Abstract: The aim of this study was to determine the sequence of the 16S rRNA gene and thus to conduct the phylogenic position of the naturally occurring wild type strain of Streptomyces QU66C. Here we show conclusively the full sequence of the 16S rRNA gene of a novel wide type strain of Streptomyces coelicolor which has been isolated from the soil of Qatar and thus characterized on the basis of its phenotypic and genotypic features. In comparison with the homologous strains in GeneBanks, the phylogenic position of the isolate is in between S. coelicolor A3 (2) (Y00411) (NC003888) and S. coelicolor (C) (EF371438). The sequence of present strain has been deposited in the International Nucleotide Sequence Databases) (INSD) in the GenBanks/DDBJ/EMBL/NCBI) and assigned an accession number of AB588124 and thus the strain is being known as Streptomyces coelicolor (AB588124) (QU66C-2002). Present strain shows a similarity and identities of 99.40, 99.40, 99.40, 99.33 and 99.31% with a score value of 2693, 2693, 2687, 2673 and 2673 bits for Streptomyces coelicolor A3(2) (Y00411) (NC003888), S. violaceoruber AF503494 (ancient name for S. coelicolor and S. lividans), S. lividans AF503498, S. lividans AB184826 and S. coelicolor (C) (EF371438), respectively. In comparison with the S. coelicolor clones in Sanger database the strain shows a positive high scoring alignment similarity of 99.4% with score of 6907 with five clones of S. coelicolor A3 (2) (AL939116, AL939119, AL939124, AL939114, AL939108) and a 99% similarity with a score of 6889 for AL939110. Similarly, the BLAST search on EMBL GeneBAnk shows a 99.40% similarity with 2 strains of S. violaceoruber (AF503494, AF503492) with high score of 2738.
INTRODUCTION
There is a world wide growing demand for production of a new generation of antibiotics particularly due to the increase of resistant pathogens, evolution of novel diseases and toxicity of the currently used compounds (Silbergeld et al., 2008; Hakvag et al., 2008). The problem of multi-resistance is being progressively increasing due to the misuse of the available antibiotics (Harrison and Svec, 1998; Larson, 2007; Marino, 2008; Hawkey, 2008). The Streptomyces genus is the most diversified groups of Eubacteria, widely spread around all environments particularly in the desert soil where most common habitats occurs (Dunbar et al., 1999). The biotechnological importance of Sterptomyces as producers for the majority of antibiotics in use today keeps them as the main natural stock for screening programs (Berdy, 2005; Bull and Stach, 2007; El-Sherbiny et al., 2009; Akanji et al., 2011; Sinha et al., 2011). In addition, empirical screening using various assays has revealed that Streptomyces are capable to produce antibiotics with a broad spectrum activity in both human and veterinary medicine such as antibacterial, antifungal, anti-cancer, anti-parasitic and anti-viral (Atta and Ahmad, 2009; Morakchi et al., 2009) as well as some immune-suppressants (Watve et al., 2001) and several enzymes important in the food and other industries. There are two main approaches being used for discovery of new antibiotics; screening programs for natural strains and mutation protocols (Busti et al., 2006; Fiedler et al., 2008). As a result of this, more than 500 species of Streptomyces have been isolated (Zaitlin et al., 2003; Euzeby, 2008; Hakvag et al., 2008; Morakchi et al., 2009). Among those was the model organism Streptomyces coelicolor A3 (2) of which its complete genome was published (Bentley et al., 2002) making the species as the most studied member of the genus world-wide and is becoming the a genetic paradigm for the actinomycetes (Swiercz et al., 2008; O'Rourke et al., 2009; Xu et al., 2010). Further more, biotechnology researchers have begun using Streptomyces species for heterologous expression of proteins over the traditionally known Escherichia coli, as the latter was not capable of protein glycosylation or folding (Payne et al., 1990; Brawner et al., 1991; Hopwood, 1999; Hu et al., 2000; Sioud et al., 2009).
