iosr journal of pharmacy and biological sciences (iosr-jpbs)
e-issn: 2278-3008, p-issn:2319-7676. volume 10, issue 4 ver. iii (jul - aug. 2015), pp 53-67
www.iosrjournals.org
doi: 10.9790/3008-10435367 www.iosrjournals.org 53 | page
detection of streptomycin production gene from actinomycetes spp. isolated from agricultural soils in hillah city /iraq ehsan f. hussain eman m. jarallah*, ali.h.al-saadi babylon university /college of science /dept. of biology abstract: one hundred of agricultures soil samples have been collected from different locations in hillah city. twenty one streptomyces isolates were obtained from these samples. these isolates have been cultured and purified on international streptomyces project type-2 (isp-2). antibacterial agents were extracted from these fermentations cultures and tested against gram positive bacterial species (staphylococcus albus, staphylococcus aureus and streptococcus pyogenes) and gram negative species (klepsilla pneumonia, escherichia coli, pseudomonas aeruginosa, serratiamarcescens and aeromonashydropinghila) on muller hinton agar. tenstretopmycesspp from these isolates were found have antibacterial activity, these isolates were named as symbols such as s.k-5, s.m.a -17, s.n-22, s.m-34, s.s-46, s.h-52, s.h.a-65, s.k-72, s.k.a-83 and s.a-98. these isolates were identified by molecular method by used pcr for identified the 16s rdna gene as well as the morphological methods, which included light microscope for study the aerial and substrate mycelia and electron microscope for study of spores surface morphology and the results revealed all these isolates were belonged tostreptomtces spp. in addition to used pcr for identification ofstra streptomycin phosphortransferase gene which responsible for streptomycin production. keywards: actinomycetes , electron microscope, genes
i. introduction
actinomycetes are gram positive bacteria that are distributed in water and some colonizing plants but widely found in soil, the types and species of these microorganisms in ecosystem are determined by numerous of physical, chemical and biological factors such as showing marked chemical and morphological diversity, also they produce branching mycelium, which are two kinds, substrate mycelium and aerial mycelium, in addition they are potential source of many bioactive compounds, which have large clinical effects and important applications in medicine (varghese et al., 2012). consequently, the of isolating of novel actinomycetes strains from the soils that used in searching for novel products has switched in way to obtain rarer genera of actinomycetes or to well characterized ones that are found in unusual environments, however, novel genera can be isolated by taking into account several factors during the isolation procedures, such as, selection of ecological locations for sample collection, chemical and physical factors of the collection samples, like use of specific selective media, of culture conditions and genus or specific isolation methods (khannaet al., 2011). actinomycetes, which are phylogenetically defined as a group within the high guanine (g) and cytosine (c) gram positive bacteria and they are one of the major populations of microbial communities in soils and more types of which have been isolated from this part of environment and also these microorganisms have been isolated from decaying and sound stone (abdulla et al., 2008 pan et al., 2013). marine environment form 70 % of the earth’s surface which provides the largest inhabitable space for living organisms, particularly microbes, and as largely source for the isolation of new microorganisms with potentiality to produce active secondary metabolites, such actinomycetes (baskaranet al., 2011).
