2010( hassan

B a k u , A z e r b a i j a n | 73

INTERNATIONAL JOURNAL Of ACADEMIC RESEARCH Vol. 2. No. 6. November, 2010, Part I

GENETIC MODIFICATION OF BACILLUS THURINGIENSIS VAR. KURSTAKI HD-73 TO OVERPRODUCE MELANIN, UV RESISTANCE

AND THEIR INSECTICIDAL POTENTIALITY AGAINST POTATO TUBER MOTH

Hassan Abdel-Latif A. Mohamed1*, Mohamed Ragaei Abd El-Fatah2, Magda M. Sabbour2,

Asmaa Z. El-Sharkawey 3, Rasha Samy El-sayed2

1Department of Microbial Genetics, 2Department of Plant Protection, National Research Center, 3Zoology Department, Faculty of Science, Al-Azhar University, Cairo (EGYPT)

* Corresponding author: [email protected] ABSTRACT Mutants of Bacillus thuringiensis var. kurstaki HD-73 (parent strain) producing a melanin were obtained after

treatment with the mutagenic agent ethyl-methane-sulfonate. Several microbiological, biochemical and molecular properties of the mutant isolates were analyzed through; estimation of spore yielding and crystal protein production; the sensitivity to the artificial sunlight (i.e. UV); insecticide activity against potato tuber moth (P. operculella) as well as a dark brown pigment production, extraction, isolation and characteristics also plasmid profile and analysis of the crystal protein by SDS-PAGE Gel Electrophoresis. Generally, the selected mutant (B.t.EMS-M3) showed significant efficiency than the other mutant isolates or their parent strain.

Key words: Mutation; EMS treatment; dark-brown pigment; Phthorimaea operculella; Photoprotection;

plasmid pattern; protein finger printing 1. INTRODUCTION Bacillus thuringiensis is a sporogenic gram-positive insect pathogen that is extensively used in biological

pest control [1]. It produces parasporal crystals composed of proteins called delta-endotoxins or Cry proteins, which are toxic to various insects on ingestion [2]. This toxin is highly diverse and displays a wide range of activity spectra useful in pest management and is particularly active against dipteran, coleopteran, and lepidopteran species [3,4]. The persistence of B. thuringiensis var. kurstaki and its potency as a biological control agent to reduce the losses caused by Phthorimaea operculella in potato stores where shady conditions existed, the persistence of P. operculella (Potato tuber moth) was longer compared to its short persistence in the field where UV radiation was the main factor which reduced persistence [5-8]. However, the use of B. thuringiensis as insecticide is limited in field applications because of the rapid inactivation of toxins and spores after exposure to sunlight [9-12]. Ultraviolet is mainly responsible for this inactivation because of its impact on cells by direct DNA damage (e.g. pyrimidine dimmers, cross-linking with proteins) or by producing reactive oxygen-derived free radicals [12-15]. So, the insecticidal activity of the B. thuringiensis formulation is less stable and less long-lasting than that of chemical pesticides in the field. It is therefore relatively more expensive because repeated spraying is necessary. Photoprotection of the mosquitocidal activity of B. thuringiensis var. israelensis, using melanin produced by Streptomyces lividans also found that melanin is an excellent photoprotective agent [16]. Many reports have described melanin-producing microorganisms such as; Bacillus [17], Pseudomonas [18], Azotobacter [19], Mycobacterium [20], Rhizobium [21], Vibrio [22], Micrococcus [23], Gluconobacter [24], and Azospirillum [25]. Several research groups have obtained B. thuringiensis mutants producing melanin from B. thuringiensis sub sp. kurstaki and other strains by inducing mutagenesis utilizing ethyl methane sulphonate (EMS) [26-28]. Compared with their parent strains, these B. thuringiensis mutants were more resistant to UV irradiation [29].

In this study, we report a mutant isolates of B. thuringiensis subsp. kurstaki "after treatment with EMS" producing melanin with highly insecticidal activities against potato tuber moth, P. operculella. The biochemical and molecular characterization of the mutant B. thuringiensis isolates were examined.

2. MATERILS AND METHODS Bacterial strain and growth conditions: Bacillus thuringiensis var. kurstaki HD-73 was tested in this study. Bacterial strain was obtained from the

insect rearing laboratory, Pests and Plant Protection Department, National Research Centre. The bacterial strain was cultured onto Luria Bertani agar media (LB-agar media) plates over night at 30ºC. To prepare one litre from this medium, Tryptone (10.0 g), Yeast extract (5.0 g), Sodium chloride (5.0 g) were dissolved in one litre distilled water and solidified by adding 20 g agar. A single colony was transferred, using a sterile loop, from agar media to 250 ml conical flask containing 100 ml Luria Bertani broth media (LB-media), the flasks were shaken at 200 rpm for 3 days at 30ºC. The composition of LB-medium is the same as LB-agar medium with the exception of agar which excluded from medium.

