Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 64-73
http://www.ijcmas.com
Original Research Article
Genetic Identification of Three Streptomyces Strains having Enzymatic
Activities Isolated from Soil-Taif, KSA
Mohamed H. Sonya1, 2, Altalhi A.D. 1, El-Zahrani Gh. S. Badria1 and Sadik A.S.1, 3*
1
Department of Biology, Faculty of Science, Taif University, P.O. Box 888, Taif, KSA
Soil, Water and Environmental Research Institute, Department of Agricultural Microbiology,
Agricultural Research Center, P.O. Box, 12619, Giza, Egypt
3
Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, P.O.
Box 68, Hadayek Shobra 11241, Cairo, Egypt
2
*Corresponding author
ABSTRACT
Keywords
SoilStreptomycetes,
DNA Markers,
RAPD-PCR,
16S rRNA
Gene
Several studies have documented the use of RAPDs for the typing of fungal as well
as bacterial species and strains. In this study molecular identification of three soilstreptomycetes having enzymatic activities isolated from Taif regions was aimed.
At the level of DNA fingerprinting, data showed that 72 fragments (65
polymorphic and 7 monomorphic) were amplified from the DNA of the three
identified Streptomyces strains (10, 12 and 13). These fragments distributed among
the seven RAPD-PCR primers as follows: 8, 5, 11, 16, 6, 12 and 14 for OPA11,
OPB15, OPC07, OPC18, OPD04, OPE05 and OPO14, respectively. Polymorphic
and monomorphic fragments were observed. Three primers named OPB15, OPC07
and OPO14 did not show any monomorphic fragments. Using the seven RAPDPCR primers 64 (5, 5, 11, 15, 5, 9 and 14) represent 88.88% were considered as
unique DNA markers, and were amplified using the OPA11, OPB15, OPC07,
OPC18, OPD04, OPE05 and OPO14, respectively. Data showed similarity matrix
between the three identified streptomycete strains based on RAPD-PCR analysis
ranged from 19.0 to 34.4%. The 16S rRNA gene was partially isolated and
sequenced from the DNA of S. lateritius isolate 10. Results showed that the percent
identity between the isolate of this study and the compared Streptomyces lateritius
(strain LMG 19372) was 81.0%. This isolate could be classified as a new strain of
the red S. lateritius, and we suggested to be named Streptomyces sp. BSAA-KSA
strain.
Introduction
are Gram-positive bacteria endowed with a
remarkable genetic repertoire for producing
secondary metabolites. Despite their
potential, only a few studies had been
reported on their esterase and lipolytic
Actinomycetes are the most economically
and biotechnologically valuable prokaryotes,
they responsible for the production of half of
the
discovered
bioactive
secondary
metabolites (Berdy, 1989). Streptomyces
64
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
activities (Sztajer et al., 1988; Bormann et
al., 1993). Several reports has been studied
using the RAPD-PCR technique for
screening the genetic variation within
several species of streptomycetes (ElDomyati and Mohamed Sonya, 2004; Saleh
et al., 2012; Shori Ghadeer et al., 2012;
Mohamed Sonya et al., 2013). RAPD-PCR
was also used by Mohamed et al. (2001) for
identification of some streptomycetes. Data
revealed that Streptomyces strain Si-1 had
similarities ranging from 60.9 to 65.1% with
the red series isolates and 58.5% with the Si9 violet series isolate.
Streptomyces QU66C (Mahasneh et al.,
2011).
At KSA, the DNA of the identified
streptomycete isolate was extracted and used
for amplification of 16S rRNA gene using
PCR followed by determination of its
nucleotide sequences (Mohamed Sonya et
al., 2012; Mohamed Sonya et al., 2013;
Mohamed Sonya et al., 2014).
This study aimed at identifying some soilstreptomycetes, having enzymatic activities,
at Taif region, KSA based on their
molecular characters.
The dissimilarities between the three red
series isolates were 33.3, 46.9 and 50.3%.