ii. materials and methods
one hundred soils samples have been collected from different locations in hilla city, about one gram of the soil samples were taken from soils top about 5 to 10 cm in depth. the soil samples when collected from these regions are taken with an auger and placed in dry and sterile polyethylene tubes and stored at 4°c until use. soil samples were pretreated with calcium carbonate to reduce the number of vegetative bacterial cells and allowing streptomyces spores to survive, this method was required for inhibiting unwanted bacteria and remain only test bacteria (pordeliet al.,2013). isolation and purification of streptomyces spp from soil samples
the air dried samples are mixed and passed through the 2mm sieve filter to remove gravel, large stone and debris. after this the samples were incubated at 55°c in an incubator for 5 min. 1g of soil was dissolved in 9ml of distilled water and successive dilutions was made up to 105. serial dilutions were spread plated on isp-2 media (international streptomyces project type-2 media) using dilution plate technique. antibiotics like
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 54 | page
nalidixic acid and nystatin were added to minimize microbial contamination. all the plates were incubated at 30°c in an incubator for 5-7 days. after incubation, the actinomycete growing colonies were selected and purified by subculturing on isp-2 agar medium plates according to type and forms of these colonies. then the purified colonies examined under light microscope at (10x). after this, the typical growing colonies of streptomyces cultured on international streptomyces project type-2 agar slants and stored at 4°c for further uses (deepthiet al.,2012). amplification of 16s rdna gene of streptomyces spp tow 16s rdna genes primes, forward and reverse were designed as show in the following table. table: 1. primers sequence for identification streptomyces app nucleotides sequence bases no. annealing temp. references
f: 5´-aagccctggaaacggggt-3´
18 bp
55 °c
dehnadet al., 2010
r: 5´-cgtgtgcagcccaagaca-3´
18 bp
gradient amplification of 16s rdna gene at different annealing temperatures for streptomyces spp pcr amplification was performed using master mix (bioneer, korea), (2 ?l) primers, (1 ?l) for forward primer and(1 ?l) reverse primer, (1.5 ?l) chromosomal dna, and the final volume was reached to (20 ?l). byadded (16.5 ?l) of demonized water. the pcr amplification was performed using the thermal cycler program as follows: 94?c for 5 min as a primary denaturation step, 35 cycles of 94?c for 1 min as denaturation step, (52.3 °c, 55 °c, 57.5 °c and 60 °c) for 1 min as annealing temperature and final extension was 72?c for 10 min. the pcr products were visualized using gel electrophoresis on 1% agarose and compared with 1 kb dna ladder. amplification of 16s rdna gene at (55 ?c) annealing temperatures for streptomyces spp pcr amplification was performed using master mix (bioneer, korea), (2 ?l) primers, (1 ?l) for forward primer and(1 ?l) reverse primer, (1.5 ?l) chromosomal dna, and the final volume was reached to (20 ?l). by added (16.5 ?l) of demonized water. the pcr amplification was performed using the thermal cycler program as follows: 94?c for 5 min as a primary denaturation step, 35 cycles of 94?c for 1 min as denaturation step, (55 ?c) for 1 min as annealing temperature and final extension was 72?c for 10 min. the pcr products were visualized using gel electrophoresis on 1% agarose and compared with 1 kb dna ladder. amplification of stra gene that codes for biosynthetic streptomycin phosphortransferase gene of streptomyces spp tow stra streptomycin phosphortransferase genes primes, forward and reverse were designed as show in the following table:. table: 2. primers sequence for identificationstra gene nucleotides sequence basesno. annealing temp. refer.
stra-f: 5´-atgagttcgtcggaccacat-3´
20 bp
59.1°c
lim et al., 1989
stra-r: 5´-tcagggcttcgccagcgctt-3´
20 bp
gradient amplification of stra gene at different annealing temperatures for streptomyces spp pcr amplification was performed using master mix (bioneer, korea), (2 ?l) primers, (1 ?l) for forward primer and(1 ?l) reverse primer, (1.5 ?l) chromosomal dna, and the final volume was reached to (20 ?l). by added (16.5 ?l) of demonized water.
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 55 | page
the pcr amplification was performed using the thermal cycler program as follows: 94?c for 5 min as a primary denaturation step, 35 cycles of 94?c for 1 min as denaturation step, (52.8 °c, 57 °c, 59.1 °c and 61.3 °c) ?c for 1 min as annealing temperature and final extension was 72?c for 10 min. the pcr products were visualized using gel electrophoresis on 1% agarose and compared with 1 kb dna ladder. amplification of stra gene at (59.1 °c) annealing temperatures for streptomyces spp pcr amplification was performed using master mix (bioneer, korea), (2 ?l) primers, (1 ?l) for forward primer and(1 ?l) reverse primer, (1.5 ?l) chromosomal dna, and the final volume was reached to (20 ?l). by added (16.5 ?l) of demonized water. the pcr amplification was performed using the thermal cycler program as follows: 94?c for 5 min as a primary denaturation step, 35 cycles of 94?c for 1 min as denaturation step, (59.1 °c) ?c for 1 min as annealing temperature and final extension was 72?c for 10 min. the pcr products were visualized using gel electrophoresis on 1% agarose and compared with 1 kb dna ladder.
