Molecular Identification of Local Isolated Streptomyces Species from North Region soil in Iraq

In this study Streptomyces were isolated from 50 bacterial isolates taken from 30 soil samples, these samples were collected from various locations in Iraq's various regions. The species of Streptomyces were isolated using starch casein agar and diagnosed microscopically and morphologically by Gram staining and glass slide. The sequence analysis 16S rRNA is used to report 11 Streptomyces. 10 bands of DNA gene, a result of specific polymerase chain reaction PCR, are elected from bacterial local isolates where 1000 base pairs within one volume, The PCR products of DNA samples were chosen from 11 local isolates based on nitrogenous base sequences. These organisms are revealed as a result of the study and by using DNA Blast NCBI as fellows; Streptomyces gancidicus, S. werraensis, S. griseorubens, S. hawaiiensis, S. thermocarboxydus, S. cyaneus, S. misionensis, S. bellus, S. parvulus, S. labedae,


Introduction
The species of streptomyces are widely prevalent in soil, It is the largest genus of the Actinomycetes, Gram-positive bacteria. Their colors mostly are grey but few are red, green, and white, while the blue is the rarest [1]. The genus Streptomyces is considered from Actinomycetes diameter (0.  µm. It is also grown Aerial mycelium carrying many spores, that are arranged in chains and take different shapes, from the spiral form, Rectus-flexibilis form, Retinaculum-Apertum form [2] [3]. The Streptomyces genus is the most important species from the group of filaments. The Novel organisms can produce secondary metabolites (Antibiotics) and (Enzyme) production used to control many pathogenic bacteria and their inhibitory impact on harmful microbes [4] [5]. The higher rate of GC is ∼70% of the content of Streptomyces spp. To separate Streptomyces from other bacterial such as Actinobacterial, there are distinguishing features such as 16S rDNA analysis and DNA-DNA hybridization [5] [6]. Geosmin, a scented substance produced by Streptomyces is responsible for the distinctive odor of soil.. This research aims to identify and isolate Streptomyces from the soil of northern Iraq (Nineveh, Duhok, Erbil) as well as diagnose them using microscopic and morphological experiments and a PCR test based on 16S rRNA to establish their genetic sequence. In addition, DNA Blast NCBI was used.

Collecting of Samples
Thirty soil samples were collected from various farm locations in Iraq's northern area, ranging in depth from 5 to 15 cm, and after being collected the samples were treated with calcium carbonate CaCO3 (1:10) and dried at 40-45 °C for four days. The samples were then placed in polyethylene bags and tightly sealed before being placed in the refrigerator until needed. (1 gm) was thoroughly mixed in tubes of 15 ml distilled water, followed by a series of dilutions until the sixth dilution was reached. On it (which was cooled to 45°C), the culture medium (starch-casein medium) was filtered. and 1ml of the last dilution was put in a sterile petri dish. This was done three times per sample. Several solitary colonies were used for re-culture in the same medium for pure culture after the plates were selected with (10-35) colonies [7] [8] [9].

Characteristics of Streptomyces
According to Bergy's manual of systematic bacteriology, second edition, the Actinobacteria, Part A [10] . The characteristics of Streptomyces are tasted based on the pattern of formation, Gram stain, and colony morphology.

Streptomyces detection
Based on the morphology and color of the colonies, the isolates were known As well as grow on Streptomyces as well as the Tryptone yeast extract glucose Agar, Glycerol Asparagine Agar, and Nutrient Agar. The aerial and medial twigs, as well as the arrangement of spores, were studied using the Slide culture technique [11].

Nutrient agar medium
This medium was made by melting (23 gm) of nutrient agar in (1 liter) of distilled water with a pH of 7.2 and sterilizing everything in the autoclave, as directed by the supplier company (Lab M Neogen Culture media). This medium was used in the isolation and identification of bacteria..

DNA from Streptomyces purification and acquisition
The DNA from the Streptomyces samples was extracted using Geneaid's kit analysis.

PCR Reactions
The Tri-EDTA (TE buffer) solution was used to fine-tune the DNA concentration in all of the isolated samples in order to achieve the optimal concentration for PCR reactions, and it worked well (50 nanogram per microliter). The master reaction for each PCR reaction was made by combining the DNA sample, the gene's specific primer, and the appender pre-mix in a (0.2ml) Eppendorf tube provided by the British company (bio). Using distilled water, the reaction volume was reduced to 20 microliters, and the components were then combined in a microfuge for (3)(4)(5) seconds. After that, the tubes were put in a thermal cycler to perform the polymer reactions, which were regulated by special software for each reaction. After that, the samples were electrophoresed in wells of a 2 percent agarose gel for 60-70 minutes. [3] [4][16]. In (Fig. 4) The samples with a similar length ( 1000 base ) pairs generated from the Streptomyces reaction DNA-specific polymer show 11 strands of purified DNA. The presence of these strands demonstrates that these isolates' genomic DNA contains shared sequences of nitrogenous bases that can join with the primer and continue the reaction, resulting in new DNA strands of the same length. These findings are similar to those of [23]. As the sequences were obtained from the nitrogenous bases of the DNA samples as follows:

Sample MU1 (Streptomyces gancidicus)
These sequences were entered into a program DNA BLAST to show their types and how close they are to sequences in the Gene Bank, as the result of the analysis showed a similarity of (99%) between these sequences and the sequences of bacterial isolates registered in the Gene Bank with the number (MT588801.1) Fig. (5). The partial 16S rRNA sequences deposited in GenBank were analyzed using DNA Blast ( Figure  5, 6,7,8,9,10,11,12,13,14,15). It was discovered in that all isolates belong to a Streptomyces species with (77-99%) resemblance to the 16S rRNA series of closely related species. 16S rRNA, bacterial genetic maker often In chromosomal DNA, there is a multigene family or operons. The role of this gene has changed dramatically over time, and its size is now large enough for informatics purposes. [24].