Molecular identification of different actinomycetes isolated from East Black Sea region plateau soil by 16 S rDNA gene sequencing

In order to obtain different actinomycetes isolates from the East Black Sea Region plateau soil, an isolation study was carried out by using sucrose gradient method and different growth media. A molecular taxonomic method, 16S rDNA gene sequencing, was applied to obtain different actinomycetes isolates from East Black Sea region plateau soil. Phylogenetic dendrograms based on 16S rDNA nucleotide sequences were constructed by using neighbour-joining algorithm. In the light of phylogenetic analyses, it was determined that out of 15 organisms, two belong to Actinomadura, three Kribbella, three Nocardia, six Micromonospora and an organism of Microbacterium. Soil isolates obtained from this study contribute to taxonomy of actinomycetes and also many species will emerge.


INTRODUCTION
Most common groups of actinomycete have the capability to produce secondary metabolites with biological activities such as anticancer, antibiotic, antiviral, antifungal, enzyme, immunosuppressant and other industrially beneficial compounds (Baltz, 2007;Kekuda et al., 2010;Naine et al., 2011).Antibiotics have been isolated from almost all the suborders of actinomycetes.Considering increase in antibiotic resistance against widely used medicines, there is still a source of continuous novel antimicrobial agents from actinomycetes isolated from aquatic and terrestrial habitats (Baltz, 2006;Yang et al., 2011).
Actinomycete taxonomy was formerly thought to be associated with morphology, which is inadequate in differentiating between different species of many genera.The use of phylogenetic and molecular evolutionary approaches has been of great importance to the classification methods (Babalola et al., 2009;Hozzein and Goodfellow, 2011).Some organisms that are in advertently placed in an ineligible group are now classified appropriate owing to the advent of molecular techniques (Zhi et al., 2009).Recently, the identification of the species and phylogenies are commonly derived from 16S rDNA and the use of polymerase chain reaction (PCR) for sequence analyses (Wood et al., 2007;Zhi et al., 2009).
Majority of actinomycetes are commonly found in both aquatic and terrestrial habitats (Macagnan et al., 2006).The best known actinomycetes in soil is the genus Streptomyces although others, like Norcardia, Microbispora, Micromonospora, Actinomyces, Actinoplanes and Streptosporangium, have also been isolated from soil.The number and variety of actinomycetes present in any soil sample would be *Corresponding author.E-mail: kamilis@omu.edu.tr.significantly influenced by geographical location, soil temperature; type and pH, organic matter content, agricultural activities, aeration, nutrient availability, moisture content and soil vegetation (Arifuzzaman et al., 2010).Presently, different antimicrobial compounds likely to be obtained are decreasing day by day from actionmycete groups.Nowadays, obtaining new antibiotics from rare actinomycetes is industrially important (Hayakawa, 2008).
Actinomadura produce a stable, extensively branched substrate mycelium and aerial hyphae, which when formed, differentiate into chains of spores (Lechevalier and Lechevalier, 1970).Members of genus Actinomadura have a mesophilic structure and growth occurs at 25-40°C.Some types of thermophilic Actinomadura require 50-65°C optimum temperature.Actinomadura was established by Lechevalier and Lechevalier (1970) and was placed in the family Thermomonosporaceae containing four other genera, Actinocorallia, Spirillospora, Thermomonospora and Actinoallomurus (Zhang et al., 1998;Trujillo and Goodfellow, 2003).
The genus Nocardia is a member of the family Nocardiaceae and the order Actinomycetales (Stackebrandt et al., 1997).Nocardia form part of the soil microflora and play a role in the turnover of organic matter (Orchard, 1981); there is also evidence that some strains produce secondary metabolites of potential industrial value (Kinoshita et al., 2001).Nocardia grow slowly on non-selective culture media, and are strict aerobes with the ability to grow in a wide temperature range.Some species are non-pathogenic while others are responsible for nocardiosis (Conville and Witebsky, 2010).
Kribbella was established by Park et al. (1999) to accommodate nocardia form of actinomycetes that contain LL-diaminopimelic acid in the cell wall.Kribbella is aerobic, Gram-positive, motionless and vegetative mycelium appears cream in colour with highly branched hyphae, which fragment in both liquid and agar cultures (Urzi et al., 2008).
The genus Micromonospora included in Micromonosporaceae family are distributed widely in different environments, such as soils, water, sandstone and root nodules.Micromonaspora is presumably the second most prolific producer of antibiotics.This genus enables the production of gentamycin micromonosporin, megalomycin, maduramycin, halomycin, quinolidomycin, arisostatin A and B, and anthraquinone antibiotics (Qiu et al., 2008).
Microbacterium includes a diverse collection of Grampositive, non-spore-forming rods that were isolated from various environmental habitats including water, soil, steep liquor, milk products and also from humans.Members of the genus Microbacterium were charac-terized largely by their marked heat resistance, presence in dairies, and production of small amounts of L(+) lactic acid from glucose (Brennan et al., 2001;Young et al., 2010) Our literature search showed that there was no isolation study of rare actinomycetes of East Black Sea region in Turkey until now.Therefore, the present preliminary study was aimed to identify rare actinomycetes strains isolated from different localities of plateau soil and to place them in phylogeny by 16S rDNA gene sequencing.

