Streptomyces Activities Isolated from Soil-Taif, KSA H. Sonya1, et al.pdf · Original Research Article Genetic Identification of Three Streptomyces Strains having Enzymatic Activities

Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 64-73

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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*

1Department of Biology, Faculty of Science, Taif University, P.O. Box 888, Taif, KSA 2Soil, Water and Environmental Research Institute, Department of Agricultural Microbiology,

Agricultural Research Center, P.O. Box, 12619, Giza, Egypt 3Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, P.O.

Box 68, Hadayek Shobra 11241, Cairo, Egypt *Corresponding author

A B S T R A C T

Introduction

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

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

ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 64-73 http://www.ijcmas.com

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 soil-streptomycetes 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 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. 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.

K e y w o r d s

Soil-Streptomycetes, DNA Markers, RAPD-PCR, 16S rRNA Gene

http://www.ijcmas.com


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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 (El-Domyati 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 Si-9 violet series isolate.

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 RAPD-PCR 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%) RAPD-PCR 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

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 soil-streptomycetes, having enzymatic activities, at Taif region, KSA based on their molecular characters.

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


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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).

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 PCR-isolation 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.

Results and Discussion

DNA fingerprinting of the three identified streptomycetes using RAPD-PCR

DNA fingerprinting of several Streptomyces sp. were determined using RAPD-PCR

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.

16S rRNA gene

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

http://www.ncbi.nlm.nih.gov/


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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 Blue-GSA-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

Type of fragment Isolate 13 Isolate 12 Isolate 10 No.

PU(+) 1 0 0 OPA11-01 M 1 1 1 OPA11-02

PU(-) 0 1 1 OPA11-03 PU(+) 0 0 1 OPA11-04 PU(-) 0 1 1 OPA11-05

M 1 1 1 OPA11-06 PU(-) 1 0 1 OPA11-07

M 1 1 1 OPA11-08 PU(+) 0 0 1 OPB15-09 PU(+) 1 0 0 OPB15-10 PU(+) 0 0 1 OPB15-11 PU(-) 0 1 1 OPB15-12 PU(-) 0 1 1 OPB15-13 PU(+) 0 0 1 OPC07-14 PU(+) 1 0 0 OPC07-15 PU(+) 0 1 0 OPC07-16 PU(-) 0 1 1 OPC07-17


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Type of

fragment Isolate 13 Isolate 12 Isolate 10 No.

PU(+) 0 0 1 OPC07-18 PU(+) 0 1 0 OPC07-19 PU(-) 1 0 1 OPC07-20 PU(+) 0 1 0 OPC07-21 PU(-) 1 1 0 OPC07-22 PU(+) 0 1 0 OPC07-23 PU(-) 1 0 1 OPC07-24 PU(+) 0 1 0 OPC18-25 PU(-) 1 1 0 OPC18-26 PU(+) 0 1 0 OPC18-27

M 1 1 1 OPC18-28 PU(+) 1 0 0 OPC18-29 PU(+) 1 0 0 OPC18-30 PU(+) 1 0 0 OPC18-31 PU(+) 0 0 1 OPC18-32 PU(+) 0 1 0 OPC18-33 PU(-) 0 1 0 OPC18-34 PU(-) 1 0 1 OPC18-35 PU(-) 1 0 1 OPC18-36 PU(+) 0 1 0 OPC18-37 PU(-) 1 1 0 OPC18-38 PU(+) 1 0 0 OPC18-39 PU(+) 1 0 0 OPC18-40 PU(+) 1 0 0 OPD04-41 PU(+) 1 0 0 OPD04-42

M 1 0 1 OPD04-43 PU(+) 0 1 0 OPD04-44 PU(-) 0 1 1 OPD04-45 PU(-) 0 0 1 OPD04-46 PU(+) 0 0 1 OPE04-47 PU(+) 1 0 0 OPE04-48 PU(+) 1 0 0 OPE04-49

M 1 1 1 OPE04-50 PU(+) 0 0 1 OPE04-51 PU(-) 0 1 1 OPE04-52 PU(+) 0 1 0 OPE04-53 PU(+) 0 0 1 OPE04-54 PU(+) 0 1 0 OPE04-55 PU(-) 0 1 1 OPE04-56

M 1 1 1 OPE04-57 M 1 1 1 OPE04-58

PU(+) 0 1 0 OPO14-59 PU(+) 0 0 1 OPO14-60 PU(+) 0 1 0 OPO14-61


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Type of

fragment Isolate 13 Isolate 12 Isolate 10 No.

PU(-) 0 1 1 OPO14-62 PU(+) 0 1 0 OPO14-63 PU(+) 1 0 0 OPO14-64 PU(+) 0 1 0 OPO14-65 PU(-) 1 0 1 OPO14-66 PU(-) 1 0 1 OPO14-67 PU(-) 1 0 1 OPO14-68 PU(-) 0 1 1 OPO14-69 PU(+) 1 0 0 OPO14-70 PU(+) 0 0 1 OPO14-71 PU(+) 0 0 1 OPO14-72

+: 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 Isolate 10 Isolate 12 Isolate 13

S. lateritius isolate 10 30.6 34.4 100

S. cacaoi subsp. asoensis 19.0 100

S. phaeopurpureus 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 Total score

Query coverage (%)

Max identity (%)

JQ962978.1 Uncultured Streptomyces sp. clone Blue-GSA-KSA 16S rRNA gene, partial

sequence

881 81 80

GQ494994.1 Uncultured Streptomyces sp. clone neau-D50 16S rRNA gene, partial sequence

870 79 81

JQ358608.1 Uncultured Streptomyces sp. clone ASC834 16S rRNA gene, partial

sequence

876 79 81


sequence

887 79 81


sequence

898 79 81

AJ781326.1 Streptomyces lateritius strain :LMG 19372 16S rRNA, complete sequence

911 79 81


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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


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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)

AGAATGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAGATGAGGTGCTTGCACCTTATCTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTCCGAAAGGAATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTGAGACTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGATTTACTGGGCGTAAAGCGTGCGTAGGCGGCTTTTTAAGTCGGATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGGAAGCTAGAGTATGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAATACTGACGCTGAGGTACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATACTAGAAACTTTCCAGAGATGGATTGGTGCCTTCGGGAATCTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTACTTGCCAGCATTTCGGATGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACACAGCGATGTGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGTAGGTAGTCTAACCGCAAGGAGGACGCTTACCACGGTGTGGCCGATGACTGGGGTGAAGTCGTACAGGG

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


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Streptomyces Activities Isolated from Soil-Taif, KSA H. Sonya1, et al.pdf · Original Research Article Genetic Identification of Three Streptomyces Strains having Enzymatic Activities