The Pharma Innovation Journal 2020; 9(1): 18-24

ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.03 Molecular Identification of Str A gene responsible for TPI 2020; 9(1): 18-24 © 2020 TPI production from isolates www.thepharmajournal.com Received: 11-11-2019 Accepted: 15-12-2019 Bayader Abdel Mohsen, Mohsen Hashim Risan and Asma G Oraibi

Bayader Abdel Mohsen College of Biotechnology, Al- Abstract Nahrain University, Iraq The present study was aimed for molecular identification of Str A gene from . Twenty-four isolates were identified as Streptomyces sp. based on their morphological and biochemical Mohsen Hashim Risan characteristics. Twelve isolates were positive in the PCR technique. Performing PCR reactions using College of Biotechnology, Al- primer pair on DNA. The results of Str A gene detection clarify that Two isolate of Streptomyces isolates Nahrain University, Iraq gave a positive result and carrying Str gene, while 10 of Streptomyces isolates were lacking the gene. Be1 and B3-4 isolates gave DNA bands 700 bp in length. The results indicated that the Be1 and B3-4 isolates Asma G Oraibi are very close to the Streptomyces griseus responsible for producing streptomycin. College of Biotechnology, Al-

Nahrain University, Iraq Keywords: , Streptomyces, Str A, streptomycin, Iraq

Introduction The largest of Actinomycetes and the type genus of the family is (Kampfer, 2006, Al-Rubaye et al., 2018a, Risan et al., 2019) [13, 2, 28]. Over 600 species of [11] Streptomyces bacteria have been described (Euzeby, 2008) . As with the other , Streptomyces are Gram-positive, and have genomes with high guanine and cytosine content. Most Streptomyces produces spores, and are noted for their distinct "earthy" odor that results from the production of a volatile metabolite, geosmin (Madigan and Martinko 2005) [16]. They produce over two-thirds of the clinically useful of natural origin (e.g., neomycin [14] and chloramphenicol) (Kieser et al., 2000) . Streptomyces are recognized as a main number of antimicrobial derived antibiotics. Two species of Streptomyces by the name of Streptomyces griseus and Streptomyces coelicolor are used for the industrial production of Streptomycin and novel antibiotics such as dihydrogranticin respectively, doxorubicins are anticancer agents (Mukhtar et al., 2012; Al-Rubaye et al., 2018b) [21, 3] and Rapamycins are immunomodulatory [38] agents are secondary metabolites produced by Streptomyces (Ying and Marta, 2001) . Streptomyces are capable of producing secondary metabolites such as antibiotics, anthelminthic enzymes, herbicides, anticancer drugs, growth factors like vitamin B12 and immune-modulators (Hasani et al., 2014; Amin et al., 2016; Risan et al., 2016; Qasim and Risan 2017) [12, 4, 25, 24]. Streptothricin is the first antibiotic obtained from Streptomyces spp. [12] (Hasani et al., 2014) . Streptomycin gene cluster is composed of 27 genes and occupies a region of 32.6 Kb in length (Tomono et al., 2005) [34]. The cluster genes encoding enzymes for the synthesis of the streptomycin subunits are not found in subpathway-specific operons; instead, they are found in mixed operons which may indicate the need for strictly coordinated expression of the cluster [23] genes (Piepersberg and Distler, 1997) . The Streptomyces glaucescens hydroxy- streptomycin cluster has a number of genes that are homologous to the streptomycin gene cluster, however, their gene sequence identiy ranges from 80% to less than 60% and the gene order of the two clusters differs as well (Distler et al., 1992) [8].

Materials and Methods Collection of samples: Thirty samples were collected by using core samples from the city of Baghdad at various depths. They were maintained at ambient temperature and brought to the laboratory in sterile polypropylene bags for isolation of actinomycetes.

