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Introduction and Aim: There are centers for antibiotics in the nature to treat disease by looking for different sources, for example, actinomycetes, are approximately two-thirds of naturally occurring antibiotics, having much of medical importance, have been isolated from actinomycetes. The aim of the present study was to isolate and to identify the actinomycetes having antagonistic activity. Materials and Methods: An actinomycetes strain isolated from dam soil sample collected from Alnajl dam of Hadhramout Yemen, showed antibacterial activity against selected microbial pathogens. The nutritional requirements and cultural conditions for maximal growth to produce secondary metabolites have been optimized under shake flask conditions. The growth and production of secondary metabolites were maximal with the use of SCA medium supplemented at pH 7.0, and incubation temperature and time of 37℃ and 20 days respectively. Results: Based on morphological, biochemical, physiological and phylogenetic characterization, the strain was identified as Actinoplanes digitatisDnj-2 (MN567713). The ethyl acetate extract (1g/mL) obtained from the isolate showed significant antibacterial activity against Gram-negative bacteria-Escherichia coli (21 cm) and Pseudomonas aeruginosa (29 cm) and Gram-positive bacteria-Bacillus subtitles (31 cm) and Staphylococcus aureus (22 cm) when compared with penicillin G 10 units. Conclusion: In conclusion, the isolated strain has broad spectrum of antagonistic activity against Gram-positive and Gram-negative bacteria.


INTRODUCTION
atural products stay to be the most hopeful source of antibiotics. There are approximately 32,500 natural products reported from microbial sources (1). In addition, at present, twothirds of natural antibiotics are obtained from actinomycetes and they serve as substitutional sources of biologically active substances (2). Actinomycetes from the genera Actinoplane, Streptomyces, and Actinopolyspora have been reported to produce over 300 broad-spectrum antibiotic substances (3,4) and representatives of these genera are widely abundant in aquatic ecosystems.
The genus Actinoplanes was classified under the group Actinoplanetes in Bergey's Manual of Determinative Bacteriology (5). Genera in this group were distinguished from each other based on the shape of the sporangium, number, shape, and arrangement of spores in the sporangium, motility, and flagellation of the spores and other such characters. The isolate was identified as belonging to Actinoplanes. Identification of isolate to the species level was done based on the morphology of sporangium, presence or absence of aerial mycelium and assimilation of carbon sources as well as a molecular genome. Species belonging to this genus were identified as Actinoplanes digitatis. At the time of writing report, the genus involves 45 species with validly published names, including as those of late described Actinoplanes lichenis (6), Actinoplanes subglobosus (7), Actinoplanes subg lobosus (8), Actinoplan-es ramoplaninifer (9) and Actinoplanes sediminis (10). A study of the morphology, biochemical characters in agreement with 16S rRNA gene sequence-based phylogenetic analysis, showed later that a member of a second species of the genus Actinoplanes related to this strain had been found. This strain has been described previously as Actinoplanes digitatis (11).
The Hadhramout-Yemen has not been studied extensively with respect to the antagonistic properties of actinomycetes. In the course of our screening program for new antagonistic actinomycetes, the strain, Actinoplanes digitatisDnj-2 (MN567713) was isolated from the soil sample collected from Rahbat bin Junaid Hadhramout Yemen capable of producing antibiotics that strongly inhibit the growth of Grampositive and Gram-negative bacteria. In the present study, we have reported the antimicrobial activity of Actinoplanes digitatisDnj-2 (MN567713).

MATERIALS AND METHODS
This study was carried out during December 2018 to June 2019 in the Jnana Kaveri Post Graduate Centre, N

Microorganisms and culture conditions
The strain Dnj-2 was isolated from a soil sample collected from Alnajl dam, Hadhramout -Yemen. It was subjected to serial dilution (up to 10 6 dilution) by adding 1 g of the soil sample in 10 mL of distilled water. About 1.0ml of diluted sample plated on starch casein agar containing 10 g starch, 1.0 g casein, 0.2 g calcium carbonate, 0.01 ferrous sulfate (anhydrous), 2.0g potassium nitrate, and 20g agar in distilled water at pH 7.2, incubated at 37°C for two weeks (12).

Cultural and morphological characterization
Cultural characteristics of strain Dnj-2 were compared on the base of observations made after 14 days incubation starch casein agar, nutrient agar sabouraud dextrose agar, yeast malt extract agar, glycerol asparagines agar, tyrosine agar, Czapeck's dox thom agar, glycerol yeast extract agar and starch yeast extract agar (13). Morphology was examined by light microscope.

Physiological characterization
The utilization of carbohydrate was investigated with a basal carbon nutrient medium (13,14). Methods and media used for physiological tests were as described by Williams et al., (15). All cultures were incubated at 37℃ for 10 days. The assay for enzymatic activity was performed (16)(17)(18).

