My Scientific Blog - Research and Articles: September 2009

Kiran Babu Tiwari^1*, Mamata Khatri², Yurika Rajbhandari², Shruti Regmi², Junu Hamal², Upendra Thapa Shrestha¹ and Vishwanath Prasad Agrawal^{1, 2}

¹Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal

²Universal Science College, Pokhara University, Maitidevi, Kathmandu, Nepal

Abstract

Sodium azide was employed to generate mutants of Actinomucetes from Sagarmatha National Park, Jorsela, Kalapatthar and Namche region located in Mount Everest base camp. Pure cultures of Actinomycetes were inoculated on Starch Casein Agar plates containing 10ppm or 50ppm or 100ppm of sodium azide. Actinomycete isolates were categorized as highly sensitive (lethal dose 10ppm), moderately sensitive (lethal dose 50ppm) and tolerant (lethal dose 100ppm). Out of total 38 Actinomycete isolates, two (5.3%) were highly sensitive, 23 (60.5%) moderately sensitive and 13 (34.2%) tolerant. To generate mutants, Actinomycetes were treated with sublethal concentration and mutants were analyzed morphologically and biochemically. Altogether nine loss-of-function (LOF) mutants with respect to single substrate, two LOF mutants with respect to two substrates, one gain-of-function (GOF) with respect to single substrate, one GOF mutant with respect to two substrates and five mutants having GOF as well as LOF with respect to substrates.

Introduction

Actinomycetes are diverse groups of soil inhabiting microbes. They are aerobic Gram- positive bacteria that form branching, usually nonfragmenting hyphae and asexual spores. They comprise 63 genera and are relatively well documented because of their value in drug discovery (Williams et al. 1989). Streptomyces spp., a major group of Actinomycetes, are 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 (Williams et al. 1989). Streptomyces spp. produce most of the known antibiotics and, hence, are considered as model research system that has therapeutic importance. Hence, investigation of actnomycetes from different ecological niches may yield novel isolates having more useful properties.

In Research Laboratory for Biotechnology and Biochemistry (RLABB), Bhattarai et al. (2007) were the first to observe sodium azide induced mutation of Actinomycetes. Further, enhanced antibacterial property in a Streptomyces strain - a Gain-of-Function (GOF) mutation was reported by Bhattarai et al. (2007) and Tiwari et al. (2008). Hence, the study was further conducted to explore the mutagenic effects of sodium azide to the cold tolerant Actinmycetes from Mt. Everest region. The objective of this work was to classify Actinomycetes based on their sensitivities to different concentrations of sodium azide.

Abbreviations: Ara = Arabinose, Fru = Fructose, Gal = Galactose, Glu = Glucose, GOF = Gain-of-Function, GOF = Gain-of-Function mutation, Jor = Jorsela, Kal = Kalapatthar, LOF = Loss-of-Function, Man = Mannose, Nam = Namche, ppm = parts per million, RLABB = Research Laboratory for Biotechnology and Biochemistry, SCA = Starch Casein Agar, SNP = Sagarmatha National Park

Materials and Methods

Collection of soil samples: Soil samples were collected randomly from Jorsela (2800m), Namche (3450m), Sagarmatha National Park (around 5000m) and Kalapatthar (5545m) in Mt. Everest base camp (Fig. 1) . Soil samples were taken from 6-10 cm depth into sterile plastic bags, sealed and transported to RLABB and the samples were air dried in room temperature.

Isolation of Actinomycetes: Altogether 38 Actinomycetes were isolated from 17 soil samples by dilution plate technique using SCA media(Singh and Agrawal 2003).

Macroscopic methods: Actinomycetes were 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 was used to purify Actinomycetes (Williams and Cross 1971; Singh and Agrawal 2003).

Categorization of Actinomycete isolates on the basis of sodium azide sensitivity: Individual pure colonies were streaked on SCA plates containing varying concentrations (10, 50 and 100ppm) of sodium azide, incubated at 28ºC for 5-6 days. Based on the complete inhibition of the growth of Actinomycetes, three categories were assigned respectively as (a) Highly sensitive (<10ppm),>50ppm).

Generation and selection of mutants: Actinomycete isolates were streaked on the Starch-Casein agar plates containing sublethal concentrations of sodium azide and incubated at 28ºC for 5 - 6 days. The resulting colonies will be characterized morphologically. The colonies having morphology different from the wild type will be considered as the mutants.