Corresponding Author: Isolation of actinomycetes: Actinomycetes were isolated by spread plate technique following Bayader Abdel Mohsen the serial dilution of soil samples on Yeast extract-malt extract agar (YEME) with Tetracyclin College of Biotechnology, Al- Nahrain University, Iraq ~ 18 ~ The Pharma Innovation Journal http://www.thepharmajournal.com

50 μg/L and Nystatin 50 μg/L, (Williams et al., 1993; Oskay al., 2018) [31, 17, 27], and some the biochemical tests described et al., 2004; Risan et al., 2017) [37, 22, 26]. by Bergey's Manual of Systematic Bacteriology 2nd Edition Volume 2 (2005). Identification of streptomyces: Light microscopical studies were carried out by cover slip culture technique and the DNA extraction NA primers characters such as aerial mycelia, spores arrangement are The DNA primers used in this study are represented in table observed under microscope. The isolated Streptomyces were (1): also cultured on ISP2 and their morphological characters were also observed. Table 1: DNA primers used in the study.

Primer Tm Seq. Characterization of Streptomyces: The Streptomyces name Size isolates selected from screening methods were characterized strA_F 5`-GCG GCT GCT CGA CCA CGA C-3` 63: by morphological and biochemical methods. Streptomyces strA_R 5`-CCG TCC TCG ATG TCC CAC AGG G-3` 570bp isolates were differentiated by their colony characters strA_f2 5`-AGG CCT CCC TCG TGS TGC-3` 58: (Kawato and Sinobu 1959; Aghighi et al., 2004) [15, 1]. Various strA_R2 5`-SGT CAG CAG GTC GAA GCG-3` 615bp biochemical tests performed were catalase, citrate utilization, Melanin production, Carbon Utilization, Indole production Kits used for gene expression test (Shirling and Gottlieb, 1966; Macfadden, 2000; Risan et The kits used for gene expression are represented in table (2):

Table 2: DNA primers used in the study.

Kits Company/ Origin TRIzol Reagent Thermo Scientific, USA GoTagqPCR Master Mix, GoTaq® 1-Step RT-qPCR System, Promega, USA MgCL2, Nuclease Free Water, Quantiflor RNA System. Isopropanol, 70% Ethanol ROMIL pure chemistry, UK Chloroform LiChrosolv, Germany Primers Macrogen, Korea

DNA primers for gene expression 9. Diluted DNA transfer to a clean tube containing 600μl of The DNA primers for gene expression in this study are room temperature isopropanol. After mixing gently, represented in table (3): centrifuge as in “Pellet Cells” above, and supernatant was decanted. Table 3: DNA primers for gene expression used in the study. 10. From room temperature 70% ethanol, 600μl was added

Primer Name Seq. then centrifuged for 2 minutes at 13,000 rpm. strA_F2 5 5`-AGG CCT CCC TCG TGS TGC-3` 11. Ethanol was then aspirated and air-dried the pellet. strA_R 5`-SGT CAG CAG GTC GAA GCG-3` 12. DNA pellet was rehydrated in 100μl of Rehydration 16s rRNA-F 5`-CTCTTTCAGCAGGGAAGAAG-3` Solution for 1 hour at 65°C 16s rRNA-R 5`-GAGCTCTTTACGCCCAATAA-3` Quantitation of DNA Genomic DNA was isolated from bacterial growth according Quantus Florometer was used to detect the concentration of to the protocol of Wizard. Genomic DNA Purification Kit, extracted DNA in order to detect the goodness of samples for Promega as the following steps: downstream applications. For 1 μl of DNA, 199 μl of diluted 1. For pellet calls, 1ml of overnight culture for 2min at Quanty Flour Dye was mixed. After 5min of incubation at 13000 rpm. Supernatant was then discarded. room temperature, DNA concentration values were detected. 2. For gram positive bacteria, 100μl from DNA rehydration plus 100μl from Lysozyme solution were added to pellet Optimization and vortex. To examine the optimum annealing temperature of primer, the 3. Incubated in water bath for 30 min. at 37 oC. DNA template was amplified with the same primer pair, 4. After incubation, samples were centrifuged for 2min at (Forward) (Reverse), at annealing temperatures of 55, 58, 60, 13000 rpm. Supernatant then discarded. 63 and 65°C. PCR amplifications were performed with 20μl 5. From Nuclei Lysis Solution, 600μl. was added and volumes containing 10μl GoTaq Green Master Mix (2X); 1μl pipeted gently for mixing. for each primer (10pmol); 6μl nuclease free water and 2μl of 6. All mixtures were Incubate for 5 minutes at 80 °C, then template DNA.PCR cycling was performed with PCR Express cooled to room temperature. (Thermo Cycler, BioRad, USA) with the following 7. For RNA lysis, 3μl of RNase Solution was mixed, then temperature program: denatured at 94 °C for 4 min followed incubated at 37 °C for 15 minutes. by 30 cycles of denaturation at 94 °C for 30 sec; annealing at 8. For Protein Precipitation, 200μl of Protein Precipitation 55, 58, 60, 63 or 65 °C for 30 sec; and extension at 72 °C for Solution was add to cell lysate, mixed well by vortexing, 30 sec. A final extension incubation of 7 min at 72 °C was then incubated by deep freeze (-30 °C). After that, it included, followed by a 10 min of incubation at 4 °C to stop centrifuged at 13,000 rpm for 5 minutes. the reactions.