Selection of soil samples and isolation of microorganisms
For this study, in the six provinces of the Eastern Black Sea region, soil samples obtained from seven different localities were put in sterile container with sterile plastic bags.Collected samples were taken to the laboratory, numbered and stored at 4°C.Each soil sample weighed 20-25 g and was added to 100 ml beaker.Sufficient amount of distilled water was added and after 24 h, pH was measured with pH meter for each soil samples and values are also shown in Table 1.
Sucrose gradient method was applied as a selective isolation method (Yamamura et al., 2003).This application achieved 20% sucrose gradients to help in selection of different soil bacteria.20% solution of sucrose was prepared in a screw cap centrifuge tube (105 mm).
This solution was added to 1 ml of purified spore suspensions of the test actinomycete strains prepared by using conventional technique and the tube was centrifuged (room temperature, 30 min, 240 g) in a swinging bucket rotor.After centrifugation, each sucrose layer (1 ml) was transferred sequentially from the top of the gradient using a different sterile pipette, and then diluted in a 10fold series in sterile Ringer"s solution (Merck, Darmstadt, Germany) (Hayakawa and Nonomura, 1987;Yamamura et al., 2003).Numbering according to Escherichia coli numbering system (Brosius et al., 1978).
Isolates putatively assigned to the different actinomycetes on the basis of colony morphology, notably aerial spore mass colour, substrate mycelial pigmentation and the colour of diffusible pigments, were sub-cultured on glucose yeast extract agar, glucose yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3) and tryptone-yeast extract agar and incubated at 28°C for 10 days.Observed morphological characteristics were compared with those of type strains by light microscopy under described conditions (Brennan et al., 2001).
Spore suspensions and mycelial fragments of the isolates were preserved in 20% glycerol (v/v) at -20°C until required.

Culture conditions and DNA extraction
Test strains were maintained on glucose-yeast extract agar (GYEA; Gordon and Mihm, 1962) slopes and as glycerol suspensions (20%, v/v) at -20°C.Before DNA extraction, biomass were grown in shake flasks containing modified tryptone-yeast glucose extract (TYG; Blackall et al., 1989) broth; these cultures were incubated for 6 to 8 days at 28°C.Chromosomal DNA was isolated by using a method by modifying "guanidine thiocyanate DNA isolation method" of Pitcher et al. (1989).
In order to assess the identities of the strains isolated, the nearly full-length sequences of the 16S rDNA gene were analyzed.The PCR products of the selected isolates were purified (QIAquick purification kits, Qiagen, Hilden, Germany) and sequencing was performed by ABI PRISM BigDye Terminator Cycle Sequencing kits (Macrogen, Netherland) with previously described oligonucleotide primers (Table 2).
The resulting 16S rDNA gene sequences (1.325-1.435nucleotides) were used to search the GenBank/EMBL/DDBJ database with the BLAST program (http://www.ncbi.nlm.nih.gov/) to determine relative phylogenetic positions.Phylogenetic analysis was conducted using MEGA 5.0 (Tamura et al., 2011) by first generating a complete alignment of 16S rDNA gene sequences of the isolates and type strains of all valid species.A phylogenetic tree was inferred using neighbour-joining tree algorithms (Jukes and Cantor, 1969).16S rDNA datasets were cooperatively analysed using MEGA 5.0 (Tamura et al., 2011) which was used to calculate evolutionary distances and similarity values.Topography of the constructed tree was evaluated by bootstrap analysis with 1000 replicates (Saitou and Nei, 1987).Only nodes with bootstrap values over 50% were considered to be significant.