Actinomycete Nomenclature system in RLABB: Wild strains were represented by a capital initial and next two small letters denoting sampling site followed by a serial number for different sample number from the same region. Small letter following the serial number represents different Actinomycete isolates from the same soil sample. e.g., Snp16d is the fourth Acinomycete isolate from sixteenth soil sample from SNP. For mutants, respective wild type name is followed by the concentration of sodium azide after a dot. Two or more mutants from a wild strain from same concentration of sodium azide exposure are represented by small Roman numbers after a dot. e.g., Snp16d.10.i is the first mutant for 10ppm of sodium azide.

Biochemical characterization: Different physiological and biochemical tests (viz., starch hydrolysis, casein hydrolysis, Tween 20 hydrolysis, urea- hydrolysis, sugar assimilation, citrate utilization test, and catalase test) were performed to characterize wild and correspomding mutants of actinomycete isolates (Williams et al. 1989; Singh and Agrawal 2003).

Result

Categorization of Actinomycetes: Nine wild isolates from SNP, 17 isolates from Jorsela, 10 isoates from Kalapatthar and two isolates from Namche were treated with sodium azide concentrations of 10, 50 and 100ppm to categorize the isolates in highly sensitive, moderately sensitive and tolerant groups (Table 1).

Table 1: Categories of the actinomycetes (n=38) based on their sensitivity to sublethal concentration of sodium azide

Sampling sites	Highly sensitive	Moderately sensitive	Tolerant	Number of isolates^@
Sagarmatha National Park (Snp)	-	Snp16b, Snp16d, Snp18a, Snp20a, Snp21b	Snp1b, Snp1c, Snp1e, Snp19b	9 (6)
Jorsela (Jor)	Jor13e	Jor11g, Jor11h, Jor14b, Jor14d, Jor14e, Jor14g, Jor16c	Jor11d, Jor11e, Jor11j, Jor11k, Jor13a, Jor13b, Jor13c, Jor16b, Jor16f	17 (4)
Kalapatthar (Kal)	Kal8b	Kal6b, Kal16h, Kal25a, Kal25b, Kal48c, Kal70a, Kal70c, Kal70e, Kal100b	-	10 (6)
Namche (Nam)	-	Nam29a, Nam29b	-	2 (1)
Total isolates	2	23	13	38 (17)

^@Figures in parentheses represent total number of corresponding soil samples.

Of the total 38 Actinomycete isolates, two (5.3%) were highly sensitive, 23 (60.5%) were moderately sensitive, and 13 (34.2%) were tolerant. As for example, for three isolates from Jorsela, the results have been graphically presented in Fig. 2.

Figure 2 Relative growth of Jor13e, Jor14b and Jor13c upto sublethal concentration of sodium azide

Generation of mutants: Altogether 23 mutants were generated exposing eight wild strains to sublethal concentration of sodium azide (Table 2 and Fig. 3).

Table 2: Wild type strains and their corresponding mutants generated with sublethal concentration of sodium azide

Wild types*	Lethal Concentration	Mutants
Snp16d (3)	30ppm	Snp16d.10.i, Snp16d.10.ii, Snp16d.20.i
Snp18a (4)	40ppm	Snp18a.10.i, Snp18a.10.ii, Snp18a.20.i, Snp18a.30.i
Jor11g (2)	30ppm	Jor11g.10.i, Jor11g.20.i
Jor11h (1)	20ppm	Jor11h.10.i
Kal70c (2)	20ppm	Kal70c.10.i, Kal70c.10.ii
Kal70e (5)	50ppm	Kal70e.10.i, Kal70e.10.ii, Kal70e.20.i, Kal70e.30.i, Kal70e.40.i
Nam29a (4)	40ppm	Nam29a.10.i, Nam29a.10.ii, Nam29a.20.i, Nam29a.30.i
Nam29b (2)	20ppm	Nam29b.10.i, Nam29b.10.ii

*Figures in parentheses represent number of mutants corresponding to wild type strains

Figure 3 Growth pattern of Nam29a and Nam29b in plates containing different concentrations of sodium azide (a = Nam29b and Nam29a in 10ppm agar, b = Nam29b and Nam29a in 20ppm agar, c = Nam29a and Nam29b in 30ppm agar)

Characterization of mutants: All wild type strains and their corresponding mutants were able to hydrolyse starch, Tween 20 and casein. All were urease and catalase positive. GOF mutations with respect to Arabinose (Ara), Galactose (Gal), Mannose (Man) and Sucrose (Suc) assimilation were observed among mutants from Snp18a (two, Ara), Kal70e (two, Gal; one, Man), Nam29a (one, Ara) and Nam29b (two, Suc).