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Table 4: Reaction Setup and Thermal Cycling Protocol

Gene: strA2 No. of Reaction 12 rxn Annealing temperature of primers 58°C Reaction Volume /run 20 μl Length of PCR product (bp) 615 Safety Margin 5 % No. of PCR Cycles 30

Master mix Components Stock Unit Final Unit Volume 1 sample 12 samples Master Mix 2 X 1 X 10 121.000 Forward primer 10 μM 1 μM 1 12.1 Reverse primer 10 μM 1 μM 1 12.1 Nuclease Free Water 5 60.5 DNA 10 ng/μl 10 ng/μl 3

Total volume 20 Aliquot per single rxn 17 μl of Master mix per tube and add 3 μl of Template

Table 5: PCR Program agar media carefully to obtain a pure isolate which was

Steps °C m:s Cycle characterized as colored in aerial and substrate mycelium, Initial Denaturation 95 05:00 1 dried, rough\ smooth, with an irregular/regular margin; Denaturation 95 00:30 generally convex colony. Most colonies that were isolated

Annealing 58 00:30 30 possess earthy odors as described by Williams et al., (1983) Extension 72 00:30 [36]. Final extension 72 07:00 1 Hold 10 10:00 Selection by streaking a plate for single colonies As observed in figure (1), a single colony was formed by the Agarose Gel electrophoresis streak plate method, to purify cultures of actinomycetes. This After PCR amplification, agarose gel electrophoresis was plating technique serially dilutes the number of bacteria in adopted to confirm the presence of amplification. PCR was each streak, the first streak probably has a very high completely dependable on the extracted DNA criteria. concentration of bacteria since it comes from a concentrated stock. By dragging a new (or freshly sterilized) tool across Solutions only one small part of the initial line, we spread a small part 1 X TAE buffer, loading dye, DNA ladder marker, Ethidium of the initial line out over a much larger area (the second bromide (10mg / ml). line). This second line has less bacteria, and therefore increases the chances of seeing individual colonies. The Preparation of agarose dilution was repeated many times by streaking the entire plate . 100 ml of 1X TAE was taken in a beaker. from the initial concentrated streak, so somewhere on the . 1 gm (for 1%) agarose was added to the buffer. plate a single isolated colony was picked as reported by . The solution was heated to boiling (Using micro wave) Williams et al., (1993) [37] and Singh and Agrawal (2003) [32]. until all the gel particles were dissolved. . 1μl of Ethidium Bromide (10mg/ml) was added to the agarose. . The agarose was stirred in order to get mixed and to avoid bubbles. . The solution was allowed to cool down at 50-60 °C.

Casting of the horizontal agarose gel The agarose solution was poured into the gel tray after both the edges were sealed with cellophane tapes and the agarose was allowed to solidify at room temperature for 30 minutes.