iii. results and discussion
isolation of streptomyces from agricultures soils one hundred agricultures soil samples have been collected from different locations in babylon city, twenty one actinomycetes isolates obtained from these samples. these isolates have been cultured and purified on international streptomyces project type-2 (isp-2) (table: 3). table: 3. number of actinomycetes isolates that obtained from agricultures samples no. site of collection no. of site soils samples sequence of site samples actinomycetesno. samples (%)
1
al-kothar
10
1 - 10
3
14.286%
2
al-mahaweel
10
11 - 20
2
9.524%
3
al-nile
10
21 - 30
2
9.524%
4
al-mussaiab
10
31 - 40
4
19.046%
5
al –sadda
10
41 - 50
3
14.286%
6
al-hashmia
10
51 - 60
1
4.762%
7
al-hamza
10
61 - 70
1
4.762%
8
al-kasim
10
71 – 80
2
9.524%
9
al-karama
10
81 – 90
1
4.762%
10
al-annana
10
91 – 100
2
9.524%
total
100
1 - 100
21 isolates
21 %
secondary screening test for actinomycetes isolates against pathogenic bacteria the size of inhibition zones of activity of stretopmycessppcultures extracts againstpathogenicgram negative bacteria(klepsilla pneumonia, escherichia coli, pseudomonasaeruginosa ,serratiamarcescens and aeromonashydropinghila (measured by milliliter and found the rage of these zones from zero mm to 22.5 mm ,this mean thecultures extracts of this bacteria have no or moderate or strong effect against these pathogenic bacteria (table: 4).and in comparison to the size of inhibition zones of activity of stretopmycessppcultures extracts against pathogenic gram positive bacteria (staphylococcusaureus ,streptococcuspyogenes and staphylococcusalbus)measured by milliliter and found the rage of these zones from zero mm to 27.5 mm ,this mean the cultures extracts of this bacteria also have no or moderate or strong effect against these pathogenic bacteria (table: 5).
table: 4. measurements of inhibition zones formed by actinomycetes extract against pathogenic gram positive bacterial species mean of inhibition zones formed by actinomycetes extract against pathogenic gram positive bacteria measured by millimeter (mm)±se antibacterial extracts of actinomycetes s. aureus s. pyogenes s. albus
s.k-5
-
-
7.5±0.3
s.m.a -17
15.5±0.2
17.5±0.2
-
s.n-22
14.5±0.1
24.5±0.3
-
s.m-34
19.5±0.3
19.5±0.1
6±0.2
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doi: 10.9790/3008-10435367 www.iosrjournals.org 56 | page
s.s-46
6±0.2
-
-
s.h-52
25±0.3
-
8.5±0.5
s.h.a-65
-
5±0.4
8.5 ±0.2
s.k-72
27.5±0.2
20.5±0.1
24.5±0.2
s.k.a-83
-
5±0.2
10.5±0.4
s.a-98
24.5±0.2
21±0.1
11±0.3
*each record represents mean of threereplicate this table explained the test of secondary screening activity of actinomycetesspp extract against pathogenic gram positive bacteria and results showed the range was from zero mm to 27.5 mm. table: 5. measurements of inhibition zones formed by actinomycetes extract against pathogenic gram negative bacterial species inhibition zone formed by actinomycetes extract against pathogenic gram negative bacteria measured by millimeter (mm)* ±se antibacterial extracts of actinomycetes k. pneumonia e. coli p. aeruginosa s. marcescens a. hydropinghila
s.k-5
-
20.5±0.1
2.5±0.2
6.5±0.5
-
s.m.a -17
-
-
-
13±0.3
-
s.n-22
9.5±0.2
9±0.1
-
3.5±0.1
1±0.1
s.m-34
2±0.1
-
2 ±0.1
6±0.2
2±0.1
s.s-46
6.5±0.2
-
6.5 ±0.2
11±0.2
8.2±0.2
s.h-52
1±0.2
13±0.5
15 ±0.2
-
2±0.2
s.h.a-65
-
8±0.5
1.5 ±0.3
-
-
s.k-72
9.5±0.1
10±0.2
16±0.2
19.5±
22.5±0.4
s.k.a-83
2.5±0.1
20±0.2
5.5 ±0.2
-
-
.a-98
6.5±0.3
11±0.3
22.5±0.1
21±0.1
9±0.6