Unique and specific PCR fragments were
obtained for each strain; therefore, the
results of that work paid an attention to
encourage the use of RAPD-PCR technique
as a molecular tool for identification of
actinomycetes. The DNA fingerprinting of
five halotolerant Streptomyces using RAPDPCR analysis was carried out. A total of 327
DNA bands generated by 16 random 10-mer
primers were detected in which 311 (about
95%) were considered as useful markers for
the five Streptomyces strains used in the that
study. A very few number of bands were
common (monomorphic) for all strains. As
high as 192 out of the 327 (59%) RAPDPCR
markers
were
strain-specific
(polymorphic) (Mahfouz and Mohamed
Sonya, 2002). An actinomycete isolate from
Riyadh, KSA was classified as Streptomyces
spororaveus RDS28 according to analysis of
16S rRNA gene sequence. 16S rRNA was
amplified in a thermocycler by using
universal primers of 27F and 1525R under
the following condition: 94°C for 5 min, 35
cycles of 94°C for 60 s, 55°C for 60 s, 72°C
for 90 s and final extension at 72°C for 5
min (Al-Askar et al., 2011). The sequence of
the 16S rRNA gene was determined and
thus to conduct the phylogenic position of
the naturally occurring wild type strain of
Materials and Methods
To reach the aim of this study, the DNA of
the selected streptomycete isolates was
extracted and subjected to determination of
DNA fingerprinting using a molecular tool,
i.e., RAPD-PCR). The nucleotide sequence
of the 16S rRNA gene of a selected
streptomycete isolate was also determined.
Determination of DNA Fingerprinting of
the Identified Streptomyces Strains
Based on the method of Mahfouz and
Mohamed (2002), DNA from the pulverized
streptomycete cells of Streptomyces strains
(# 10, 12 and 13) was prepared. The RNA
and protein molecules were removed using
the enzyme ribonuclease (DNase-free
RNase) and the enzyme proteinase K as
reported by Mohamed et al. (2012). The
method of Brown (1990) was applied for
purifying and concentrating the DNA
extracts.
For RAPD-PCR and its analysis, a group of
seven RAPD-PCR primers (OPA11,
OPB15, OPC07, OPC18, OPD04, OPE05
and OPO14) were used for determination of
DNA fingerprinting of DNA streptomycete
strains as described by El-Domyati and
65
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
Mohamed (2004) in a total volume of 25 l
as described by Bagheri et al. (1995) in a
GeneAmp 2400 PCR machine using the
program reported by Mohamed et al. (2012).
The DNA polymorphisms were resolved by
electrophoresis (Sambrook et al., 1989) in a
1.2% agarose gel at 60 volts for 2.5 h with
1X TAE buffer. The presence or absence of
each sizes class was scored as 1 (present) or
0 (absent), respectively. Bands of the same
mobility were scored as identical. The
similarity coefficient (F) between isolates
was defined by the formula of Nei and Li
(1979). A dendrogram was derived from the
distance by un-weighted paired-group
method (Rohlf, 1990).
technique isolated from soil either in Egypt
(Mohamed Sonya et al., 2001; Mahfouz and
Mohamed Sonya, 2002; Abdel-Fattah, 2005)
or in KSA (Shori Ghadeer et al., 2012;
Mohamed Sonya et al., 2012; Mohamed
Sonya et al., 2013; Mohamed Sonya et al.,
2014; Saleh et al., 2011).