Phylogenetic analysis
The current version of the EzTaxon-e (http://eztaxone.ezbiocloud.net/;Kim et al., 2012) server provides multiple sequence alignment by CLUSTAL W 1.8 (Thompson et al., 1994).The resultant sequence alignment can then be used for the neighbour-joining (Saitou and Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods using the MEGA 5.0 (Tamura et al., 2011) package; all were implemented within the server.The alignment can be exported for use by external programs including MEGA 5.0 (Tamura et al., 2011) and BioEdit (version 7.2.0,Hall, 1999).An evolutionary distance matrix was generated for the neighbour-joining as described by  Jukes and Cantor (1969).The resultant tree topology was evaluated by a bootstrap analysis (Felsenstein, 1985) with 1000 resamplings from the neighbour-joining dataset using Seqboot and Consense from the Phylip package (Felsenstein, 1993).

RESULTS
Many studies have been performed using several selective isolation techniques in order to assess the diversity of actinomycetes in natural habitats (Goodfellow and Fiedler, 2010).In this study, various pretreatment techniques and selective media have also been used for actinomycete isolation from plateau soil samples.Totally one hundred and one strain were isolated on humic acidvitamin (HV), tryptone-yeast glucose extract (TYG) and glucose-yeast extract (GYEA) agars supplemented with nalidixic acid, rifampicin and cycloheximide (Yamamura et al., 2003) and incubated at 28°C for about two to three weeks (Figure 1).
The organisms obtained by using humic acid-vitamin agar (HV; Hayakawa and Nonomura, 1987) were more easily observed than by using tryptone-yeast glucose extract agar (Blackall et al., 1989).The organisms selected based on colony morphology were inoculated on oatmeal agar medium.Sporulating colonies with grey pigmentation on this medium were eliminated due to having high probability of being members of the genus Streptomyces.
Isolates putatively assigned to the rare actinomycetes on the basis of colony morphology-notably spore mass colour, substrate mycelial pigmentation and the colour of diffusible pigment were subcultured on oatmeal agar (Küster, 1959) and tryptone-yeast glucose extract agar (Blackall et al., 1989) and incubated 28°C for 10 days (Table 3).Indication of different pigmentation of fifteen rare actinomycetes colonies were selected to study further molecular techniques.All of the strains were maintained on glucose-yeast extract agar (GYEA; Gordon and Mihm, 1962) at 28°C and as glycerol suspensions (20%, v/v) stored at -20°C for future work.
Genomic DNA from bacteria likely to be involved in different actinomycetes groups is obtained by modifying "guanidine thiocyanate DNA Isolation Method" (Pitcher et al., 1989).The 16S ribosomal RNA gene was amplified by using the PCR method with Taq DNA polymerase and primers 27f and 1525r.The molecular-based methods using polymerase chain reaction (PCR) is suitable for taxonomic studies.Samples of PCR product patterns of some representatives of test strains are given in Figure 2.During the analysis based on 16S rDNA gene sequencing 800R, MG3f, MG5f, 1115r and 1492r primers were also used to get a nearly complete 16S rDNA sequence data of test starins.
16S rDNA gene sequence data was determined for the fifteen test strains according to Blast analysis and a 1309 nucleotide base pair was used for phylogenetic analysis and compared with 16S rDNA gene sequences of tree representatives of closely related type strains of Actinomycetes (Figure 3).Obtained results of nucleotide similarities (%) and differences based on 16S rDNA gene sequences data are given in Table 4.
Plateau soil samples Actinomadura sp.ART34 shares same 98.2 and 97.5% of 16S rDNA gene sequence similarity with the type strains of A. glucoflavus and A. mexicana, value corresponding to 18 and 25 nt difference.Actinomadura sp.ART64 shares 97.8% and 97.1% 16S rDNA gene sequence similarity with the type strains of Actinomadura glucoflavus and Actinomadura mexicana, value corresponding to 22 and 29 nt difference.Actinomadura sp.ART34 also shares 99.6% 16S rDNA gene sequence similarity with Actinomadura sp.ART64, value corresponding to 4 nt difference, respectively (Figure 3 and Table 4).
Soil sample Nocardia sp.BY352 shares 99.1% of 16S rDNA gene sequence similarity with the type strain of Nocardia rhamnosiphila, value corresponding to 8 nt difference.Nocardia sp.GR130 shares 99.6 and 98.0% of 16S rDNA gene sequence similarity with the type strain of N. cyriacigeorgica and N. carnea, value corresponding to 3 and 19 nt difference.Nocardia sp.GR14 shares 99.7 and 98.1% of 16S rDNA gene sequence similarity with the type strain of Nocardia cyriacigeorgica and Nocardia carnea, value corresponding to 2 and 19 nt difference, respectively (Figure 3 and Table 4).