*each record represents mean of three replicate this table explained the test of secondary screening activity of actinomycetesspp extract against pathogenic gram negative and the results showed range was from zero mm to 22.5 mm. when comparison of these results with the results of close study for prabhahar and his group (2014) were isolated of actinomycetes from different locations in muthuppettai mangrove areas in india and test antibacterial activity of these isolates against pathogenic bacteria, this found the size of inhibition zone range from zero mm to 18 mm against pathogenic negative bacteria and from zero mm to 23 mm against pathogenic gram positive bacteria (prabhaharet al., 2014). and incomparison of the results with the results of different study for singh and his workers that isolation of actinomycetes from different soils samples in n delhi in india and test antibacterial activity of these isolates against pathogenic bacteria, this found the size of inhibition zone range from zero mm to 14 mm against both of gram positive and negative pathogenic bacteria (singh et al., 2012). morphological identification of actinomycetes isolates the morphological identification of streptomycesspp involved the study of textures and colors of colonies on international streptomyces project (isp) systems as well as the study of aerial and substrate mycelia morphology under light microscope at 1000 x, in addition, the study of the morphological characteristics of spores with scanning electron microscope for identifications of spores surface properties.
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a. shape of aerial mycelium on isp-2 agar b. shape of substrate mycelium on isp-2 agar
figure: 1. morphological properties ofs.k-5 isolate figure (1. a) showed the colors of aerial mycelium and figure (1. b) explained substrate mycelium in on isp-2 agar, in addition figure (1. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (1. d) explained the shape and surface morphology of spores under electron microscope at 21000 x.
d. shape of spores under electron
microscope at 21000 x
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
c. shape of mycelium under light
microscope at 1000 x with gram stain
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
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figure: 2. morphological properties ofs.m.a-17isolate figure (2. a) showed the colors of aerial mycelium and figure (2. b) explained substrate mycelium in on isp-2 agar, in addition figure (2. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (2. d) explained the shape and surface morphology of spores under electron microscope at 14500 x.
figure: 3. morphological properties ofs.n-22 isolate
c. shape of mycelium under light
microscope at 1000 x with gram stain
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on
c. shape of mycelium under light microscope at 1000 x with gram stain
d. isp-2 agar shape of spores under electron microscope at 27000 x
d. shape of spores under electron
microscope at 14500 x
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
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figure (3. a) showed the colors of aerial mycelium and figure (3. b) explained substrate mycelium in on isp-2 agar, in addition figure (3. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (3. d) explained the shape and surface morphology of spores under electron microscope at 27000 x.
figure: 4. morphological properties ofs.m-34 isolate figure (4. a) showed the colors of aerial mycelium and figure (4. b) explained substrate mycelium in on isp-2 agar, in addition figure (4. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (4. d) explained the shape and surface morphology of spores under electron microscope at 14500 x.