Data in Table 1 and illustrated by Figure 1
show that a total number of 72 fragments
(65 polymorphic and 7 monomorphic) were
amplified from the DNA of the three
identified Streptomyces strains (# 10, 12 and
13). These fragment distributed among the
seven RAPD-PCR primers used as follows:
8, 5, 11, 16, 6, 12 and 14 for OPA11,
OPB15, OPC07, OPC18, OPD04, OPE05
and OPO14, respectively. Three primers
named OPB15, OPC07 and OPO14 did not
show any monomorphic fragments. Using
the seven RAPD-PCR primers 64 (5, 5, 11,
15, 5, 9 and 14) represent 88.88% were
considered as unique DNA markers, and
were amplified using the OPA11, OPB15,
OPC07, OPC18, OPD04, OPE05 and
OPO14, respectively. A number of 41
unique fragments were recorded as present
(+) DNA markers, while 23 were recorded
to be absent (-) DNA markers. Results in
Table 2 showed similarity matrix between
the three identified streptomycete strains
based on RAPD-PCR analysis ranged from
19.0 to 34.4%. Two clusters were found in
the dendogram deduced from the
statistically analysis of the data (Figure 2).
The first includes both of Streptomyces
isolates 10 and 12.
Molecular
confirmation
of
the
Streptomyces strain using the 16S rRNA
Gene
The DNA of the selected Streptomyces
isolate 10 was used as a template for PCRisolation of 16S rRNA gene using two
universal primers (518F & 800R) as
described by Mohamed et al. (2012). The
PCR-amplified 16S rRNA gene was sent to
Macrogen® (908 world meridian venture
center, #60-24, Gasan-dong, Geumchun-gu,
Seoul 153-781, Korea) for determining its
nucleotide sequences. The DNA sequences
of the PCR product of the 16S rRNA gene
of the selected Streptomyces strain were
aligned with the nucleotide sequences of the
universal
isolates
collected
from
http://www.ncbi.nlm.nih.gov/,
using
BLASTN 2.2.23+ software (http://www.
ncbi.nlm.nih.gov/blast/) against the isolates
collected from the database for genotyping.
16S rRNA gene
Results and Discussion
Actinomycetes were molecularly identified
using the 16S rRNA gene by some
investigators (Shori Ghadeer et al., 2012;
Al-Askar et al., 2011; Mahasneh et al.,
2011; Mohamed Sonya et al., 2012;
Mohamed Sonya et al., 2013; Mohamed
Sonya et al., 2014; Moran et al., 1995; Cook
DNA fingerprinting of the three identified
streptomycetes using RAPD-PCR
DNA fingerprinting of several Streptomyces
sp. were determined using RAPD-PCR
66
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
and Meyers, 2003; Song et al., 2004; Jose et
al., 2011). Partial sequences of the 16S
rRNA of S. lateritius isolate 10 was
determined. Results showed that 972 and
1206 nts were obtained from forward and
reverse sides. The sequences from position 1
to 620 of both forward and reverse sides are
determined (Data not shown). The final
sequence shown in Figure 3 was compared
with 6 universal bacterial isolates or clones
(Uncultured Streptomyces sp. clone BlueGSA-KSA 16S rRNA gene partial sequence,
JQ962978.1; Uncultured Streptomyces sp.
clone neau-D50 16S rRNA gene, partial
sequence,
GQ494994.1;
Uncultured
Streptomyces sp. clone ASC834 16S rRNA
gene, partial sequence, JQ358608.1;
Uncultured Streptomyces sp. clone ASC835
16S rRNA gene, partial sequence,
JQ358606.1; Uncultured Streptomyces sp.
clone ASC825 16S rRNA gene, partial
sequence, JQ358604.1 and S. lateritius
strain: LMG 19372 16S rRNA, complete
sequence, AJ781326.1).
Results in Table 3 showed that the percent
identity between the final sequence of the
isolate of this study (S. lateritius isolate 10)
and the compared S. lateritius (strain LMG
19372) was 81.0%.
The phylogenetic tree in Figure 4 showed
the S. lateritius isolate 10 was felled in a
separate cluster. Therefore, this isolate could
be classified as a new strain of the red S.
lateritius, and we suggested to be named
Uncultured Streptomyces sp. gene for 16S
ribosomal RNA, partial sequence, clone:
BSAA (GeneBank: AB915914). As a
conclusion, one can recommend use of
RAPD-PCR and 16S rRNA molecular tools
for genetic identification of microorganisms.