Types of mutations in carbon metabolism: Various LOF mutations were observed among mutants from Snp16d (two, Gal), Snp18a (five, Man), Jor11g (two, Gal), Kal70c (two, Ara and Gal each), Nam29a (four, Gal) and Nam29b (two, Ara) (Table 3).

Table 3: Biochemical characterization of eight moderately sensitive actinomycetes to sodium azide and their corresponding mutants

Wild Type	Colony color		Colony Texture	Carbohydrate Utilization Test
Mutants	Upper	Lower	Colony Texture	Ara	Glu	Man	Gal	Fru	Lac	Suc
Snp16d	OW	CB	Scr	-	+	-	+	+	+	+
Snp16d.10.i Snp16d.20.i	LCW	LGY	Bri	-	+	-	-	+	+	+	LOF (Gal)
Snp16d.10.i Snp16d.20.i	W	W	Bri	-	+	-	-	+	+	+	LOF (Gal)
Snp18a	CW	DB	Scr	-	+	+	+	+	+	+
Snp18a.10.i Snp18a.10.ii Snp18a.20.i Snp18a.30.i	DW	LB	Bri	-	+	-	+	+	+	+	LOF (Man)
	DW	LB	Bri	+	+	-	+	+	+	+	GOF (Ara) + LOF (Man)
	DW	OW	Bri	+	+	-	+	+	+	+	GOF (Ara) + LOF (Man)
	DW	OW	Bri	-	+	-	+	+	+	+	LOF (Man)
Jor11g	CW	LB	V bri	-	+	-	+	+	+	+
Jor11g.10.i Jor11g.20.i	DW	LY	Scr	-	+	-	-	+	+	+	LOF (Gal)
Jor11g.10.i Jor11g.20.i	W	W	Scr	-	+	-	-	+	+	+	LOF (Gal)
Jor11h	G	GY	L bri	+	+	-	-	+	+	+
Jor11h.10.i	Y	Y	Bri	+	+	-	-	+	+	+	No mutation in CU
Kal70c	GW	B	Bri	+	+	-	+	+	+	+
Kal70c.10.i Kal70c.10.ii	GW	CB	L bri	-	+	-	-	+	+	+	LOF (Ara,Gal)
Kal70c.10.i Kal70c.10.ii	GW	CB	L bri	-	+	-	-	+	+	+	LOF (Ara,Gal)
Kal70e	GW	YB	L bri	-	+	-	-	+	+	+
Kal70e.10.i Kal70e.20.i Kal70e.30.i Kal70e.40.i	DGW	CB	Bri	-	+	-	+	+	+	+	GOF (Gal)
	CW	LY	L bri	-	+	-	-	+	+	+	No mutation in CU
	CW	LY	L bri	-	+	+	+	+	+	+	GOF (Man) + GOF (Gal)
	OW	OW	Bri	-	+	-	-	+	+	+	No mutation in CU
Nam29a	DW	PG	Scr	-	+	-	+	+	+	+
Nam29a.10.i Nam29a.10.ii Nam29a.20.i Nam29a.30.i	WG	OW	L bri	+	+	-	-	+	+	+	GOF (Ara) + LOF (Gal)
	WG	OW	L bri	+	+	-	-	+	+	+	GOF (Ara) + LOF (Gal)
	OW	OW	L bri	-	+	-	-	+	+	+	LOF (Gal)
	OW	OW	Bri	-	+	-	-	+	+	+	LOF (Gal)
Nam29b	W	DW	Bri	+	+	-	+	+	+	-
Nam29b.10.i Nam29b.10.ii	CW	BY	Scr	-	+	-	-	+	+	+	GOF (Suc) + LOF (Ara,Gal)
Nam29b.10.i Nam29b.10.ii	CW	BY	Scr	-	+	-	+	+	+	+	LOF (Ara)

B = Brown, Bri = Brittle, BY = Bright Yellow, CB = Chocolaty Brown, CB = Creamy Brown, CU = tested carbohydrate utilization, CW = Chalky White, DB = Dark Brown, DGW = Dirty Greenish White, DW = Dirty White, G = Grey, GW = Greenish White, GY = Grayish Yellow, L Bri = Less Brittle, LB = Light Brown, LCW = Light Creamy White, LGY = Light Greenish Yellow, LY = Light Yellow, OF = Off-white, PG = Parrot Green, Scr = Scraped, V Bri = Very Brittle, WG = White Green, Y = Yellow, YB = Yellowish Brown

Discussion

For last seven years, RLABB has been studying diversity of actinomycetes in soil samples of Mount Everest Base Camp under CNR, Italy supported program. More than 600 Actinomycetes isolates were characterized and 40% of them are Streptomyces spp.(Singh and Agrawal 2003).