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
c. shape of mycelium under light microscope at 1000 x with gram stain
d. shape of spores under electron microscope at 14500 x
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
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figure: 5. morphological properties ofs.s-46 isolate figure (5. a) showed the colors of aerial mycelium and figure (5. b) explained substrate mycelium in on isp-2 agar, in addition figure (5. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (5. d) explained the shape and surface morphology of spores under electron microscope at 11000 x.
figure: 6. morphological properties ofs.h-52 isolate figure (6. a) showed the colors of aerial mycelium and figure (6. b) explained substrate mycelium in on isp-2 agar, in addition figure (6. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (6. d) explained the .shape and surface morphology of spores under electron microscope at 14500 x.
c. shape of mycelium under light microscope at 1000 x with gram stain
d. shape of spores under electron microscope at 11000 x
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
c. shape of mycelium under light microscope at 1000 x with gram stain
d. shape of spores under electron microscope at 14500 x
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
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figure: 7. morphological properties ofs.h.a-65 isolate
figure (7. a) showed the colors of aerial mycelium and figure (7. b) explained substrate mycelium in on isp-2 agar, in addition figure (7. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (7. d) explained the shape and surface morphology of spores under electron microscope at 9800 x. figure: 8. morphological properties ofs.k-72 isolate
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
c. shape of mycelium under light microscope at 1000 x with gram stain
d. shape of spores under electron microscope at 9800 x
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 62 | page
figure (8. a) showed the colors of aerial mycelium and figure (8. b) explained substrate mycelium in on isp-2 agar, in addition figure (8. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (8. d) explained the shape and surface morphology of spores under electron microscope at 18500 x. figure: 9. morphological properties ofs.k.a-83 isolate
figure (9. a) showed the colors of aerial mycelium and figure (9. b) explained substrate mycelium in on isp-2 agar, in addition figure (9. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (9. d) explained the shape and surface morphology of spores under electron microscope at 24000 x.
c. shape of mycelium under light microscope at 1000 x with gram stain
d. shape of spores under electron microscope at 18500 x
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
c. shape of mycelium under light microscope at 1000 x with gram stain
d. shape of spores under electron microscope at 24000 x
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 63 | page
figure: 10. morphological properties ofs.a-98 isolate
figure (10. a) showed the colors of aerial mycelium and figure (10. b) explained substrate mycelium in on isp-2 agar, in addition figure (10. c) show the color and morphology of mycelium under light microscope at 1000 x with gram stain, while figure (10. d) explained the shape and surface morphology of spores under electron microscope at 20000 x. the present results of colonies characteristics approximately agreed with the results of zin and his group (2011) were isolated the streptomycesspp from the soil of peripheral area in university putra in malaysia and study the shapes of colonies and aerial with substrate mycelia colors for these microorganisms on isp-2 media (zinet al., 2011).also the presented results approximately agreed with the results of balasubramaniam and his students (2011) were isolate the actinomycetes from soils in india and study the morphological characteristics for these bacteria (balasubramaniamet al., 2011). the current results of mycelia morphology agreed with the results of rakshanya and his students (2011) were isolated the actinomycetes from soils and study the antibacterial against different human pathogen bacteria and, also study the mycelia morphological characteristics with oil immersion under light microscope for these bacteria (rakshanyaet al., 2011). also the current results of mycelia morphology agreed with the results of janardhan and his group (2014) were collected the actinomycetes from soil in nellore region of andhra pradesh in india, and screened for these ability to produce bioactive compounds and, also study the morphological characteristics of mycelia for these bacteria (janardhanet al., 2014). the results of scanning electron microscope approximately agreed with the atta and his group (2012) were isolated streptomyces form soils in egypt and examined of spores morphology with scanning electron microscope at magnification bower 15000 x, and el-batal and his student (2014) were isolated streptomyces form sediments in egypt and examined of spores with scanning electron microscope at magnification bowers 8500 x and 14000 x , both of them found these spores have smooth surface (atta et al.,2012 el-batalet al.,2014).