The comb was carefully removed and the gel was placed in the gel tray. The tray was filled with 1X TAE-electrophoresis Fig 1: Single colony formation of Streptomyces spp. cultured on buffer until the buffer reached 3-5 mm over the surface of the ISP2 formed by streak plate method. gel. Identification and characterization of Streptomyces spp. DNA loading Morphological characterization PCR products were loaded directly. For PCR product, 10μl The isolates of Streptomyces were identified according to the was directly loaded to well. Electrical power was turned on at variability in their colony morphology and microscopic 100v/m Amp for 75min. DNA moves from Cathode to plus characteristics like the aerial and substrate mycelium, soluble Anode poles. The Ethidium bromide stained bands in gel were pigment, spore chain arrangement (table 6). Some visualized using Gel imaging system. Streptomyces isolates produced diffusible pigment in the surrounding media in accordance with the aerial mycelium Results and Discussion colour. Soluble pigment was also observed in 15 isolate. Isolation, purification and identification of Streptomyces Figure (2) shows distinctive yellowish (isolate 30) series isolates established in the Bergey’s manual of determinatives Suspected Actinomycetes colonies were sub-cultured in ISP2 bacteriology (Buchanan and Gibbons, 1974) [5] and in the ~ 20 ~ The Pharma Innovation Journal http://www.thepharmajournal.com

Bergey’s manual of systemic Bacteriology\ category 4.

Fig 2: Streptomyces spp. cultured on glycerol yeast extract media at 28 °C for 7-10 days. Left isolate without pigment, right isolate with yellow pigment

Table 6: Morphological characteristics of Streptomyces isolates.

Substrate Mycelium Mycelium Soluble Spore chain Isolate No. Isolates Colony morphology Arial mycelium Reverse side pigments surface pigment morphology 1. B3-2 Irregular edge-circular Light gray Light brown smooth brown straight 2. B12 Regular edge-circular Light gray Light brown smooth Light brown straight 3. B1-3 Irregular edge-circular Light gray Light brown smooth Light brown straight 4. B3-4 Regular edge-circular Light gray Yellowish Smooth Yellow straight 5. B1-4 Regular edge-circular gray Darck brown Rough No pigment straight 6. B18 Irregular edge-circular Light gray Darck brown smooth Light yellow straight 7. BT6 Regular edge-circular gray brown rough Light brown straight 8. B25 Irregular edge-circular White gray Light brown smooth No pigment spiral 9. BT5 Irregular edge-circular gray Darck brown rough No pigment straight 10. BH14 Regular edge-circular Light gray Light brown rough light yellow straight 11. B1 Regular edge-circular Light gray Light brown smooth dark straight 12. 1-3C Regular circular White gray Brown smooth No pigment spiral 13. 4-3C Regular edge-circular Light gray Light brown smooth Dark yellow straight 14. B2-4 Irregular edge-circular gray Light brown smooth No pigment straight 15. B3 Irregular edge-circular White gray brown rough Light yellow straight 16. B21 Regular edge-circular gray Light brown smooth No pigment spiral 17. B4-4 Regular edge-circular gray brown smooth Light yellow straight 18. B1-4 Regular circular gray brown smooth No pigment rectiflexible 19. B3-3 Irregular circular Light gray Light brown smooth Dark brown straight 20. B5 Iregular circular gray Brown smooth Light yellow Straight 21. B5-5 Irregularcircular White Light yellow rough Light yellow straight 22. BM3 Regular Gray brown smooth No pigment straight 23. B23 Irregular circular White gray Light brown rough Light yellow rectiflexible 24. B5-2 regular gray Light brown smooth Light brown straight

As shown in figure (3 a), a colony morphology showed brown while one isolate showed a dark green figure (3 b). different Streptomyces isolates with regular edge and irregular Twenty four isolates that grew on ISP2 media belong to the edge. The mycelium surface is shown in some species with genus Streptomyces since colonies were slow growing, rough surface and smooth surface in others. The aerial aerobic, glabours or chalky, folded. Most colonies produce D mycelium colour either white, dark, pale gray or greenish an earthy odour and they possessed aerial and substrate gray. Substrate mycelium was either dark brown or light mycelia with different colors.

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Fig (3a): Arial mycelium of Streptomyces grown in ISP2 media at 28 °C for 7-10 days.

source, using the biochemical test to analyze was reported by Vijayalakshmi et al., (2011) [35].