DISCUSSION
Conventional culture methods are not satisfactory for analysis of actinomycetes.Therefore, 16S rDNA gene studies became the most popular, routine and convenient method for many research laboratories.16S rDNA part of the ribosomes are required by all prokaryotic organisms to synthesize proteins.The gene sequence encoding 16S rDNA is a very suitable molecular marker to clarify phylogenetic relationships.16S rDNA gene sequences have played a vital role in microbiology and can be utilized in many ways in various disciplines, notably taxonomy and ecology.It has led to a wealth of information concerning prokaryotic diversity (Zhi et al., 2009;Tindall et al., 2010).
The order Actinomycetales is considered the most promising source of bioactive natural products.The recent researchers screened intensively the marine, plant and medicinal plant, sediment and soil environments of actinomycetes to elucidate the structures of bioactive molecules produced by them (Qin et al., 2009;Kumar et al., 2010;Baskaran et al., 2011;Khanna et al., 2011;George et al., 2012;Kaewkla and Franco, 2013).Using 16S rDNA sequencing, in this preliminary study, we aimed at isolating rare actinomycetes, which could have high possibility of gene clusters for bioactive secondary metabolites.The strains from the genera Actinomadura, Kribbella, Microbacterium, Micromonospora and Nocardia obtained in our work might have great capacity for bioactive secondary metabolites such as antibiotics, antitumor agents, etc.In order to reveal biotechnological capabilities of these strains, further molecular and chemotaxonomic studies will be carried out.
In conclusion, preliminary isolation studies shows that the obtained results are in harmony with literature of Yamamura et al. (2003) and also indicate that isolation of many novel actinomycetes from plateau soil of East Black Sea region have a great capacity to be added as a merit species to the literature from Turkey.

a
Degeneracies according to lane (1991): Y = C:T; R = A:G; W = A:T.b Binding site on the 16S rDNA molecule:

Figure 3 .
Figure 3. Neighbour-joining tree (Lane, 1991) based on nearly complete 16S rDNA gene sequences (1309 bp length) showing relationships between test isolate and closely related type strains of the genus Actinomadura, Kribbella, Micromonospora, Microbacterium and Nocardia.Numbers on branch nodes are bootstrap values (1000 resamplings; only values over 50% are given).Bar: 0.005 substitutions per nucleotide position.GenBank accession numbers are also given in parenthesis.

Table 1 .
Sources and strain histories of the test organisms.

Table 2 .
Oligonucleotide primers used in the PCR amplification and sequencing of 16S rDNA.

Table 3 .
Growth and cultural characteristics of the test organisms in different culture.