a. shape of aerial mycelium on isp-2 agar
b. shape of substrate mycelium on isp-2 agar
c. shape of mycelium under light microscope at 1000 x with gram stain
d. shape of spores under electron microscope at 20000 x
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 64 | page
molecular identification of streptomyces spp isolates by amplification of 16s rdna gene and optimization of amplification conditions for this gene :after identification of streptomyces by morphological characteristics, these isolates then subjected to molecular identification depended on amplification of 16s rdna gene by polymerase chain reaction. pcr performed at different annealing temperatures for amplification of genomic dna by using the (st-f) and (st-r) primers in order to determine the appropriate annealing temperature for identification of streptomycesisolates. the results revealed the 55 °c was the suitable annealing temperature. after this, all isolates then identified by amplification of thegenomic dna at 55 °c. the gradient amplification of 16s rdna genefor three streptomycesisolates were s.k-5, s.h-52 and s.a-98 at four different annealing temperatures were done in order to determine the optimal amplification temperature. four temperatures were tested (52.3 °c, 55 °c, 57.5 °c and 60 °c). the results exhibited the (55 °c) it specific temperature for amplification 16s rdna gene, (figurer: 11). the amplification of 16s rdnagenefor streptomyces spp isolates at(55 °c)annealing temperatures by the polymerase chain reaction and the product was approximately (1500 bp).however, this used for identification of streptomyces species isolates,(figurer: 12).
figure: 11. gradient amplification of 16s rdna genes for three streptomyces isolates at different annealing temperatures
figure: 12. amplification of 16s rdna genes for all streptomyces isolates at (55°c) annealing temperatures
dna-ladder
55°c
55°c
55°c
55°c
55°c
55°c
55°c
55°c
55°c
55°c
~1500 bp
isolates
temp.
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 65 | page
these results similar to maleki (2013) who used same the 16s rdna gene (st-f) and (st-r) primers for identification of high antimicrobials stretopmycesspp isolates, were isolated from soils in northwest of iran and these gave dna bands ~1500 bp in length (malekiet al., 2013). however, in the genetic application the 16s rdna gene is more simple, specific and yet efficient in identification of new streptomyces strains (anderson andwellington.,2001). in addition, the 16s rdna gene has less changes and transformation through evolution, and for these it is deemed for taxonomic studies as well as the 16s rdna gene containing of five variable regions which are ?, ?, ?, ?, and particularly variable ? part which shows relatively high polymorphism at the 5? end of its structure (stachet al., 2003). therefore, the identification of new strains of streptomyces by using amplification of hyper variable regions have been frequently described (pandeyand mishra., 1995).the 16s rdna gene is mainly used for molecular identification of bacteria, some regions of this gene are highly conserved and common to all types of bacteria while others are specific to species of these microorganisms (jihaniet al.,2012). and for this the 16s rdna analysis has great advantage for the molecular identification of the actinomycetes(provost et al., 1997). the 16s rdna used for molecular identification of new strain of streptomyces with high capacity for antibiotic production (oh et al., 2005). molecular identification ofstra gene for streptomyces spp isolates and optimization of amplification conditions for this gene the streptomyces isolates then subjected to molecular identification of the stra streptomycin phosphortransferase genes by polymerase chain reaction. pcr performed at different annealing temperatures for amplification of genomic dna by using the (stra-f) and (stra-r) primers for known appropriate annealing temperature. the results found the 59.1 °c was the suitable annealing temperature. after this, all isolates then identified by amplification of thegenomic dna at 59.1 °c. the results showed the gradient amplification of stra genefor tow streptomyces spp isolates were s.k.a-83and s.a-98 at four different annealing temperatures in order to determine the optimal amplification temperature. these temperatures were (52.8 °c, 57 °c, 59.1 °c and 61.3 °c). the amplification results found the (59.1 °c) was specific temperature for amplification of stragene, (figurer: 13). the results revealed the amplification of stra genefor streptomyces isolates. the best annealing temperatures was (59.1 °c). the polymerase chain reaction product approximately (920 bp).however, this used for identification ofstra streptomycin phosphortransferase gene for streptomyces species isolates and found the s.a-98 isolate gave dna bands (~920 bp)in length, (figurer: 14).
