A proposal on
Study of DNA polymorphisms of Streptomyces mutants developed by sodium azide exposure

Principle Investigator: Saroj Sapkota
Student from eighth semester
Universal Science College
Pokhara University
Maitidevi, Kathmandu2007

Supervisor: Kiran Babu Tiwari
Universal Science College
Pokhara University
RLABB
Maitidevi, Kathmandu

Research Lab
Research Laboratory for AgriculturalBiotechnology and Biochemistry (RLABB)
Maitidevi, Kathmandu

Introduction:
Actiomycetes are the gram positive organisms and has been identified as one of the major groups of soil population. These organisms are assumed to be the transition group between fungi and bacteria. Actinomycetes are also called as antibiotic producing bacteria and such antibiotics are diverse in chemical structure. Actinomycetes belong to the order Actinomycetales ( Superkingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual of Systematic Bacteriology (1989), they are divided into eight diverse families: Actinomycetaceae, Microbacteriaceae, Actinoplanceae, Frankiaceae, Dermatophilaceae, Nocaveliaceae, Streptomycetaceae, Micromonosporaceae. Based on 16sr RNA classification system they have recently been grouped in ten suborders: Actinomycineae, Cornebacterineae, Frankieae, Micromonosporineae, Propionibacterineae, Pseudocardinieae, Streptomycineae.

Sodium azide is a mild mutagen and its toxicity is compared to cyanide. It has molecular formula NaN3 which consists of positive sodium ion and negative azide ion when mixed with water changes to gas. Thus it has very unstable configuration and tends to attain stable configuration of nitrogen gas. This property of molecule to attain stable configuration makes it to carry out different bio chemical manipulations which include the inhibition of enzymes, inhibition of oxidase, mutation, and also activate some biochemical pathways in living organisms.

Background:
In, RLABB, Bhattarai and Tiwari (2006) developed a prototype methodology to explore mutagenic effects of sodium azide in Streptomyces spp., viz. both for loss of function and gain of function effects. The study should be extended in order to cover more parameters of actinomycetes, which helps to understand their physiology.

General objectives:To develop mutants using sodium azide and study of DNA polymorphisms among wild type and mutants

Specific Objectives:
To isolate and purify streptomycete from soil sample
To characterize the isolate for colonial, microscopical and biochemical properties
To develop mutants by treating the isolate with sodium azide in different concentrations
To characterize the mutants for colonial, microscopical and biochemical properties
To extract total DNA from corresponding strains
To study the genetic polymorphisms among mutants and the wild type using
RAPD-primer 261 and 262 separately
RAPD primers 261 and 262 in combination

Methodology:
Isolation of actinomycetes: Soil samples will be obtained from Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). Isolation of actinomycetes will be performed by soil dilution plate technique using Starch-Casein Agar ( Singh and Agrawal, 2002 & 2003 ). Actinomycetes on the plates will be identified as colored, dried, rough, with irregular/regular margin; generally convex colony as described by Williams and Cross (1971).

Purification of actinomycetes: Streak plate method will be used to purify cultures of actinomycetes (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

Morphological characterization: Morphological examination of the actinomycetes will be done by using cellophane tape and cover slip-buried methods (Williams and Cross, 1971; Singh and Agrawal 2002; Agrawal 2003). The mycelium structure, color and arrangement of conidiospore and arthrospore on the mycelium will be examined under oil immersion (1000X). The observed structure will be compared with Bergay’s manual of Determinative Bacteriology, Ninth edition (2000) for identification Streptomyces spp.

Biochemical characterization: Different biochemical tests will be performed to characterize the Streptomyces spp. The tests generally used are gelatin hydrolysis, starch hydrolysis, urea- hydrolysis, acid production from different sugars, resistance to NaCl, temperature tolerance test, hydrogen sulphide production test, motility test, triple sugar iron (TSI) agar test, citrate utilization test, indole test, methyl red test, voges-proskauer (Acetoin Production) test, catalase test, oxidase test (Holt 1989; Singh and Agrawal 2002; Agrawal 2003).

Exposure to sodium azide (Bhattarai et al. 2007): The isolate will be streaked on the Starch-Casein Agar plates containing varying concentrations (5-100ppm) of sodium azide, incubated at 28C for 5-6 days. Lethal concentration of sodium azide will be designated for that concentration that totally inhibits the growth. Mutants will be screened inintially based on differed colonial characteristics. Macroscopical, microscopical morphologies and biochemical characteristics of the corresponding mutants will be studied as described for wild strain.

Storage of wild and mutants strains: The mutants will be subcultured on azide free SCA (incubation at 28C for 5-6 days) plates and the strains will be stored in 15% glycerol containing Nutrient agar (G-NA) and keeping in deep freeze. The cultures will be revived as per the requirement.

DNA extraction: Individual strains will be mass cultured in SC-broth by incubation the broth in shaker water bath for 5-6 days at 28C. Total DNA from corresponding strains will be extracted as described by Sambrook et al (1990).

DNA polymorphisms: DNA polymorphisms among the strains will be studied by RAPD-PCR using (a) Primer 261 (b) Primer 262 and (c) Combination of both primers (261+262). In the PCR mixture, 1U of Taq polymerase and 1% of DMSO will be 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 will be separated on a 1.5% agarose gel (0.5 µg/ml ethidium bromide) made in TAE buffer (pH 8.0).

Expected Outcome: It has been found that azide induces gain of function and loss of function (Bhattarai and Tiwari, 2006). The mutants may develop different biochemical properties which will be of importance to undersatand more about streptomycetes’ physiology. Understanding such pathways and mutation of genes will give new idea for to further manipulate the organism.

References:
Agrawal, V. P (2003) Biodiversity of Khumbu Region : Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Enhanced antibacterial activity of sodium azide treated mutant Streptomyces strain. Journal of Nepal Association for Medical Laboratory Sciences, 8(1): 67-8.

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Loss-of-function (LOF) and gain-of-function (GOF) mutation of sodium azide in Streptomyces spp. Journal of Nepal Biotechnology Association. (accepted).

Holt, J.G., (1989) Bergey’s manual of systematic bacteriology, vol. 4, ed. S.T.Williams and M.E.Sharpe, Baltimore, Md : Williams and Williams

Pandey, B., Ghimire, P. and Agrawal, V.P. (2004 ) Studies on Antibacterial Activity of Soil from Khumbu Region of Mount Everest, a paper presented in International Conference on The Great Himalayas : Climate, Health, Ecology, Management and Conservation, Kathmandu, January 12 -15, 2004 ( organized by Kathmandu University and The Aquatic Ecosystem Health & Management Society, Canada )

Roberts MA and Crawford ( 2000) Use of Randomly Amplified Polymorphic DNA as a Means of Developing Genus- and Strain-Specific Streptomyces DNA Probes. Appl Environ Microbiol vol 66 2555–2564.

Singh, D. and Agrawal, V.P. (2002) Microbial Biodiversity of Mount Everest Region, a paper presented in International Seminar on Mountains - Kathmandu, March 6 – 8, 2002 ( organized by Royal Nepal Academy of Science and Technology )

Singh, D. and Agrawal, V.P. (2003) Diversity of Actinomycetes of Lobuche in Mount Everest I Proceedings of International Seminar on Mountains – Kathmandu, March 6 – 8, 2002 pp. 357 – 360.