Recently there have been world wide interest on the molecular techniques (Abu Bakar et al., 2010; Onasanya et al., 2010; Sifour et al., 2010; Zolgharnein et al., 2010) particularly the 16S rRNA phylogenic analysis which provides a great impact on Streptomyces systematic and minimizing wrong identification (Anderson and Wellington, 2001; Kim et al., 2004; Jiang et al., 2007; Singh et al., 2009). Taxonomically, S. coelicolor A3 (2) belongs to the species of S. violaceoruber and not a validly described separate species which should not to be mistaken for the actual S. coelicolor (Muller). The present Streptomyces strain QU66C has been isolated from the soil of Qatar during screening program and was selected as presumable strain of S. coelicolor. The aim of this study was to determine the phylogenetic position of the naturally occurring wild type strain of Streptomyces QU 66C which has proven to have high potential of production a novel antibiotic.
MATERIALS AND METHODS
Bacterial strain and culture conditions: The investigated strain was isolated from desert soil of Qatar (Abu-Smra area) during a screening program for a novel antibiotic during the year 2002. The strain was then deposited in the Qatar University Culture Collection (QUCC) as strain QU66C and later on (during the partial sequence of the 16S rRNA gene) it was deposited in the NCIMB Ltd, Aberdeen, Scotland, UK as strain NCSQ 17869 during 2003. Phenotypic characteristics of the strain were determined after examination as described previously (Garrity, 2010; Williams et al., 1983a, b). Cultures of strain from different growth stages were examined using both phase-contrast microscope and electron microscope.
The screening for optimum growth of the strain revealed that the Nutrient Agar (NA) and Nutrient Broth (NB) (each supplemented with 1% starch) were the optimum media, known hereinafter as NAS and NBS, respectively. The pure culture was then maintained in NBS medium at 37°C and culture conditions were kept as described previously (Kieser et al., 2000).
Assay for antibiotics: The antimicrobial assay for the strain antibiotics was conducted using both of inhibition zone method and the minimum inhibition concentration. The production of antibiotics by present strain was determined as described earlier (Kieser et al., 2000) and as follows: The Actinorhodin (Act) was extracted using the 1 N KOH for pH adjustment to 8.0 before A640 was measured. The undecylprodigiosin (Red) was extracted using 0.5 M HCl for acidification before A530 was measured. The calcium-dependent lipopeptide antibiotics (CDA) was detected as normally assayed (Kieser et al., 2000). For production of the antibiotic droplets on the surface of the colonies, strain was cultured on Potato Dextrose Agar (PDA) and incubated for several days.
Analysis of 16S rRNA gene: During 2007, the 16S rRNA gene sequence was determined. The genomic DNA of the Streptomyces QU66C strain was extracted using the PrepMan Ultra Sample Preparation Reagent kit (No: 4367554) according to the protocol stated by manufacturer (Applied Biosystem, USA). The 16S rRNA gene was amplified using the MicroSeq full gene 16S rRNA Bacterial Identification kit (No: 4349155) and the PCR Amp system 9700 according to the manufacturer protocol (Applied Biosystem, USA). The PCR program consisted of an initial denaturation at 95°C for 10 min to activate the AmpliTaq DNA polymerase, then 30 cycles (30 sec at 95°C, 30 sec at 60°C , 45 sec at 72°C for denaturation, annealing and extension, respectively), followed by 10 min at 72°C for final extension. Then, the PCR product was purified using wizard PCR preps DNA purification system kit (No: A7231) according to the manufacturer (Promega, USA). The sequencing mix then were mixed and run on the PCR for 25 cycles (10 sec at 96°C, 5 sec at 50°C, 4 min at 60°C for denaturation, annealing and extension, respectively). The reaction was terminated with a final extension at 72°C for 5 min. The excess dye terminators and primer were removed from the sequencing mix using DyeEX® 2.0 spin kit (No: 63204) according to the manufacturer (Qiagen, USA).