figure: 13. gradient amplification of stra genes for two streptomyces isolates at different annealing temperatures
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 66 | page
figure: 14. amplification of stra genes for all streptomyces isolates at (59.1°c) annealing temperatures. these results were agreed with huddleston and his group (1997) were collected fifty-three isolates from rhizosphere soil in brazilian and found approximately half of these strains were streptomycin resistant, these streptomyces griseus strains then screened for the presence of stra by using same our (stra-f) and (stra-r)primers for identification of streptomycin producing and found only twelve of thesestrains produced streptomycin and gave dna fragments at ~ 920 bp in length (huddleston et al., 1997). the stra genes were identified by number of pcr methods (piccard et al., 1992). these genes were isolated from streptomyces (tolbaet al., 2002). anumber of s. griseus strains possess the stra streptomycin resistance gene within the streptomycin biosynthetic gene cluster to avoid suicide (laskariset al., 2010). the production of streptomycin associated with determine of streptomycin phosphotransferasestra gene that the presence in streptomycesgriseus, there were also significant differences in the stra of homologue groups between different soil sites, demonstrated that the composition of the populations of these genes varies across different regions (laskariset al., 2012). references
[1]. abdulla, h. may, e. bahgat, m and dewedar, a. (2008). characterization of actinomycetes isolated from ancient stone and their potential deterioration. polish journal of microbiology. 57(3): 213-220.
[2]. anderson, a. s. and wellington, e. m. h. (2001). the taxonomy of streptomyces and related genera. int j systevolmicrobiol. 3: 797-814.
[3]. atta, h . m. el-sayed, a.s. el-desoukey, m.a. hassan, m. and el-gazar, m. (2012). biochemical studies on the natamycin antibiotic produced by streptomyces lydicus: fermentation, extraction and biological activities. journal of saudi chemical society. http://dx.doi.org/10.1016/j.jscs. 04.001 : 1-12.
[4]. balasubramaniam, v. ganesh, s. karunaithi, v and perumal, p. (2011). improved culturing, screening and fermention of soil actinomyctes for antimicrobial agents. int. j. pharm and ind. res. 1(2): 153-159.
[5]. baskaran, r.vijayakumar, r. and mohan, p. m. (2011). enrichment method for the isolation of bioactive actinomycetes from mangrove sediments of andaman islands, india. malaysian journal of microbiology. 7(1): 26-32.
[6]. deepthi, m. k.,sudhakar. m. s., and devamma, m. n(2012).isolation and screening of streptomyces spp. from cornicamangrove soils for enzymes production and antimicrobial activity. international journofpharma. chem. and bio. scin. 2(1), 110-116.
[7]. el-batal, a. i. el-sayed, m. h. refaat, b. m. and aska a. a. z. (2014). marine streptomyces cyaneusstrain alex-sk121 mediated eco friendly synthesis of silver nanoparticles using gamma radiation. british journal of pharmaceutical research. 4(21): 2525- 2547.
[8]. huddleston, a. s. neil ,c. m. cristina, p. n. john, e. b. simon b. d. and elizabethm. h. w. (1997). molecular detection of streptomycin producing streptomycetes in brazilian soils. appl. environ. microbiol. (63) 4: 1288–1297.
[9]. janardhan, a. kumar, a. p. viswanath, b. saigopal, d. v. r. and d. v. r. narasimha, d. v. r. (2014). production of bioactive compounds byactinomycetes and their antioxidant properties. biotechnology research international. http://dx.doi.org/10.1155/2014/217030: 1-8.
[10]. jihani, s. brodolin, k. iraqui, m. ibnsouda, s and haggoud, a(2012). isolation and molecular identification of antibiotic-producing actinomycetes from an old house in the medina of fez, morocco. african journal of microbiology research. 6 (47): 7370-7376.
biotinyl-tyramide-based in situ hybridization signal patterns in the detection of high-risk human
doi: 10.9790/3008-10435367 www.iosrjournals.org 67 | page
[11]. khanna, m.solanki, r. and lal, r .(2011). selective isolation of rare actinomycetes producing novel antimicrobial compoundes. international journal of advanced biotechnology and research issn 0976-2612. 2(3): 357-375.