Table 7: Biochemical tests of Streptomyces spp

No Test Reaction Result 1. Melanin Black to brown Negative 2. Catalase Bubbles Positive 3. Citrate Utilization Deep blue color Positive

Fig (3b): Substrate mycelium of Streptomyces grown in ISP2 media 4. Indole production No color zone Negative at 28 °C for 7-10 days. 5. Sugar utilization Growth Positive

All the isolates were examined under a microscope after 7-10 Molecular detection of Str A gene responsible for days of incubation to see the hyphae. The spore chain streptomycin production morphology was observed after 2 weeks of incubation, DNA Concentration (ng/μl) showing various arrangements either straight, spiral or Genomic DNA was isolated from bacterial growth according flexuous depending on the Streptomyces species. Most strains to the protocol of Wizard Genomic DNA Purification Kit and were with straight chain arrangement, except three strains the DNA concentration ranged from 1 to 3 ng/ μl as shown in with spiral chain arrangement and two with rectiflexible table (8). arrangement. The same results were reported by Sakiyama et [29] Table 8: DNA Concentration (ng/μl) al., (2014) . Streptomyces are chemo hetero organotrophs. They make a No. isolates Concentration large class of Gram positive bacteria, forming hyphae like that 1 BH14 1.32 in fungi with a growing temperature and pH at 28 °C and 8, 2 B2-4 2 respectively. They produce a characteristic “earthy” smell of 3 B12 1.2 soil by the production volatile low molecular weight 4 Be1 2 compounds called geosmins. They can utilize complex 5 B3-4 3 organic materials in the soil and use them as sources for 6 B4-3 1.3 carbon and energy making these bacteria essential for the 7 B25 2 production of fertile soil. Streptomyces belong to the order 8 B21 2.4 Actinomycetales, characterized by the formation of substrate 9 BT5C 1.2 10 B18 2.23 and aerial mycelium on solid media, presence of spores. The 11 B1-3 1 majority of soil actinomycetes form a very important class of 12 B3-3 1.5 bacteria since they produce numerous natural products such as antibiotics and enzymes. More than 70% of the known natural The PCR amplification of strA gene appeared in among the 2 antibiotics produced are from Actinomycetes (Berdy, 2005) [5] Streptomyces isolates in the current study using specific . primer were positive results were only shown in 4 and 5 Streptomyces isolates only (Be1 and B3-4 respectively). The Biochemical test negative results 10 of Streptomyces isolates did not had strA Biochemical results of Streptomyces spp are shown in table gene. According to the statistical analysis, there were highly (7). The Streptomyces have the ability to produce enzymes significant differences at p values p˂0.01 among the positive like catalase. Simmon’s citrate utilization was positive while isolates of Streptomyces isolates to this gene. This PCR indole production was negative. Sugar utilization was products amplification gave an amplicon size of (700) bp as represented by growing of Streptomyces in media shown in Figure (4) on the gel of electrophoresis and supplemented with Dextrose or starch or Glycerol as a carbon comparing its molecular size with 100bp DNA Ladder.

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Fig 4: Agarose gel electrophoresis of PCR products (1% agarose, 100v/mAmp for 75min) for str A gene product using DNA template of Streptomyces isolates. Lane (M): (100 bp DNA Ladder). Lanes (4 and 5) of Streptomyces isolates show positive results (amplified size 700 bp).