The 16S rRNA gene sequence was conducted using the genetic analyzer (ABI Prism GA310, Applied Biosystem, USA). The GA310 analyzer is equipped with a compatible a data collecting software (v 3.0) and a Microbial Identification Software MicroSEQ® ID (v 2.0). The software allows the user to create consensus sequences and to compare it with the microbial library of the full gene of 16S rRNA, available in the Applied Biosystem (http://www.appliedbiosystems.com).
Pairwise sequence alignment: The full gene sequence of present strain QU66C was aligned automatically using the BLAST against the gene library available for Streptomyces species in the NCBI (www.ncbinlm.nih.gov), Sanger Institute (http://www.sanger.ac.uk), DDBJ (http://www.ddbj.nig.ac.jp) and EMBL-EBI GeneBank (http://www.ebi.ac.uk).
Multiple sequence alignment: The phylogenetic analysis was constructed using Nighbor-Joinin tree (Saitou and Nei, 1987; Dopazo, 1994; Tamura et al., 2007) of the isolated strain using the BLAST and CLUSTAL W (1.83) available in the DDBJ GeneBank. The closely related homologous strains were identified, retrieved and compared to the sequence of the strain QU66C, using CLUSTAL W (version 3.2) available on the Biology StudyBench (http://woekbench.sds.edu).
Statistical criteria for species identification: Identification of species through sequence similarity was determined based on the criteria used by Bosshard et al. (2003) where if the difference between the query and the compared strain is 1-1.5% (14-22 bp), 1.5-5.0% (23-72 bp) and 5.-0-7.0% (72-98 bp), then the query strain should be given to the same species, genus or a different genus, respectively.
Genebank accession number: The complete sequence (1404 bp) of the 16S rRNA gene of QU66C strain has been deposited in the International Nucleotide Sequence Databases (INSD) (DDBJ GenBank), EMBL-EBI Bank (European Bioinformatics Institute and the European Molecular Biology Laboratory) and the National Center for Biotechnology Information (NCBI).
RESULTS
Physiological and biochemical features: The physiological and biochemical characteristics of the isolate are given in Table 1. The examined features of the investigated strain QU66C showed the typical morphology of Streptomyces on various agar plates, aerobic, gram-positive, non-motile, non-acid fast.
The morphology of colonies were typically similar to that of S. coelicolor where it showed the actinorhodin production and then its conspicuous red color diffusion in the media after 48 h of growth. The isolate was exposed to the most notable test for the S. coelicolor is that the red-blue acid-base indicator.
| Table 1: | Physio-biochemical features and antimicrobial profile of the isolate strain streptomyces coelicolor AB588124 (QU66C-2202). Bacterial strains used as control (*) |
Thus the colonies that become red-purple because of actinorhodin production will rapidly turn blue on fuming the colonies with ammonia, consistent with the known pH indicator properties of the compounds.
The screening for optimum growth of the strain revealed that the NAS and NBS were the optimum solid and liquid media respectively. For production of the antibiotic droplets on the colonies surface, isolate was cultured on Potato Dextrose Agar (PDA) for 5 days before several droplets (3-5) appeared on the top of each colony.
The isolate has the ability to utilize all tested carbon sources (D-glucose, D-xylose, D-arbinose, D-manitol, D-lactose, D-succurose, D-sorbitol, D-starch). The isolate showed a positive for catalase, amylase, nitrate reductase, starch hydrolyses, gelatin hydrolyses, casein break down but was negative for ornithine, indole and urease as well as hydrogen sulfide production.
Antimicrobial profile of strain QU66C: The antimicrobial profiles of the isolate is given in Table 1. The antibiotic of the strain showed antimicrobial activity against some local medical isolates (all brought from Hamad Hospital, Qatar) such as: Escherichia coli, Bacillus cerus, Micrococcus luts, Pseudomonas aeruginosa, Staphylococcus aureus, S. epidermis and Klebsilla sp.
| Fig. 1: | The full sequence of 16S rRNA gene of the novel streptomyces coelicolor AB588124 (isolate QU66C) with a length of 1404 bp. IUPAC codes: R (A or G), Y (C or T), M (A or C), S (G or C), W (A or T), K (G or T ) |
It also showed antibacterial activity against the control organisms (brought from the Central Laboratory, Ministry of Health, Jordan) such as: Staphylococcus aureus MRSA ATCC 25023, Pseudomonas aeruginosa ATCC 27853, Escheria coli ATCC 25922. The isolate showed an activity against Candida albicans but not against Penicillum IM56, Penicillium IM 65/3).