Genotypic characterization
DNA was isolated from Actinoplane strains using the method of Healy and Lambert (19). DNA extraction, PCR amplification, Gel verification, purification, sequencing, assembling, phylogenetic tree.

Fermentation and preparation of crude extract
The actinomycetes are inoculated on starch casein agar plates and incubated at 37℃ 7 days. The spores were harvested in sterile 0.01% (v/v) Tween-20 in distilled water. 300 ml SCB medium was inoculated with 10% (v/v) spore's suspension of actinomycetes, the flasks incubated at 37℃ on rotary incubator shaker, at 200 rpm for 14 days, and OD of the growth was recorded every day at A540. Fermentation broth was centrifuged at 10,000 rpm for 20 minutes to separate the mycelial biomass. Ethyl acetate was added to the supernatant for extraction of antibiotics, in 1:1 proportion. Solvent-supernatant mixture was agitated for 45 minutes with homogenizer. Solvent was separated from broth by separating funnel. Solvent was centrifuged at 5,000 rpm for 10 minutes to remove traces of fermentation broth and crude antibiotic stored in Eppendorf tubes at 4℃ (20).

Microbial pathogens
Bacterial pathogens, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus were used.

Method of antimicrobial activity
The antibacterial activity of secondary metabolites (1g/ml) extracted by the ethyl acetate solvent was tested by the agar diffusion method. The plates were incubated at 37℃ for 24 h during which activity was evidenced by the presence of a zone of inhibition surrounding the well. Each test was repeated three times and the antibacterial activity was expressed as the mean of a diameter of the inhibition zones (mm) produced by the secondary metabolite when compared to controls.

Characterization of Actinoplanes digitatis Dnj2 isolates
The strain Dnj-2 was isolated from Alnajl dam on SCA. The early work had shown that the morphological and physiological characteristics of the strain Dnj-2 was observed using agar cultures incubated for 21 days at 37 o C on the various media described by Shirling and Gottlieb (13): YMA, GAA and TA, but we added various media as SCA, PDA, NA, SYA, and GYA (Table 1). The nucleotide sequence of the 16S rRNA gene of strain Dnj-2 showed 99.59% similarity to Actinoplanes digitatis. The phylogenetic tree (Fig.1)   By comparing the results between strain Dnj-2 and closely related species of the genus, Actinoplanes revealed that it is similar to Actinoplanes digitatis strain OKG1 (KF447933.1) in morphological, cultural, and physiological characteristics (table 2). The aerial mycelium of strain Dnj-2 was initially white and become orange brown on CDTA, produce diffusible pigment as brownish in YMA. A similar pattern of results was obtained in the aerial mycelium of Actinoplanes digitatis as white and soluble pigments were produced. Overall, these findings are in accordance with findings reported by Stackebrandt and Kroppenstedt (6). Notably, all prior carbon source utilization was determined by the methods described by Shirling and Gottlieb (13) and Locci (21). Nitrogen source utilization was determined according to Williams et al., (22). In line with previous studies, our results were casting a new light on carbon sources. Therefore, the Dnj-2 strain of Actinoplanes has the same physiological characters such as the ability to utilize sole carbon and nitrogen sources: D-glucose, Dmannose, L-arabinose, glycerol, D-maltose, sucrose, and lactose but not sodium acetate and sucrose (sole carbon sources) and glycerol, alanine and L-asparagine (sole nitrogen sources). Growth of strain Dnj-2 was observed at a wide range of temperatures (20-45°C), although the optimal temperature range was 37°C. The initial pH range for which growth of strain Dnj-2 was observed between pH 7-10; however, the optimal pH value for growth was determined to be 7.0. Strain Dnj-2 was also capable of growth in the presence of 4% NaCl. In addition, the strain Dnj-2 reduced nitrate to nitrite and hydrogen sulfide was not produced. Starch, casein, gelatin, starch, chitin, lecithin, pectin and urea were degraded by Dnj-2 but not, keratin and lipid. Based on the genotypic and phenotypic evidence, it is suggested that strain Dnj-2 is a species of the genus Actinoplanes, for which the name is Actinoplanes digitatis Dnj2 under accession number MN567713.

CONCLUSION
Our studies will establish the rich bioactive actinomycete of the Hadhramout region. Therefore, further intensive studies are required on the actinobacterial diversity of bioactive compounds in Hadhramout-Yemen, which could form an important input into pharmaceutical industries.

ACKNOWLEDGMENT
We wish to genuinely thank Mangalore University for providing the necessary facilities and support for this study.

CONFLICT OF INTEREST
Authors declare no conflict of interest.