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Insect pest: Insects from laboratory colonies of P. operculella (potato tuber moth). They were obtained from the insect

rearing laboratory, Pests and Plant Protection Department, National Research Centre, Dokki, Giza, Egypt. A disease-free culture of the test insect was maintained under controlled conditions (26±2°C and 70±5% R.H.) and used for the experiments.

Mutation induction: Ethyl methane sulphonate (EMS) was used as a chemical mutagenic agent to mutagenized Bacillus

thuringiensis var. kurstaki HD-73 as described by [28] with slightly modifications. The vegetative cells of B. thuringiensis var. kurstaki HD-73 were grown in L-broth for 16 hours at 30 ºC. Ten ml aliquots of the produced culture were centrifuged at 3000 rpm for 10 minutes; the pellet was washed 3 times with saline (0.1 % NaCl) and then recentrifuged at 3000 rpm for 10 minutes. The pellet was resuspended in appropriate volume of saline. 600 μl of EMS and 2 ml of EMS buffer (0.1M of sodium dihydrogen phosphate pH. 6.9) were added to only one part of the suspension and shaken for 3 hours at 100 rpm, the suspension was centrifuged at 3000 rpm for 10 minutes at 30 ± 3ºC. The pellet was washed 3 times with EMS buffer and resuspended in 5 ml saline, while the other part of the suspension (untreated with EMS) were diluted serially and appropriate dilution was made to determine the optical density (O.D.) of the control and plated onto LB-agar medium over night at 30 ± 2ºC and then the colonies was counted by the counter. Part of the treated suspension was diluted serially and appropriate dilution was made to determine the optical density (O.D.) of the treatment and the arising colonies from the diluted EMS-treated suspension were counted. Appropriate dilutions of EMS-treated suspension were plated on BP-media (To prepare one litre from this medium, peptone (7.0g/L); glucose (10.0 g/L); KH2PO4 (6.7 g/L); MgSO4·7H2O (0.3 g/L); ZnSO4·7H2O (0.02 g/L); FeSO4·7H2O (0.02 g/L); MnSO4·7H2O (0.02 g/L); and CaCl2 (0.1 g/L); dissolved in 1000 ml distilled water. The pH of the medium was adjusted to 7.2 ± 0.1 using NaOH [27], for the melanin production this medium must contain yeast extract (10.0g/L); tyrosine (1.0g/L) and CuCl2 (0.017g/L) as illustrated by Liu et al. [16] over night at 30 ± 2ºC and dark colonies were isolated randomly.

Estimation of spore and crystal protein production: The dark coloured isolated mutants were grown in 50 ml of liquid T3-media (sporulation media) [30]. To

estimate the number of spores formed per millilitre of the culture medium, one ml of the culture broth was placed in a 1.5-ml tube then heated at 60°C for 20 minutes [29, 31], diluted and plated onto LB-agar media [27], then colonies were counted after 12 hours of growth at 30 ± 2ºC. The amount of crystal protein synthesized by each mutant isolate was quantified primarily by microscope examination to calculate crystal numbers.

The effect of exposure to the artificial sunlight (i.e. UV): The selected B. thuringiensis mutants and their parent strain were cultured in LB-media and shacked for 3

days at 200 rpm at 25 ± 2ºC. Biomass was collected using the Lactose–acetone co-precipitation method as described by Dulmage et al. [32]. The tested bacterial strains were irradiated by UV source (a set of two 275 W, high intensity, and mercury sun lamps). Spore-crystal preparations of B. thuringiensis samples were sprayed onto Petri dishes opened under the UV source at a distance of 47 cm from the source of UV and irradiated for 0, 4, 8, 12, 16, 20 and 24 hours.

Insecticide activity: The irradiated spores-crystal preparations of B. thuringiensis were bioassayed against potato tuber moth (P.

operculella) as described by Ridgway et al. [33]. Insect bioassay were performed with 1st instars larvae of P. operculella which fed on potato slices covered with 1.5 % agar containing B. thuringiensis samples at 500 µg/ml. Fifty larvae were tested for each treatment, ten in each replicate then, the larvae were placed in plastic cups and incubated at 30 ± 2ºC. Mortality was scored after 72 hours. The percentage of observed larval mortality reported and corrected according to Abbott's formula [34]: Corrected percent mortality = [(T-C) / (n-C)] х100

Where: (T =number of dead larvae in treated replicates. C = number of dead larvae in control replicates and n = the original number of the larvae used). The choice of the selected strain was based on its potential activity against the target insect

Pigment production, extraction and isolation: The growth of the tested isolates cells cultured in BP medium [16] and the pigment production were

determined by measuring the culture broth absorption in a spectrophotometer at the wavelength of 600 nm, centrifuged and the pellet precipitate discarded; absorptions of supernatants were then measured at 540 nm [35].