Molecular Identification of Streptomyces isolates PCR treated soil. Evidence of horizontal gene transfer was products were separated and the isolation of genomic DNAs recorded in isolates identified as Streptomyces platensis was verified with electrophoresis on 1% agarose gels (w/v) recovered from CR and AR sites which had acquired a and run in 1x Tris-borate buffer, pH 8.3. Genomic DNAs streptomycin resistance gene homologous to that found in were seen as a smear of different molecular weight DNAs Streptomyces griseus. Members of the latter species were the under UV light. The ampilified DNA bands were visualised most abundant streptomycin resistant Streptomyces isolated after ethidium bromide staining and photographed under from all . (Tolba et al., 2002) [33]. ultraviolet light. The results show the DNA of isolated strains was extracted successfully. The results of PCR amplification References which was performed on the DNA extracted from all isolates 1. Aghighi S, Bonjar GHS, Rawashdeh R, Batayneh S, were confirmed by the electrophoresis analysis. Two isolates Saadoun I. First report of antifungal spectra of activity of (Be1 and B3-4) were positive in the PCR technique. Iranian Actinomycetes strains against Alternaria solani, Performing PCR reactions using primer pair on DNA from Alternaria alternate, Fusarium solani, Phytophthora Streptomyces isolates produced a single 700 bp band in all megasperma, Verticillium dahliaeand, Saccharomyces tested isolates (Fig 4). Two species of Streptomyces by the cerevisiae, 2004. name of Streptomyces griseus and Streptomyces coelicolor 2. Al-Rubaye TS, Risan MH, Al-Rubaye D, Radi OR. are used for the industrial production of Streptomycin and Characterization of marine Streptomyces spp. bacterial novel antibiotics (Mukhtar et al., 2012) [21]. Streptomycin isolates from Tigris river sediments in Baghdad city with gene cluster is composed of 27 genes and occupies a region of Lc-ms and 1 HNMR, Journal of Pharmacognosy and 32.6 Kb in length (Tomono et al., 2005) [34]. The cluster genes Phytochemistry. 2018a; 7(5):2053-2060. encoding enzymes for the synthesis of the streptomycin 3. Al-Rubaye T, Risan MH, Al-Rubaye D, Radi OR. subunits are not found in subpathway-specific operons; Identification and In vitro antimicrobial activities of instead, they are found in mixed operons which may indicate Marine Streptomyces spp. Bacteria from Tigris River the need for strictly coordinated expression of the cluster Sediments in Baghdad City. World Journal of genes (Piepersberg and Distler, 1997) [23]. Three streptomycin Pharmaceutical and Life Sciences. 2018b; 4(10):120-134. (SM) production genes from Streptomyces griseus (Distler et 4. Amin SM, Risan MH, Abdulmohimin N. Antimicrobial al., 1987) [10] Actinomycetes produce a variety of natural and Antioxidant Activities of Biologically Active Extract products that are of major importance in the pharmaceutical from Locally Isolated Actinomycetes in Garmian Area, J industry. Antibiotic production genes in actinomycetes are Garmian University. 2016; 1(10):625-639. gener- ally organized in a single large gene cluster on the 5. Bérdy J. Bioactive Microbial Metabolites A Personal chromosomes or, rarely, on plasmids (Seno and Baltz 1989) View. J Antibiot Buchanan and Gibbons, Bergey’s [30]. Some years ago we and others began to clone and analyse manual of determenaives bacteriology. 1974-2005; the genes for the biosynthesis of streptomycin (SM)-type 58:126. aminoglycoside antibiotics in Streptomyces griseus (Distler et 6. Birch A, Häusler A, Vögtli M, Krek W, Hütter R. al., 1985) [9] and S. glaucescens (Mayer et al., 1988) [20]. The Extremely large chromosomal deletions are intimately cluster of str genes was found in genetically highly unstable involved in genetic instability and genomic regions in S. glaucescens and appeared to be very stable in S. rearrangements in Streptomyces glaucescens. Mol Gen griseus (Birch et al., 1989) [6]. The genes analysed so far from Genet. 1989; 217:447-458. an approximately 11 kb DNA fragment in S. griseus include 7. Buchanan RE, Gibbons NE. Bergey’s manual of the strDR-aphD-strB1FGHIK genes, which represent a determenaives bacteriology, 8th ed. Williams and mixture of resistance, regulatory and biosynthetic genes Wilkins Co., Baltimore, Md, 1974. (Mansouri and Piepersberg 1991) [19]. Streptomyces isolates 8. Distler J, Mansouri K, Mayer G, Stockmann M, were screened for the streptomycin resistance gene, strA, and Piepersberg W. Streptomycin biosynthesis and its flanking biosynthesis gene, strB1. Distribution of these genes regulation in streptomycetes. Gene. 1992; 115(1-2):105- indicated the prevalence of str genes in the streptomycin 11. ~ 23 ~ The Pharma Innovation Journal http://www.thepharmajournal.com

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