[12]. laskaris, p.sekine, t. and wellington, e. m. h. (2012). diversity analysis of streptomycetes and associated phosphotranspherase genes in soil. plos one. (7) 4: 1-9.
[13]. laskaris, p.tolba, s. calvo-bado, l. and wellington, e. m, (2010). coevolution of antibiotic production and counter-resistance in soil bacteria. environ microbiol. 12: 783–796.
[14]. maleki, h.dehnad, a. hanifian, s. and khani, s. (2013). isolation and molecular identification of streptomyces spp. with antibacterial activity from northwest of iran. bioimpacts. 3 (3): 129-134.
[15]. oh, s. t. lee, j. j. lee, j. y. kim, j. k. yang, s. y.and kim, y. s. (2005). isolation and identification of streptomyces sp. producing anti-vancomycin resistant staphylococcus aureus substance. hangugmisaengmulsaengmyeong gong haghoeji. 2: 90-5.
[16]. pan, s. y. tan, g. y. a. convey, p. pearce, d. a. and irene k. p. (2013).diversity and bioactivity of actinomycetes from signy islandterrestrial soils, maritime antarctic. advances in polar science. 24(4): 208-212.
[17]. pandey, m. and mishra, s. (1995). cloning and expression of b-glucosidase gene from the yeast pichiaetchellsii. j ferment bioengineer. 5: 446-53.
[18]. piccard, c. ponsonnet, c. paget, e. nesme, x. andsimonet. p. (1992). detection and enumeration of bacteria in soil by direct dna extraction and polymerase chain reaction. appl. environ. microbiol. 58: 2717–2722.
[19]. pordeli, h.,hashemi, s.j., jamshidian, m., and bayat, m.(2013).isolation, molecular identification and evaluation of antifungal activity of soil streptomycesagainstdermatophytes. world research journal of biotechnology. 1(1): 04-06.
[20]. prabhahar, c.saleshrani, k.andenbarasan, r. (2014). isolatin and characterization of marine actinomycetes from muhuppettaimangroves ,tamilanu, india. international journal of recent scientific research 5(4) 906-910.
[21]. provost, f. laurent, f. camacho uzcategui, l. r. and boiron, p. (1997). molecular study of persistence of nocardiaasteroides and nocardiaotitidiscaviarumstrains in patients with long term nocardiosis. j. clin. microbiol. 35: 1157-1160.
[22]. rakshanya. j. u.shenpagam .n. h. and kanchana d. d. (2011). antagonistic activity of actinomycetes isolates against human pathogen. j. microbiol. biotech. res. 1 (2): 74-79.
[23]. singh, s. kumar, p. gopalan, n. shrivastava, b. kuhad, r. c. and chaudhary, h. s. (2012). isolation and partial characterization of actinomycetes with antimicrobial activity against multidrug resistant bacteria. asian pacific journal of tropical biomedicine. doi: 10.1016/s2221-1691(12)60375-x: s1147-s1150.
[24]. stach, j. e. m. maldonado, l. a. ward, a. c. goodfellow, m. and bull, a. t. (2003). new primers for the class actinobacteria: application to marine and terrestrial environments. environ microbiol. 10: 828-41.
[25]. tolba, s. egan, s. kallifidas, d. and wellington, e. m. (2002). distribution of streptomycin resistance and biosynthesis genes in streptomycetes recovered from different soil sites. fems microbiol ecol. 42: 269–276.
[26]. varghese, r.nishamol, s. suchithra, r. and mohamed, h. a. a.(2012). biochemical and physiological characteristics of actinomycetes isolated from high altitude shola soils of tropical montane forest.indian j. innovations dev. 1(3): 142-144.
[27]. zin, n. z. m. tasrip, n. a. mohdnasirmohddesa, m. n. m. kqueen, c. y.zakaria, z. a. hamat, r. a. and shamsudin, m. n. (2011).characterization and antimicrobial activities of two streptomyces isolates from soil in the periphery of university putra malaysia. tropical biomedicine.(28)3: 651–660.