The antibiotics production by present strain was determined as described above in the Materials and Methods section. The stain showed a high potential for antibiotics production (data in preparation for the coming study). The strain is capable of production of all antibiotics known for the S. coelicolor.
Comparative analysis 16S rRNA sequence of QU66C strain: A full sequence (1404 bp) of the gene which is given in Fig. 1. Initially a partial sequence of the gene (480 bp) was determined (data not shown) in the National Institute for Multicultural Competence (NIMC, UK) during 2002 but the library search-report at that time was not enough to determine the species level, therefore we have waited seven years until we have imported the GA310 sequencer. The full sequence (1404) of present strain was BLASTED with the microbial genome library of the full gene of 16S rRNA, available in the Applied Biosystem (http://www.appliedbiosystems.com). The library search report for the top 20 matching strains were obtained and thus the phylogenetic tree of the top 5 homologous was conducted (Fig. 3). The phylogenetic destination of the strain QU66C is in between S. coelicolor A3 (2) (Y00411) (NC003888) and S. coelicolor (C) (EF371438) (Fig. 3). The summary of the BLAST given in Table 2 shows that the query strain QU66C has a similarity and identities of 99.40, 99.40, 99.40, 99.33 and 99.31% with variable score value of 2693, 2693, 2687, 2673 and 2673 bits for Streptomyces coelicolor A3 (2) (Y00411) (NC003888), S. violaceoruber AF503494 (ancient name for S. coelicolor and S. lividans), S. lividans AF503498, S. lividans AB184826 and S. coelicolor (C) (EF371438), respectively. The positions of the mismatched between the strain QU66C and the compared strains S. violaceoruber AF503494 are given in Table 3.
The graphical BLAST and summary of the 16S rRNA sequence of the isolate QU66C are given in Fig. 2 and Table 2, respectively. The searching report out of the S. coelicolor database shows that the strain QU66C has a positive high scoring alignment similarity of 99.40% with score of 6907 with five clones of S. coelicolor A3 (2) (AL939116, AL939119, AL939124, AL939114, AL939108); whilst shows a 99.00% similarity with a score of 6889 for AL939110.
| Table 2: | Library search report of the top 5 high-scoring strains to our isolate streptomyces coelicolor AB588124 (QU66C) as BLASTED in sanger institute (http://www.sanger.ac.uk), DDBJ (http://www.ddbj.nig.ac.jp), applied biosystem (http://www.appliedbiosystems.com), EMBL-EBI Bank (http://www.ebi.ac.uk) and the NCBI (www.ncbinlm.nih.gov) |
| Table 3: | Locations of the different 17 base pairs between the full sequence of 16S rRNA gene of the Streptomyces coelicolor AB588124 (QU66C) and that of S. violaceoruber AF503494. IUPAC codes: R (A or G), Y (C or T), M (A or C), S (G or C), W (A or T), K (G or T ) |
| Fig. 2 (a-b): | Graphical BLAST of QU66C (1404 bp) as compared to the S. coelicolor complete sequence (8, 668, 907 bp) available in Sanger database (http://blast.wustl.edu, webmaster@sanger.ac.uk) (b) The visible feature range of the 16S rRNA of the isolate QU66C as appeared in EMBL-EBI Bank (http://www.ebi.ac.uk) |
| Fig. 3: | The inferred phylogenetic tree of the isolate strain (specimen) Streptomyces coelicolor AB588124 (QU66C-2002) using the Neighbor-Joining method |
A similar results was obtained when BLAST search was carried out on EMBL BLAST in addition to 2 strains of S. violaceoruber (AF503494, AF503492) with high score of 2738. Furthermore when the sequence of QU66C was BLASTED with the DDBJ database using the CLUSTAL W (1.83), the strains showed a similarity of 99.40% for the closest 3 strains of S. coelicolor EF371438, S. violaceoruber AB184833 and S. lividans AB 184695.