Sodium azide, a chemical mutagen, is being used in RLABB to generate various mutants of Actinomycetes isolates from soil samples collected from Khumbu, Mount Everest Base camp (Bhattarai et al. 2007; Tiwari et al. 2008). The preliminary work on sodium azide exposure to the Actinomycetes yielded some Gain-of-Function (GOF) mutations (Tiwari et al. 2008). The work was supported by International Foundation for Science (IFS) Grant – 2008. Hence, the Actinomycetes were required to be categorized into different groups before generating corresponding mutants. The Actinomycetes were categorized on the basis of their sensitivity to sodium azide.

Low concentrations (<100ppm) style="mso-bidi-font-style: normal">Escherichia coli (Bendahmane et al. 2002; Kleinhofs and Smith 1976; Oliver et al. 1990). Of the total isolates, two (2/38; 5.3%) were highly sensitive and could not grow on 10ppm and above concentration of sodium azide. Similarly, moderately sensitive and tolerant actinomycetes were identified as described in Table 1. The wild type actinomycetes were first classified according to their sensitivity to sodium azide and then were further used to generate their corresponding mutants.

Various LOF and GOF mutations were characterized for monosaccharides (5C and 6C) and a disaccharide sugar assimilation. GOF mutations with respect to Ara, Gal, Man and Suc assimilation were observed among mutants from Snp18a (two, Ara), Kal70e (two, Gal; one, Man), Nam29a (one, Ara) and Nam29b (two, Suc). Similarly, various LOF mutations were observed among mutants from Snp16d (two, Gal), Snp18a (four, Man), Jor11g (two, Gal), Kal70c (two, Ara and Gal each), Nam29a (four, Gal) and Nam29b (two, Ara) (Table 3). Interestingly, many biochemical mutants were recovered for monosaccharide assimilation (Ara: three GOF and four LOF; Gal: two GOF and 11 LOF; Man: one GOF and four LOF). Actinomycetes from Namche (Nam) were found to be hypermutated for carbon assimilation. Nam29a.10.i was found to be Ara assimilating but Gal negative, Nam29b.10.i was Suc assimilating but Ara and Gal negative, and Nam29b.10.ii was Suc assimilating but not Ara. Similarly, two mutants from Snp18a (Snp18a.10.ii and Snp18a.20.i) were Ara assimilating but not Man compared to their wild type. Only GOF mutations were observed for mutants from Kal70e while LOF mutations were observed for mutants from Kal70c, Jor11g and Snp16d wild types. Only two mutations were observed for Suc assimilation (as GOF in Nam29b.10.i and Nam29b.10.ii). All wild types and their mutants were able to assimilate Fructose (Fru) and Lactose (Lac).

The Actinomycetes biodiversity of Mount Everest base camp region can be studied in order to explore various GOF mutations; for example, antimicrobial activities may be developed in those wild Actinomycetes which donot posses the activity, antimicrobial activities may be enhanced in those wild Actinomycetes which posses low antimicrobial activities or are completely ineffective before sodium azide exposure. Other mutants can be obtained as spin-offs, which helps in understanding biochemical processes in Actinomycetes.

Acknowledgement

Authors are thankful to International Foundation for Science (IFS) for support this work.

Reference

Bendahmane A, Farnham G, Moffett P and Baulcombe DC (2002) Constitutive gain of function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the treatment locus of potato. Plant J 32: 195-204.

Bhattarai K, Tiwari KB and Agrawal VP (2007) Enhanced antibacterial activity of sodium azide treated mutant Streptomyces strain. J Nep Asso Med Lab Sci 8: 67-68.

Tiwari KB, Bhattarai K and Agrawal VP (2008) Gain-of-Function mutation in Streptomyces spp. induced by sodium azide treatment. Life Sci Mag 1(1). URL: http://www.lifesciencemag.edu.np/mutations.php

Kleinhofs A and Smith JA (1976) Effect of excision repair on azide-induced mutagenesis. Mutat Res 41: 233-240.

Oliver DB, Cabelli RJ, Dolan KM and Jarosik GP (1990) Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide sensitive component of the protein export machinery. Proc Natl Acad Sci USA 87: 8227-8231.

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

Williams ST, Sharpe ME and Holt JG (1989) Streptomyces and related genera. In Bergey’s Manual of Systematic Bacteriology. Vol. 4. Williams and Wilkins, USA.