statistical data analysis

Figure 2 Phylogenetic analysis of alkaline protease producing
bacteria from soil, India based on partial nucleotide sequences of
16S rDNA. The tree was constructed using the neighbor-joining
method. Percentages at nodes represent levels of bootstrap
support from 1000 resampled datasets. Bootstrap values less than
50% are not shown.
Binary data based on the presence or absence of bands were
analyzed by pair-wise comparison using Jaccard’s coefficient
[16]. The similarity matrix thus obtained was subjected to clus-
ter analysis by UPGMA and dendrogram was generated to
study the relatedness of the bacterial strain. The robustness
of the nodes of the dendrogram was tested by bootstrap anal-
ysis using 1000 resamplings. These analyses were carried out
using Free tree software [25]. UPGMA dendrogram was drawn
using Treeview [24].
3. Result
3.1. Isolation and screening

a specific fingerprinting pattern. The amplification reaction
was prepared in 25 ll volumes 25–50 ng/ll of genomic DNA,
3.5 of 10· PCR buffer (100 mM Tris–HCl, pH 8.3, 250 mM
KCl, 1.5 mM MgCl2), 0.1 mM of each dNTPs, 1.5 U of Taq
DNA polymerase (Bangalore Genei, India) and 50 pmol pri-
mer (Operon kit). The amplification was carried out in a ther-
mal cycler, programed for 4 min of initial denaturation at
A total of 178 single isolated colonies were inoculated on
alkaline agar medium and pure culture of bacterial colonies
were obtained by repeated streaking after that transferred to
skimmed milk plate for screening of protease producing
bacteria and designated as Bact-1, 2, 3 and so on. Out of
178, only 20 bacterial colonies developed a translucent zone
on skimmed milk plate assay and taken as positive for
RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria 31

Table 4 List of primer used for RAPD-PCR amplification.

S. no. Primer name Sequence (50 –30 ) GC (%) Total number of bands Poly morphic bands Mono morphic band Polymorphism (%)
1 OPA-17 GACCGCTTGT 60 7 7 0 100
2 OPN-02 ACCAGGGGCA 70 12 12 0 100
3 OPU-13 GGCTGGTTCC 70 11 10 1 90
4 OPU-15 ACGGGCCAGT 70 9 9 0 100
5 OPV-10 CAAACGTGGG 60 11 11 0 100
6 OPV-14 GGTCGATCTG 60 16 16 0 100
7 OPV-20 AGCCGTGAAA 50 14 14 0 100
8 OPW-8 GACTGCCTCT 60 14 14 0 100
9 OPW-11 CTGATGCGTG 60 14 14 0 100
10 OPW-15 ACACCGGAAC 60 16 16 0 100
11 OPW-20 TGTGGCAGCA 60 11 11 0 100
12 OPX-02 TTCCGCCACC 70 14 14 0 100
13 OPX-07 GAGCGAGGCT 70 10 10 0 100
14 OPX-11 GGAGCCTCAG 70 14 14 0 100
15 OPX-12 TCGCCAGCCA 70 15 14 1 90
16 OPY-01 GTGGCATCTC 60 13 13 0 100
17 OPY-10 CAAACGTGGG 60 6 6 0 100
18 OPY-20 AGCCCTGGAA 60 15 15 0 100
19 OPZ-01 TCTGTGCCAC 60 13 13 0 100
20 OPZ-08 GGGTGGGTAA 60 14 13 1 90
21 OPZ-10 CCGACAAACC 60 12 12 0 100