We have therefore, shown conclusively that on the basis of both phenotypic and genotypic features and according to the criteria reported earlier (Bosshard et al., 2003), the strain isolate QU66C should be assigned as Streptomyces coelicolor (QU66C-2002). The complete sequence of the 16S rRNA gene of S. coelicolor (QU66C) strain has been deposited in the International Nucleotide Sequence Databases (INSD) in the GenBank/DDBJ (connected with GeneBank/EMBL/NCBI) and assigned an accession number of AB588124 under a voucher specimen (Professor Ihsan Mahasneh QU66C-2002). The strains is therefore, hereinafter, known as Streptomyces coelicolor (AB588124) (QU66C-2002).
DISCUSSION
The present strain S. coelicolor AB588124 (QU66C-2002) has showed a marked and conspicuous phenotypic and genotypic features of S. coelicolor compared with homologous strains in different GeneBanks (Fig. 1-3; Table 2, 3) which support present results. The strain was highly (99.40%) identical to S. coelicolor A3 (2), S. violaceoruber and S. lividans. This is in full agreement with a previous result reported earlier using the 16S rRNA on the molecular taxonomy of Streptomyces collected from different GeneBanks and collections (ATCC, DSM, JCM) where both species S. lividans and S. coelicolor has given a possibility to be classified as S. violaceoruber (Chistova et al., 1995). The taxonomy of S. lividans is closely related to S. coelicolor because it produces the same four types of antibiotics but the Act and Red genes are normally poorly expressed under usual growth condition (Hosoya et al., 1998). The majority of the members of this group share highly similar phenotypes and 16S rRNA sequences and thus the biosynthetic gene clusters required for production of this antibiotic must be transferred from S. coelicolor into the S. lividans (Lai et al., 2002; Haifing et al., 2002).
Historically, it has been known for many years that the most notable indicator for the S. coelicolor is that the red-blue acid-base test (Kieser et al., 2000). Present strain started to release its pigments that are blue/green in alkali and red in acidic conditions, thereby giving the bacterial colonies/culture those colors under the respective conditions. Thus the colonies of present isolate that become red-purple because of actinorhodin production have rapidly turned blue on fuming with ammonia which is consistent with the known pH indicator properties of the compounds (Kieser et al., 2000). Based on phenotypic and genotypic data, it has been shown conclusively that the isolate QU66C is a novel wild type strain for Qatar and has been given the its name and accession of S. coelicolor AB588124 (QU66C-2002) which to be used in antibiotic production. The S. coelicolor produces pigments, complex lipids, signal molecules and four kinds of antibiotics including the cyclopentanon methylenomycin, lipoptide Calcium-Dependent Antibiotic (CDA), blue polyketide Actinorhodin (Act) and red tripyrcol undecylprodigiosin (Feitelson et al., 1986; Liu et al., 2005). The 16S rRNA sequence data provide a conclusive evidence that present isolate is phylogenetically identical to S. coelicolor A3 (2), S. violaceoruber and S. lividans.
CONCLUSION
The present results shows that the present strain is a novel wide type strain capable of antibiotics production without any genetic alteration as it appeared in other strains. Moreover, the present results provides a better understanding of the role of RNase III gene (AbsB) encoded the mRNA for the AdpA transcription factor on regulation of antibiotic production (Lee et al., 2006; O'Rourke et al., 2009; Anderson and Wellington, 2001; Payne et al., 1990).
ACKNOWLEDGMENTS
The authors are gratefully to Al-al-Bayt University for providing leave of Professor Ihsan Mahasneh to Qatar University during 2001-2005. Authors are also gratefully to both Qatar University and Al al-Bayt for providing laboratories and kits for gene analysis.