The pigment was extracted from the culture broth based on the method described by Chen et al. [35]. The protocol for isolation of melanin from the broth was illustrated in the Figure (1). At the end of incubation the cultures were adjusted at pH 8 and then filtered through Wattman filter paper, the melanin in the filtrate was precipitated by adjusting the pH to 3.0 with 5 N HCL, the melanin was collected by centrifugation at 3000 rpm for 10 minutes at 25 ºC, then washing 3 times with distilled water at pH 3, then the precipitate left for drying at room temperature in a dark place to prevent oxidation, so melanin powder was obtained. The melanin powder of each isolate was weighed to determine the amount of the produced melanin pigment from each isolate.

Pigment characteristics: To determine the light absorption spectra of the pigments, a 0.1 mg/ml pigment solution was made by

dissolving the pigment pellet in heated 4 N KOH to 60°C, and scanned for its absorbance from 200 to 700 nm wavelengths. Synthetic melanin from Sigma (USA) was used as a standard [35]. Some chemical tests were also carried out to identify this pigment [28]. One of the chemical tests was solubilitied; the criteria of Zussman et al. [36] were adopted to describe the solubility of pigment in the solvents. Pigments were designated "soluble" if they dissolved in the solvent, "slightly soluble" if the solvent became colored but the pigment did not dissolve, and "insoluble" if no color was imparted to the solvent [37].



Fig. 1. Scheme of isolation and purification procedure of melanin from fermented broth of the tested B. thuringiensis isolates

Plasmid analysis: The plasmid profile of the tested B. thuringiensis isolates were achieved after isolation by alkaline lyses of

cells [38] and analyzed the plasmids content and their relative sizes using a 1.0 % agarose gel by horizontal gel electrophoresis mini gel system (Molecular Bio. Products, inc., MBPTM) and then observing the pattern. Whereas UV-Transilluminator was used to detect DNA bands and the gel is photographed using a computerized system.

Purification and analysis of the crystal protein: The tested bacteria were grown in suspension following the methods of [39-41] with slightly modification.

Fifty milliliters of sterile LB media was inoculated into 250-ml flasks with one sterile loop of bacteria and shaken for 72 hours at 30°C (300 rpm). At the end of this incubation, the majority of the population was in the form of free spores and crystals (less than 5% vegetative cells). The suspension was centrifuged for 10 min at 10,000 rpm at 4°C; the pellet was washed twice in water and resuspended in 4 ml of water. This suspension was adjusted with water to give an absorbance of 15 at 600 nm. The supernatants of toxic samples were autoclaved (121°C for 10 minutes). The samples were resuspended into 1 ml of ice-cold 0.5 M NaCl. The suspension was centrifuged at 13,000 rpm for 5 minutes. The pellet was resuspended in 1% SDS, 0.01% mercaptoethanol, boiled for 10 minutes and recentrifuged at 13,000 rpm for 10 minutes. The supernatant was removed and analyzed by 12% SDS-PAGE. The electrophoresis was carried out using vertical slabs with dimensions 14 x 14 x 0.75 mm using E-C apparatus Corporation St. Petersburg, Florida – USA. The Buffers for SDS-PAGE were prepared according to Laemmli, [42]. Protein staining was achieved by binding with dye composed of 0.25 % Comassie Brilliant blue R250, 1 % trichloroacetic acid, 45.4 % methanol and 9.2 % glacial acetic acid to detection the protein bands. The bands appear after successive washing and rinsing with de-staining solution composed of 10 % acetic acid, 40 % methanol and 50 % distilled water with agitation. The blue protein bands appeared against a clear background of the gel after washing. Finally the separated proteins were photographed by using a Digital Camera Pattens of the unknown mutant isolate as compared with the respective patterns of the parent, and the gel documentation system image analysis gel works ID advanced software (Gel Biometral, Germany) was used for more accurate analysis. Then, comparison was made between the local strains via biochemical genetic analysis. The genetic discrimination among different B. thuringiensis isolates were based on the presence or absence of unique bands, so the polymorphic peptide bands among the isolates were scored as (+) for present and (-) for absent.