Table.1 DNA fragments (DFs) amplified by RAPD-PCR of three Streptomyces strains (S.
lateritius isolate 10, S. cacaoi subsp. asoensis isolate 12 and S. phaeopurpureus isolate 13) using
7 RAPD-PCR primers
No.
Isolate 10
Isolate 12
Isolate 13
OPA11-01
OPA11-02
OPA11-03
OPA11-04
OPA11-05
OPA11-06
OPA11-07
OPA11-08
OPB15-09
OPB15-10
OPB15-11
OPB15-12
OPB15-13
OPC07-14
OPC07-15
OPC07-16
OPC07-17
0
1
1
1
1
1
1
1
1
0
1
1
1
1
0
0
1
0
1
1
0
1
1
0
1
0
0
0
1
1
0
0
1
1
1
1
0
0
0
1
1
1
0
1
0
0
0
0
1
0
0
67
Type of
fragment
PU(+)
M
PU(-)
PU(+)
PU(-)
M
PU(-)
M
PU(+)
PU(+)
PU(+)
PU(-)
PU(-)
PU(+)
PU(+)
PU(+)
PU(-)
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
No.
Isolate 10
Isolate 12
Isolate 13
OPC07-18
OPC07-19
OPC07-20
OPC07-21
OPC07-22
OPC07-23
OPC07-24
OPC18-25
OPC18-26
OPC18-27
OPC18-28
OPC18-29
OPC18-30
OPC18-31
OPC18-32
OPC18-33
OPC18-34
OPC18-35
OPC18-36
OPC18-37
OPC18-38
OPC18-39
OPC18-40
OPD04-41
OPD04-42
OPD04-43
OPD04-44
OPD04-45
OPD04-46
OPE04-47
OPE04-48
OPE04-49
OPE04-50
OPE04-51
OPE04-52
OPE04-53
OPE04-54
OPE04-55
OPE04-56
OPE04-57
OPE04-58
OPO14-59
OPO14-60
OPO14-61
1
0
1
0
0
0
1
0
0
0
1
0
0
0
1
0
0
1
1
0
0
0
0
0
0
1
0
1
1
1
0
0
1
1
1
0
1
0
1
1
1
0
1
0
0
1
0
1
1
1
0
1
1
1
1
0
0
0
0
1
1
0
0
1
1
0
0
0
0
0
1
1
0
0
0
0
1
0
1
1
0
1
1
1
1
1
0
1
0
0
1
0
1
0
1
0
1
0
1
1
1
1
0
0
0
1
1
0
1
1
1
1
1
1
0
0
0
0
1
1
1
0
0
0
0
0
0
1
1
0
0
0
68
Type of
fragment
PU(+)
PU(+)
PU(-)
PU(+)
PU(-)
PU(+)
PU(-)
PU(+)
PU(-)
PU(+)
M
PU(+)
PU(+)
PU(+)
PU(+)
PU(+)
PU(-)
PU(-)
PU(-)
PU(+)
PU(-)
PU(+)
PU(+)
PU(+)
PU(+)
M
PU(+)
PU(-)
PU(-)
PU(+)
PU(+)
PU(+)
M
PU(+)
PU(-)
PU(+)
PU(+)
PU(+)
PU(-)
M
M
PU(+)
PU(+)
PU(+)
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
No.
