Isolation, identification and molecular characterization of streptomyces from soil in halabja, Iraq

The aim of this study is to isolate actinomycetes, particularly Streptomyces, species from soil samples were collected from Halabja at Iraq. For this aim, total 30 soil samples were collected from the different part of Halabja and physicochemical parameters of them were measured after brought the laboratory. First of all, a conventional isolation method used to isolate Streptomyces. Ten-fold dilution of soil samples were inoculated onto strach casein agar plates supplemented cycloheximide, nystatin and novobiocin and raffinose-histidine agar plates supplemented with cycloheximide and nystatin and 105 colonies having substrate and aerial mycelium was selected and purified. All isolates presumably were colour grouped according to aerial spore mass, colony reverse and diffusible pigment colours formed on oatmeal agar and on their capacity to produce melanin pigments on peptone-yeast extract iron agar and isolates were assigned to 10 colour group. 20 representatives strains of coluor groups were tested for 40 diagnostic identification test and then the results were analyzed by computer-assisted program generating a dendrogram. These 20 test strains were assigned 10 clusters and the cluster of dendrogram showed to match those obtained by manual colour-grouping of the isolates more or less. For phylogenetic analysis, 16S rDNA genes of 20 test strains were amplified with universal primers after extraction of genomic DNA and then sequenced, but 17 of them were successfully sequenced. Phylogenetic analysis of 17 test strains carried out using base sequences of 16S rDNA genes in the core genome resulted that 10 test strains were identified as Streptomyces species those were 3 Streptomyces badius NRRL B-2567, 5 Streptomyces cinereoruber subsp. cinereoruber NBRC 12756 and 2 Streptomyces enissocaesilis NRRL B-16365. In addition, 3 test strains were identified as Mesorhizobium huakuii IAM 14158 while 3 of them were Nocardiopsis synnemataformans DSM 44143, one of them was identified as Pseudomonas taiwanensis BCRC 17751T species that may be a contaminated strains. Results of phylogenetic analysis showed that test strains identified as Mesorhizobium huakuii IAM 14158 may be a new species due to 98,45% similarity level, that is differences is to high with type species. For this reason, DNA-DNA hybridization should be carried out in future to accept new species due to essential technique for description a new species.xiv Also the diaminopimelic amino acid (DAP) content of the cell wall of 4 test strains was determined and the sugars of the whole cell hydrolysates were analyzed. 3 test strains had LL-diaminopimelic acid as the diagnostic cell wall diamino acid for Streptomyces genus, and glucose, ribose and mannose were detected in the whole cell hydrolysates while one of test strains contained mezo-A2pm DAP as diagnostic for Nocardiopsis species, and galactose and ribose in the whole cell hydrolysates. The results of chemotaxonomic studies supported the phylogenetic analysis findings. Keywords: Streptomyces, Halabja, 16S rDNA, Actinobacteria.