Streptomycetes are aerobic or facultative anaerobic, gram positive, non-acid fast, non-motile with high (55%) G+C content (Olsen et al., 1993). Streptomyces spp. are the most populated and diverse soil bacteria producing various industrially and medically important secondary metabolites, such as enzymes (proteases, amylase, cellulase, lignase, chitinase etc.), chromogens and antibiotics. Streptomyces spp. produces most of the known antibiotics and, hence, is considered as the model research system that has therapeutic importance. Wild isolates may not necessarily possess beneficial properties such as potent antimicrobial activities, enzymes with economic manipulability etc.; hence, strain improvement for enhanced useful activities holds a significant importance in basic medical researches. In various researches, sodium azide, a chemical mutagen, has been found to increase interleukin-2 production (Bendahmane et al. 2002) and guanylate cyclase activity (Feldman et al., 1983) in mammals. Role of sodium azide as chemical mutagen has been studied in plants, fruit flies, rats etc.

Sodium azide is a white crystalline powder consisting of positive sodium ion and negative azide ions.  The azide ion is the interesting part of the molecule and is the part which is responsible for much of the molecules' reactivity. The azide group is made up three nitrogen atoms joined together.  This is a very unstable arrangement and consequently the azide will readily react to gain the more stable configuration of nitrogen gas.  This is the driving force of the explosive reactions of sodium azide. Several studies on loss-of-function (LOF) mutation and gain-of-function (GOF) mutation by sodium azide in different organisms could be seen, however GOF mutation is not yet reported in Streptomyces spp. Therefore, we attempted to explore on the beneficial mutations it can bring about while working with the actinomycetes isolates from Khumbu, Everest region.

MATERIALS AND METHODS

Isolation and identification of Streptomyces spp.: Soil sample (Lob. 20) was obtained from Research Laboratory for Biotechnology and Biochemistry (RLABB). Isolation  of actinomycetes was performed by soil dilution plate technique using Starch-Casein Agar ( Singh and Agrawal, 2002 & 2003 ). Actinomycetes were identified as colored, dried, rough, with irregular/regular margin; generally convex colony as described by Williams and Cross (1971). Streak plate method was used to purify cultures of actinomycetes (Williams and Cross, 1971), Singh and Agrawal 2002; Agrawal 2003). The isolate was  subcultured on other plates to obtain pure colonies. Gram staining was done for microscopical observation under oil immersion (1000X) (Bergay’s manual of Determinative Bacteriology, Ninth edition, 2000) in order to identify the isolate as Streptomyces spp.

Biochemical characterization: Different biochemical tests were performed to characterize the Streptomyces spp., viz. carbohydrate utilization test (salicin, arabinose, glucose, fructose, mannitol, galactose, sucrose, lactose, maltose), citrate utilization, hydrolysis (starch, Tween-20, casein), urease production and nitrate reduction (Holt, 1989; Singh and Agrawal, 2002; Agrawal, 2003).

Primary screening of antimicrobial activities: The strain was inoculated diagonally on the Nutrient agar (NA) plates and incubated at 28oC for a week. Antibacterial activity of the strains was determined by streaking the test bacteria perpendicularly to the Streptomyces strains (Tamrakar, 1997). The test organisms were Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi and Shigella species.

Sodium azide treatment: The isolate, then, was streaked on the Starch-Casein Agar plates containing varying concentrations (1-100ppm) of sodium azide, incubated at 28ºC for 5-6 days. Mutants were screened inintially based on differed colonial characteristics. Macroscopical, microscopical morphologies and biochemical characteristics of the corresponding mutants were studied as described for wild strain.

DNA polymorphisms: Individual strains were mass cultured in SC-broth by incubation the broth in shaker water bath for 5-6 days at 28ºC. Total DNA from corresponding strains was extracted as described by Sambrook et al. (2001). DNA polymorphisms among the strains were studied by RAPD-PCR using  Primer 261 (CTGGCGTGAC; GC%, 70; mp., 34ºC). In the PCR mixture, 1U of Taq polymerase and 1% of DMSO were used in addition to other regular components (1XPCR buffer  containing 1.5mM  MgCl2 with pH 8.3; dNTPs, 2.5mM each; 10-20ng of template DNA and primer/s, 10mM). The PCR products were separated on a 1.5% agarose gel (0.5 µg/ml ethidium bromide) made in TAE buffer (pH 8.0).

A corresponding reduction of the bacterial populations was observed on plates containing 12.5ppm, 25.0ppm, 37.5ppm sodium azide, beyond which the azide had lethal effect. These three corresponding mutants viz. S1, S2 and S3 had distinctly different phenotypic characteristics, e.g. loss of spore bearing mycelium and brown pigmentation with more brittle, irregular and diminished colony size compared to that of master strain, S0 (Table- 1). Among the test bacteria, B. subtillis, a food poisoning bacterium, was found to be markedly susceptible to S2 and S3 mutants, while S. aureus was slightly sensitive compared to the S0 (Figure- 1a).

Treatment of Streptomyces with 12.5ppm of sodium azide enabled bacteria to utilize nitrate, whereas alternate carbon sources (sucrose, mannitol and salicin) were metabolized by those bacteria treated with 37.5ppm of the chemical (Table- 2). All the strains (both wild type and mutants) were able to utilize glucose, galactose, fructose, lactose, maltose, citrate but arabinose. Similarly, urea, starch, Tween-20 and casein were hydrolyzed by all strains.

Two bands were missing in mutant S3 on agarose gel electrophoresis compared to five bands in wild strain (S0). The other mutants showed similar band patterns as of wild type (Figure- 1b).