Isolate 10
Isolate 12
Isolate 13
OPO14-62
OPO14-63
OPO14-64
OPO14-65
OPO14-66
OPO14-67
OPO14-68
OPO14-69
OPO14-70
OPO14-71
OPO14-72
1
0
0
0
1
1
1
1
0
1
1
1
1
0
1
0
0
0
1
0
0
0
0
0
1
0
1
1
1
0
1
0
0
Type of
fragment
PU(-)
PU(+)
PU(+)
PU(+)
PU(-)
PU(-)
PU(-)
PU(-)
PU(+)
PU(+)
PU(+)
+: Present. -: Absent. PU: Polymorphic (Unique). M: Monomorphic
Table.2 Similarity matrix between the three Streptomyces strains (S. lateritius isolate 10, S.
cacaoi subsp. asoensis isolate 12 and S. phaeopurpureus isolate 13) based on RAPD-PCR
analysis
Streptomyces strains
S. lateritius isolate 10
S. cacaoi subsp. asoensis
S. phaeopurpureus isolate 13
Isolate 10
30.6
19.0
100
Isolate 12
34.4
100
Isolate 13
100
Table.3 Sequences producing significant alignments between the forward direction of S.
lateritius isolate 10 and the related overseas bacterial strains
Accession
Description
JQ962978.1
Uncultured Streptomyces sp. clone BlueGSA-KSA 16S rRNA gene, partial
sequence
Uncultured Streptomyces sp. clone neauD50 16S rRNA gene, partial sequence
Uncultured Streptomyces sp. clone
ASC834 16S rRNA gene, partial
sequence
Uncultured Streptomyces sp. clone
ASC835 16S rRNA gene, partial
sequence
Uncultured Streptomyces sp. clone
ASC825 16S rRNA gene, partial
sequence
Streptomyces lateritius strain :LMG
19372 16S rRNA, complete sequence
GQ494994.1
JQ358608.1
JQ358606.1
JQ358604.1
AJ781326.1
69
Total
score
881
Query
coverage (%)
81
Max
identity (%)
80
870
79
81
876
79
81
887
79
81
898
79
81
911
79
81
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
Figure.1 Electrophoresis of RAPD-PCR using agarose gel (1.2%) stained with ethidium bromide
shows DNA polymorphisms of S. lateritius isolate 10, S. cacaoi subsp. asoensis isolate 12 and S.
phaeopurpureus isolate 13 using OPA11; OPB15; OPC07; OPC18; OPD04; OPE04 and OPO14
RAPD-PCR primers. M: 100 bp DNA Ladder
Figure.2 Genetic relationship based on RAPD-PCR analysis between the three Streptomyces
strains (S. lateritius isolate 10, S. cacaoi subsp. asoensis isolate 12 and S. phaeopurpureus isolate
13) using 7 RAPD-PCR primers
70
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
Figure.3 Final nucleotide sequence (1474 nts) of the PCR product of 16S rRNA gene amplified
from the DNA of S. lateritius isolate 10 (Uncultured Streptomyces sp. gene for 16S ribosomal
RNA, partial sequence, and clone: BSAA, GeneBank: AB915914)
AGAATGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAGATGAGGTGCTTGCACCTTATCTTA
GCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTCCGAAAGGAATGCTA
ATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCG
GATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGC
CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGG
AACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGA
GGCTACTGAGACTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGCCA
GCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGATTTACTGGGCGTAAAGCGTGCGTAGGCGGCT
TTTTAAGTCGGATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGGAAGCTAGAGTATG
GGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAG
GCAGCCATCTGGCCTAATACTGACGCTGAGGTACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGG
TAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGA
TAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGC
GGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATACTAGAAACTTTC
CAGAGATGGATTGGTGCCTTCGGGAATCTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA
GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTACTTGCCAGCATTTCGGATGGGAACTTT
AAGGATACTGCCAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAG
GGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACACAGCGATGTGATGCTAATCTCAAAAAG
CCGATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCA
GAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACC
AGAAGTAGGTAGTCTAACCGCAAGGAGGACGCTTACCACGGTGTGGCCGATGACTGGGGTGAAGTCGTAC
AGGG
Figure.4 Phylogenetic tree of nucleotide sequence of forward direction of the PCR product of
16S rRNA gene amplified from the DNA of S. lateritius isolate 10 and the related universal
bacteria strains
71
Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73
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