alkaline protease production. All the 20 bacterial isolates were
checked for variability at the molecular level
with respect to 16S rDNA sequence analysis and RAPD
analysis.
Cell growth expressed as colony forming unit (CFU) per
ml was evaluated by plating ten fold serial dilution of cul-
ture media onto alkaline agar plates and colonies were
counted after 24–72 h of incubation. All the bacterial colo-
nies were inoculated in 50 ml alkaline broth separately.
After 72 h of incubation, culture of each strains was centri-
fuged and supernatant was used for protease assay. Out of
20 alkaline protease producing bacteria, only Bact2 showed
maximum protease activity as detailed in Table 1 (23.21 U/
ml).
3.2. Phenotypic identification
Out of the 20 bacterial isolates, most of the bacterial strains
(Bact1, Bact3, Bct4, Bact6, Bact8 Bact9, Bact10, Bact11,
Bact12, Bact13 Bact14, Bact15, Bact16, Bact17, Bact18,
Bact19, Bact20) were found to be rod shaped while Bact2,
Bact4, Bact7 were coccus in shape. All the isolated bacterial
strains were aerobic. Only 2 bacterial isolates (Bact16, Bact17)
were Gram negative while rest of the isolates was Gram
positive.
3.3. Molecular characterization and identification of bacterial
isolates
A good quality genomic DNA was isolated without any signs
of smearing when observed under UV transilluminator. The
yield of genomic DNA varied from 60 to 140 ng/ll. PCR
amplification of ribosomal DNA was carried out with univer-
sal forward and reverse primers of 16S rDNA and produced a
fragment of approximately 1500 bp (Fig. 1). This size corre-
sponded to the expected size as compared to other bacteria
[6]. The variability within the amplified regions was
investigated by phylogenetic analysis. The amplified PCR
product was run on 1% agarose gel viewed under the UV
transilluminator.
Systematic study coupled with molecular study is well estab-
lished to reveal the true identity of the organisms. Since the
organisms have been potentially identified by the 16S rDNA
method, the partial 16S rRNA sequences of all alkaline protease
producing bacteria were compared with other strains of bacte-
rium from the similarity matrix, calculated by number of base
differences, depending upon the highest level of similarity
(Table 2). Bact1 (1420 bp), Bact2 (1040 bp), Bact3 (1140 bp),
Bact4 (966 bp), Bact5 (1439 bp), Bact6 (823 bp), Bact7
(248 bp), Bact8 (1439 bp), Bact9 (1442 bp), Bact10 (1431 bp),
Bact11 (1351 bp), Bact12 (13920 bp), Bact13 (1402 bp), Bact14
(1328 bp), Bact15 (1419 bp), Bact16 (744 bp), Bact17 (1102 bp),
Bact18 (1415 bp), Bact19 (1432 bp), Bact20 (893 bp) revealed
maximum score hit compared with other strains of bacterium
on the basis of similarity matrix (NCBI).
3.4. Phylogenetic analysis of bacterial isolates
The amplified PCR product of representative isolates was iden-
tified and sequenced. A BLASTN analysis carried out through
GenBank (http://www.ncbi.nlm.nih.gov) revealed that all the
isolates were members of six different genera. Among them
11 bacteria belonged to the group Bacillus, 4 bacteria belonged
to the Arthrobacter group while other bacteria belonged to dif-
ferent groups. The identified sequences of isolated strains were
submitted to NCBI (National Centre for Biotechnology Infor-
mation) to which accession numbers are already mentioned in
Table 3. The 16S rDNA based phylogenetic analysis of all bac-
terial isolates demonstrated 99.00% sequence similarity with
the blast hit as mentioned in Table 3. In phylogenetic analysis,
Cluster I was subdivided into four groups in which group I be-
longed to the family Bacillaceae, Group 2 belonged to the
Micrococcaceae family and Group 3 was identified as Arthro-
32 S. Saxena et al.

bacter sp. from different geographical locations respectively.

Bact20
Similarity coefficients (The maximum similarity is 1.0 along the diagonals and the pairwise similarity gives an idea of the relatedness of the bacterial strains to each other).
Group 4 was represented by Pseudomonadaceae which was
gram () bacteria. Cluster II was subdivided into two branches
in which one branch represents a 5th group comprising Bacil-
Bact19

0.473
lus sp. which was giving almost a similar protease activity and
other branch formed by Bact3 was identified as the least pro-
Bact18

tease producing organism (Fig. 2).

0.324
0.211
3.5. RAPD-PCR analysis
Bact17

0.205
0.198
0.200
Out of 50 primers examined, 21 primers were used for RAPD
screening to detect polymorphism of the alkaline protease pro-
Bact16

0.440
0.202
0.244
0.245
ducing bacterial isolates and used to characterize the genetic
diversity or variability analysis of 20 isolates of alkaline prote-
ase producing bacteria. RAPD analysis of the individual DNA
Bact15

0.145
0.245
0.288
0.331
0.281
samples of the bacterial isolates was done using the primer that
detected a band specific to the individual as it distinguished
from other isolates. Genomic DNA of 20 alkaline protease
Bact14

0.188
0.157
0.146
0.194
0.198
0.241
producing bacterial isolates was successfully amplified with oli-
gonucleotide primer. A total of 261 bands were scored, of
which 258 (90%) were polymorphic (Table 4). Out of 21 oligo-
Bact13

0.235
0.291
0.239
0.211
0.256
0.307
0.310

nucleotide primers, 17 primers gave 100% polymorphic band

while 3 primers gave both mono and polymorphic band pat-
terns. The amplification patterns revealed a high level of poly-
Bact12

