Enhanced Antibacterial Activity of Sodium Azide Treated Mutant Streptomyces Strain

Enhanced Antibacterial Activity of Sodium Azide Treated Mutant Streptomyces Strain

Keshav Bhattarai(1), Kiran Babu Tiwari(1, 2, 3*) and Vishwanath Prasad Agrawal(1)
1Department of Biochemistry, Universal Science College, Maitidevi, Kathmandu, Nepal
2Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal
3Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
*Address of correspondence: Kiran Babu Tiwari, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal, email: babukiran@hotmail.com, Mob.: 9841374738

Abstract
A study was conducted to establish enhanced antibacterial activity, a Gain-of-Function (GOF) type of mutation, in Streptomyces strain exposed to sodium azide. The mutagenic concentrations of the azide were from 1.25–3.75mg%, beyond which the azide had lethal effect. The mutants (viz. S1, S2 and S3) and wild strain (S0) were screened for antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtillis, B. thuringiensis, Pseudomonas aeruginosa, Klebsella pneumoniae and Proteus vulgaris. Marked increase in the antibacterial acivity was observed against B. subtillis. DNA polymorphisms were observed among the Streptomyces strains in a RAPD-PCR, which indicates mutagenic effect of sodium azide.

Keywords: Antibacterial activity, mutation, RAPD-PCR, sodium azide, Streptomyces spp.

Streptomycetes are aerobic or facultative anaerobic, gram positive, non-acid fast, non-motile with high “G+C” content (55%).1 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. The native isolate may not necessarily possesses potent antimicrobial activities; 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 production2 and guanylate cyclase activity3 in mammals. Here, Streptomyces strain was exposed to sodium azide to elucidate whether the natural antimicrobial activity of the strain is enhanced.

Streptomyces spp. was isolated from soil sample.4 The colored, dry, rough, with irregular/regular margin, convex colony was selected and subsequently purified. Starch casein agar (SCA) plates with 1.25mg%, 2.5mg%, 3.75mg%, 5.0mg%, 6.25mg%, 7.5mg%, 8.75mg% and 10.0mg% of sodium azide were prepared and 50µl of the pure culture was applied homogeneously on the surface of each plate. Control plate didn’t contain azide. The plates were incubated at 270C for 5 days. The corresponding azide resistant strains were sub-cultured onto sterile SCA plates without sodium azide. The test strains of the isolate were screened for antibacterial activities on nutrient agar plate by “Cross- streak plate” technique against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, B. thuringiensis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus vulgaris.

A corresponding reduction of the bacterial populations was observed on plates containing 1.25mg%, 2.5mg%, 3.75mg% 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. 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 (Table-1).

The genomic DNA from the corresponding Streptomyces strains was isolated5 and a primer (sequence, CTGGCGTGAC; GC%, 70; mp.,34oC) was used for Randomly Amplified Polymorphic DNA (RAPD)-Polymerase Chain Reaction (PCR)6 to observe genetic polymorphisms. PCR reactions (30mL each) were performed in 1XPCR buffer (1.5mM MgCl2) with pH 8.3; dNTPs (2.5mM each), Taq-polymerase (1U) (obtained from GENEI Company, Bangalore, India), DMSO (1%), 10-20ng of template DNA and primers (10mM, obtained from University of British Columbia). The thermal cycler programme was set as: initial denaturation at 95 oC for 5min; 39 cycles of denaturation at 94oC for 1 min, annealing at 36 oC for 2min and extension at 72oC for 3min; followed by final extension at 72oC for 10min.



Table-1: Primary screening of antibacterial activities
of the Streptomyces strains (as length of inhibition, mm)

Fig 1: RAPD-PCR band pattern of Streptomyces
(λ DNA/Hind III marker, M)




















Compared to S0, with five bands on 1% agarose gel electrophoresis (1322, 681, 596, 307, 206bp), two bands (1322 and 681) for mutant S3 were missing (Figure 1), which might be due to mutation in the primer binding region(s) ensuring no amplification of the products. Difference in morphology and antibacterial activity in S2 compared to those of the S0 could not be revealed in PCR ,possibly because mutations might have occureed outside primer binding regions necessitating use of other primer(s). Results indicate that using sodium azide as a mutagen, the antibacterial activities of Streptomyces spp. can be enhanced. This preliminary research, therefore, suggest the possibilities of strain improvement which may have wider applications in the modern chemotherapy .

Reference:
1. Williams ST, Sharpe ME, Holt JG. Streptomyces and related genera. In Bergey’s Manual of Systematic Bacteriology 1989; 4. Williams and Wilkins, USA.
2. Feldman SP, Mertelsmann R, Venuta S, Andreeff M, Welte K, Moor MAS. Sodium azide enhancement of Interleukin-2 production. Blood 1983; 61: 815-8.
3. Sano I, Mizumoto A, Sakai T, Tamura T, Itoh Z. Sodium azide induces relaxation of the canine gastric body by activating a guanylate cyclase dependant pathway. Neurogastroenterol Monit 1997; 9: 193-201.
4. Tamrakar R. Antibacterial activities of actinomycetes isolated from soils of Kathmandu valley. Thesis submitted to the Central Department of Microbiology, Tribhuvan University 1997.
5. Sambrook J, Fritsch t, Maniatis J. Molecular Cloning- A Laboratory Manual, 2nd Ed. Cold Spring Harbour Laboratory Press, London 1989.
6. Yu K, Pauls KP. Optimization of the PCR program for RAPD analysis. Nucleic Acid Res 1992; 20: 2606.