MEN AT WORK

A proposal on
Study of antibacterial properties of Streptomyces mutants developed by sodium azide exposure

Principle Investigator: Looza Shakya
Student from eighth semester
Universal Science College
Pokhara UniversityMaitidevi, Kathmandu2007

Supervisor: Kiran Babu Tiwari
Universal Science College
Pokhara University
RLABBMaitidevi, Kathmandu
Research LabResearch Laboratory for AgriculturalBiotechnology and Biochemistry (RLABB)Maitidevi, Kathmandu

Introduction:Streptomycetes are complex multi-cellular saprophytic soil bacteria. Perhaps best known for their ability to produce over two-thirds of naturally derived antibiotics currently in medical use(as well as other valuable pharmaceuticals).They are gram positive organisms and assumed to be the transition group between fungi and bacteria (Agrawal. 2003). They belong to the order Actinomycetales ( Superkingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual of Systematic Bacteriology they are divided into eight diverse families: Actinomycetaceae, Microbacteriaceae, Actinoplanceae, Frankiaceae, Dermatophilaceae, Nocaveliaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera. Based on 16sr RNA classification system they have recently been grouped in ten suborders: Actinomycineae, Cornebacterineae, Frankieae, Micromonosporineae, Propionibacterineae, Pseudocardinieae, Streptomycineae and a large member of actinomycetes are still remained to be grouped (www.ncbi.nlm.nih.gov)

Actinomycetes have characteristic biological aspects such as mycelial forms of growth that accumulates in sporulation and the ability to form a wide variety of secondary metabolites including most of the antibiotics. Complex morphological development in the genera is phenotypic ally related to secondary metabolism (Horinouchi and Beppu, 1992). Hence, investigation of actinomycetes from different ecological niches may yield novel isolates having more useful properties. Strain improvement for enhancement of antimicrobial activities holds a great significance in basic medical research.

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 (LOF) and gain of function (GOF) effects. The GOF is of particular importance and this type of work will be a good example of the foundation of applied research. 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 comparative antibacterial activities 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 screen antibacterial activities of the strains by primary screening method
To verify the antibacterial activities of the strains by secondary screening methodMethodology:

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, color 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 initially 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 sub cultured 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.

Screening of Streptomyces strains for antimicrobial activity
(a)Primary screening: The Steptomyces strains will be inoculated diagonally on the Nutrient agar plates and incubated at different temperatures, viz. 10°c,20°c, and 28°c. Antibacterial activity of the strains will be determined by streaking the test bacteria perpendicularly to the Streptomyces strains. The test organisms to be used will be Bacillus subtilis, Staphlococcus aureus, Enterobacter aerogens, E. coli, Klebsiella spp., Proteus spp., Pseudomonas spp., Salmonella typhi and Shigella spp.

(b)Secondary screening: Fresh and pure culture of each strain fron the primary screening will be inoculated in starch casein broth and incubated at accordingly for 7 days in water bath shaker. Growth of the organism in the flask wil be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flask will be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flasks will be filtered through Whatman no. 1 filter paper. The filtrate wil be used for the determination of antibacterial activity against the standard test organisms.

Expected Outcome
It has been found that azide induces gain of function and loss of function (Bhattarai and Tiwari, 2006). Enhanced antibacterial activity that they reported on primary screening (Bhattarai and Tiwari, 2006).is of particular importance; and hence, this applied aspect can be further elaborated and verified with secondary screening method. Possibly, the antibacterial activities can be created in those actinomycetes that do not posses the activity initially. Moreover, the mutants may develop different biochemical properties which will be of importance to understand more about streptomycetes physiology. Understanding such pathways and mutation of genes will give new idea for to further manipulate the organism.

Moreover, this study can possibly reveal actimomycetes species that produce novel antibiotics. It is anticipated that efforts for the isolation, characterization and the study on actinomycetes can be a milestone for the discovery of antibiotics and novel species of actinomycetes.


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 "R"
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 )
Horinouchi S, Beppu T (1992) Autoregulatory Factors and communications in Actinomycetes. Ann. Rev. Microbiol. 46:377-398
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.