0.259
0.227
0.226
0.140
0.106
0.147
0.221
0.269

morphism (Figs. 3 and 4). Fingerprinting resulted in multiple

DNA products with 03 to 10 bands ranging from 200 to
600 bp.
Bact11

0.167
0.180
0.254
0.167
0.128
0.103
0.150
0.191
0.205

3.6. Cluster analysis

Bact10

0.173
0.278
0.257
0.257
0.296
0.218
0.183
0.328
0.302
0.283

In this study, 20 native isolates of alkaline protease produc-

ing bacteria were optimized for genetic variability estima-
0.0854
0.0482
Bact9

tion. Among them 11 bacteria belong to the group

0.130
0.264
0.197
0.178
0.316
0.141

0.126
0.158
0.174

Bacillus (Bact1, Bact3, Bact5, Bact6, Bact8, Bact9, Bact10,

Bact12, Bact14, Bact19, Bact20), 4 bacteria belong to the
Bact8

0.188
0.298
0.106
0.258
0.248
0.244
0.284
0.172
0.188
0.202
0.262
0.267

Arthrobacter group (Bact11, Bact13, Bact15, Bact18), while

other isolates belong to different groups (Bact2, Bact4,
Bact7, Bact16, Bact17). On evaluating genetic profile using
0.0602
0.0893

0.0811
Bact7

0.139
0.154
0.114
0.131
0.172
0.116
0.155

0.112
0.147
0.119

FREETREE software program with Jaccord coefficient and

UPGMA (Unweighted pair group method of arithmetic)
cluster method, it showed two distinctive groups and subdi-
Bact6

0.127
0.285
0.167
0.566
0.188
0.256
0.282
0.278
0.341
0.229
0.205
0.315
0.333
0.281

vided into seven clusters (Fig. 5) thereby confirming genetic

variation. In the first group, cluster I belongs to the family
Bact5

0.373
0.120
0.279
0.169
0.379
0.223
0.250
0.295
0.229
0.346
0.205
0.236
0.399
0.566
0.346

Micrococcaceae and consisted of Bact2 strain which formed

a separate branch from all other bacterial isolates and
showed maximum protease activity (23.21 U/ml) after 72 h
0.0676
Bact4

0.173
0.295

0.177
0.117
0.194
0.133
0.132
0.191
0.148
0.191
0.138
0.180
0.136
0.128
0.112

of incubation at 37 C. Second cluster consisted of Actino-

bacteria class which was 26% similar with one other. Clus-
ters 3 and 4 belong to the genus Bacillus which was 93%
Bact3

0.218
0.340
0.309
0.176
0.262
0.176
0.219
0.167
0.220
0.299
0.229
0.313
0.244
0.227
0.259
0.291
0.270

and 100% similar with each other, respectively. Cluster 5

belongs to Bacillus flexus isolates. Cluster six belongs to
0.0600

0.0734
0.0818
0.0526

0.0686

0.0575
0.0625
0.0476
0.0440
0.0777
0.0959
Bact2

the Pseudomonadaceae family and 100% similar with each

0.161

0.104
0.175

0.122
0.103
0.101
0.149

other. They were 99% similar with uncultureable bacterial

isolates in NCBI while by morphological and biochemical
features they were 99% similar with Pseudomonas bacteria
0.0541
Bact1

0.534
0.245
0.252
0.230
0.136
0.212
0.151
0.184
0.114
0.184
0.220
0.191
0.286
0.224
0.192
0.176
0.216
0.222

and also sequenced matched in ribosomal database project

release 10 (RDP). Group II represented as into cluster se-
ven included Bact14 and Bact9 which represented the genus
Table 5

Bact10
Bact11
Bact12
Bact13
Bact14
Bact15
Bact16
Bact17
Bact18
Bact19
Bact20

Bacillus and they were 61% similar with each other while
Bact1
Bact2
Bact3
Bact4
Bact5
Bact6
Bact7
Bact8
Bact9

they were 68% dissimilar with Bact11.

RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria 33

Figure 3 RAPD profiles of 20 isolates of alkaline protease producing bacteria collected from different geographical regions of India were
subjected to PCR amplification by using 10-mer RAPD primer OPX7. Lane 1–20 indicating isolates of bacteria listed in the Table 2. L-
Represents 1 DNA Kb ladder.