Table-1:  Morphological comparison among wild and mutant strains

Strains	Colonial characteristics	Microbial Morphology
Wild (So)	Brown pigment, Sporogenous	Non fragmented, Sporogenous mycelium with curve at tips
Mutant S1 (12.5 ppm)	Little brown pigment, Sporogenous	Little fragmented, Sporogenous mycelium with curve at tips.
Mutant S2 (25.0 ppm)	Decreased colony size, Yellow pigmentation, Asporogenous, Brittle	Little fragmented, Sporogenous mycelium with curve at tips
Mutant S3 (37.5 ppm)	Yellow pigmentation, Asporogenous, Brittle	Dwarf and Irregular shaped asporogenous mycelium

Table- 2: Biochemical comparison among wild and mutant strains

Strains	Biochemical tests
	Carbohydrate utilization tests			Nitrate reductase
	Sucrose	Mannitol	Salicin
Wild (So)	-	-	-	-
Mutant S1	-	-	-	+
Mutant S2	-	-	-	-
Mutant S3	+	+	+	-

Fig- 1: Comparison of wild and corresponding mutants for (a) antibacterial properties, and (b) DNA polymorphisms

Most antibiotics that are currently used are produced by Streptomyces. Many strains produce several antibiotics derived from completely independent biosynthetic pathways and posses immense diversity of antibiotics among strains. Streptomyces spp. is considered a model system for biochemical, genetic and physiological studies.

Genetics instability is very common among Streptomyces strains and the affected strain display distinctive phenotypes (Dharmalingam and Cellum, 1996). Bacterial diversity could be induced by several mechanisms like DNA rearrangements DNA acquisition, reproductive infidelity and mutation. In the case of antibiotics synthesized by Streptomyces, the vast diversity of antibiotics produced, makes Streptomyces a particularly interesting group of organisms for studying question related to genetic variability. The large genomic size of about 8Mb in Streptomyces spp. considerably large than most well- studied metabolically versatile bacteria such as E. coli and Bacillus subtilis (ca 4.5Mb), suggests that there are probably many more genes than for metabolism sustain life (Leblond et.al., 1993). Spontaneous mutation does provide a potential genetic mechanism for the origin of these genes (Synder and Champness, 2003).

The phenotypic differences among the strains (Table- 1 & 2) clearly show the mutagenic effects of critical concentration of sodium azide beyond which the chemical has lethal effect. The morphological and biochemical changes in the present study are supported by other studies as Gehring et al. (2000), Champness (1988) and Ingram et al. (1995). Gehring et al. (2000) developed a procedure for generating insertional mutants of S. coelicolor based on in vitro transposition of a plasmid library of cloned S. coelicolor DNAs. Many of the insertions revealed previously uncharacterized morphological genes, and several caused novel mutant phenotypes, such as altered pigment production, enhanced antibiotic sensitivity, delayed or impaired formation of aerial hyphae, and a block in spore formation. Champness (1988) suggested the presence of new loci in S. coelicolor for such morphological and physiological differentiation, the colonies of which differentiate both morphologically, producing aerial spore chains, and physiologically, producing antibiotics as secondary metabolites. Single mutations, which block both aspects of differentiation, were defined as bld (bald colony) genes. Ingram et al. (1995) identified and characterized a ccrA1 mutant in S. coelicolor that affects the expression of several catabolite-controlled promoters. The result showed that the regulation of carbon utilization is of central importance in the gene expression pathways for both morphological development and antibiotic production in Streptomyces species. The ccrA1 mutants were altered (a) in expression of galP1, the glucose-sensitive galactose-dependent promoter of the galactose utilization operon, (b) in expression of the glycerol utilization operon, which is glucose sensitive and glycerol dependent, and (c) in expression of chi63, the glucose-sensitive chitin-dependent promoter of a gene involved in chitin utilization.

Genomic fingerprinting assay using Random amplified polymorphic DNA (RAPD) is useful in differentiating various strains. Relationships between species' may be determined by comparing their unique fingerprint information.  This method amplify of distinct genomic DNA sequence under low stringency conditions during annealing using an oligonucleotide primer of arbitrary sequences. The resulting amplification products of varying size can then be used as the genetic finger prints of the organism used in the assay. In order to conform the evidence of mutation, a RAPD-PCR using a decamer primer 261 (sequence, CTGGCGTGAC; GC%, 70; mp.,34oC) was selected. The PCR reaction mixture and thermal cycler progam used for DNA amplification was already optimized for Strptomyces spp. The band missing might be due to mutation in the primer binding region(s) ensuring no amplification of the products. It was concluded that point mutations on the primer-binding site resulted in the loss of bands for S3. Furthermore, similar band patterns in other mutants with S0 may be due to point mutations between primer binding flanking ends, which went undetected. Hence, additional primer evaluation is required to identify potential differences in the S1 and S2 strains.

The mutant S1 was able to reduce nitrate compared to the wild strain and other mutants. This might be due to possibly a reversion mutation in the wild strain with the lowest concentration of the azide. Azide binds to cofactors (molybdenum, iron) of the nitrate ruductase at elevated concentrations inhibiting the activity of the enzyme. Strain S3 had lower antimicrobial activity compared to that of S2. This may be attributed to the sensitivity of to higher levels of exposure of mutagen. Thus by selective levels of mutagenesis the antimicrobial activities of the organisms can be increased. Marked increase in the antibacterial activity as GOF mutation was observed against B. subtillis, a food poisoning bacterium.

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