3.7. Similarity coefficient analysis
The genetic similarity coefficient for the 20 isolates of bacteria
resulting from RAPD analysis minimum ranged from 0.0440
(between Bact2 and Bact18), 0.0476 (between Bact2 and
Bact17) followed by 0.0541 (between Bact2 and Bact3).
Among them Bact2 isolate was different from other isolates
wherein genetic similarity coefficient was maximum among
20 isolates ranging from 0.566 (between Bact5 and Bact19; be-
tween Bact6 and Bact10) followed by 0.534 (between Bact1
and Bact3). The relatedness among 4 isolates (Bact2, Bact3,
Bact19, Bact20) within 20 isolates was charted out and genetic
similarity among 20 isolates was optimized in the range of
0.112–0.257 (Table 5).
4. Discussion
The preliminary characterization of isolated strains was done
following Bergey’s manual of Systematic Bacteriology [32,27]
and further confirmation was done by 16S rDNA sequences
analysis. After that amplified sequences of isolated strains
were compared with the Genebank database using BLASTN.
Many workers have characterized and identified novel strain
by 16S rDNA analysis [21,19]. Although 16S rDNA does not
give authentic identification and differentiation of closely re-
lated Bacillus sp. such as Bacillus subtilis, Bacillus lichenifor-
mis, Bacillus cereus etc., it can be used as preliminary
relatedness. The identification of Bacillus sp. on the basis of
16S rDNA gene sequence is done by blasting it against the
available databases (NCBI, RDP). The need for developing
a tool for identifying Bacillus sp. arose due to two reasons:
(i) more than 50% of the 16S rDNA sequences deposited
in the databases have been annotated/identified only as Bacil-
lus sp. (ii) phylogenetic tree construction [26]. In the present
study, 21 oligonucleotide primers were screened (Table 1)
and used to establish the phylogenetic relationship between
alkaline protease producing bacteria. RAPD happens to be
one of the powerful tools for studying genetic variation in liv-
ing organisms as it amplifies fragments of genomic DNA

Figure 4 RAPD profiles of 20 isolates of alkaline protease producing bacteria collected from different geographical regions of India were
subjected to PCR amplification by using 10-mer RAPD primer OPV14. Lane 1–20 indicating isolates of bacteria listed in the Table 2. L-
Represents 1 DNA Kb ladder.
34
Figure 5 Dendrogram derived from cluster analysis (UPGMA) showing relationship among 20 alkaline protease producing bacterial
isolates listed in Table 2. Genetic variability was obtained by RAPD primers, using the Jaccard similarity coefficient.
which may be essentially unknown viz plants, animals and
microorganisms. The limitation associated with pedigree data
along with morphological, physiological and marker based
estimation used for assessment of genetic variability which
had largely been developed by DNA markers such as restric-
tion fragment length polymorphisms [5], random amplified
polymorphic DNA [36]. The RAPD technology is well suited
to DNA fingerprinting [34] although it suffered from a cer-
tain lack of reproducibility due to mismatch annealing [18].
The exploration of RAPD worked as a genetic marker which
improved the effectiveness of r-DNA techniques and utilized
a single, arbitrarily primer to amplify a number of DNA
fragments to generate a discrete ‘‘fingerprint’’ and resolved
by gel electrophoresis [8]. A phylogenetic tree drawn by the
UPGMA cluster method showed that Bact2 (Citricoccus
sp.) formed a separate branch in tree and showed the highest
protease producing bacteria among 20 isolates. On the basis
of genetic variability analysis, the novelty of Bact2 is proved
as alkaline protease producing bacteria being hereby reported
for the first time. There are no reports pertaining to protease
production by this genus. The genus Citricoccus, originally
proposed by Altenburger et al. [2] comprises the two species
Citricoccus muralis, isolated from a medieval wall painting [2]
and Citricoccus alkalitolerans, isolated from a desert soil sam-
ple collected in Egypt [22]. Both species were isolated from
dry samples.
5. Conclusion
In last two decades, DNA methods have brought valuable cri-
teria to the taxonomy of bacteria, phylogenetic classification,
DNA sequencing and PCR fingerprinting are applied as com-
S. Saxena et al.
mon tools for phylogenetic studies. Very few studies have been
made on genetic variability of alkaline protease producing bac-
teria. At present, PCR amplification of the rDNA analysis
method has brought a valuable tool for characterization of
bacterial genes. The RAPD analysis shows promising results
with the oligonucleotide primer (operon) giving variability in
gene patterns among the bacterial isolates. The fingerprinting
patterns, obtained by using universal random primers are sim-
ple alternatives to gene specific molecular marker determina-
tion, where they are used to obtain polymorphic patterns in
large proportion. Moreover, the RAPD markers can be ideal
tools for a large number of species, populations of bacteria
which belong to different geographical locations, morphologi-
cally different and without any prior knowledge of genetic
information. Therefore the present study with the primers
OPX-12 OPZ-8, OPV-20 and OPW-15 has been shown to be
a potential marker for studying the variability and evolution
of alkaline protease producing bacteria to delineate them into
gene-related grouping and phylogenetics to a certain extent. As
aforesaid in the context these protease producing bacterial iso-
lates emerged as a complex group due to their gene specificity.
Further, these studies may have a significant impact toward
the evolutionary development from the point of view of
biotechnology.
Acknowledgements
The authors are grateful to the Department of Biotechnology,
Babasaheb Bhimrao Ambedker University, Vidyavihar, Luc-
know 226025 (U.P.), India for providing laboratory facilities
to carry out the present work and also support by research
Grants from University Grant Commission (UGC), New Del-
hi, India.
RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria
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