Statistical analysis: The obtained data were statistically computed using the software SPSS for Windows (release 9.0.0, Dec.

18, 1998, standard version, SPSS Inc.). All treatments in the previous experiments consisted of three or more replicates.

3. RESULTS Isolation of mutants of B. thuringiensis: The arising colonies after EMS treatment was decreased to 34.67 x1010cells ml−1 as compared to 35 x 1012

cells ml−1 in the parent strain B. thuringiensis var. kurstaki, HD-73, the survival colonies percentage after EMS-treatment was 0.99% as compared to 100% in parent strain. Twelve EMS-induced mutants (B.t.EMS-M1, B.t.EMS-M2, B.t.EMS-M3, B.t.EMS-M4, B.t.EMS-M5, B.t.EMS-M6, B.t.EMS-M7, B.t.EMS-M8, B.t.EMS-M9, B.t.EMS-M10, B.t.EMS-M11 and B.t.EMS-M12) were isolated maintained to further examinations. The mutant B.t.EMS-M3 was selected based on the production of dark brown pigment on BP media plates, the rate of spore and crystal production, the persistence ability against UV irradiation and on the highly insecticidal activity against P. operculella comparing with their parent strain.

Formation rate of vegetative cells, spores and protein crystals: EMS treatment resulted in the decrease of the formation rates of vegetative cells reached to 44.44% of the

mutant (B.t.EMS-M3) and other mutants showed decrease ranged from 11.11% (B.t.EMS-M7) to 66.67% (B.t.EMS-M9) as compared to parent strain. Spore formation rates of the twelve selected mutants showed variable decrease. Where, the mutants (B.t.EMS-M6) and (B.t.EMS-M12) showed higher decrease reached to 99.33 and 98.50%, respectively, other mutants showed lower decrease ranged from 4.17% for (B.t.EMS-M4) to 66.67% for (B.t.EMS-M9) while, the mutant (B.t.EMS-M3) showed 41.67% decrease as compared to parent strain. The crystal proteins formation rate of the tested mutants showed variable decrease ranged from 27.27% (B.t.EMS-M3) to 100% (B.t.EMS-M2 and B.t.EMS-M6) as compared to parent strain (Table 1 & Figure 2).

Table 1. Formation rate of vegetative cells, spores and protein crystals of some mutant isolates after incubation on sporulation media over night at 30ºC ±2ºC

Treatments Code No. of Vegetative

cells No. of Spores

No. of Protein Crystals

B. thuringiensis var. kurstaki HD-73

Parent strain 900a 600a 110a

EMS–mutants

B.t.EMS-M1 600d 400c 4cd B.t.EMS-M2 700c 500b 0.0d B.t.EMS-M3 500e 350cd 80b B.t.EMS-M4 550cd 375cd 4cd B.t.EMS-M5 650de 370cd 3cd B.t.EMS-M6 700c 4e 0.0d B.t.EMS-M7 800b 400c 3cd B.t.EMS-M8 500e 300d 2cd B.t.EMS-M9 300f 200 1cd B.t.EMS-M10 550cd 500b 9c B.t.EMS-M11 580cd 480bc 2cd B.t.EMS-M12 600d 9e 2cd

EMS= Ethyl Methane Sulphonate. #Means within columns followed by different letters are significantly different (p<0.05).

Fig. 2. Stained smears of B. thuringiensis (HD-73) grown in LB-medium for 3 days at 30ºC show clusters of separated spores, vegetative cells and protein crystals

The insecticidal activity of the some mutant isolates after exposure to the artificial sunlight (i.e. UV): The data given in Table (2) indicated that, before exposure to artificial UV irradiation, a significant

insecticidal activity was achieved with the most tested mutants and their parent against 1st instar larvae of P. operculella.



Before the exposure to artificial UV irradiation, B. thuringiensis (HD-73) which represent the parent strain gave 88% larval mortality of P. operculella that decreased to be 4.0 % after 24 hours of exposure and the persistence of B. thuringiensis was 4.225 hours of artificial UV irradiation. The mutant (B.t.EMS-M1) presented larval mortality before exposure to artificial UV reached to 92% and to 40% after 24 hours of exposure and the persistence was 18.186 hours of artificial UV irradiation. Other the tested mutant (B.t.EMS-M3) gave larval mortality ranged from 93% before to 46% after 24 hours of exposure and the persistence was 18.186 hours of artificial UV irradiation.

Table 2. % larval mortality of P. operculella after treated with

the tested yeast strains previously exposed to artificial UV irradiation and indicates hours as well as persistence of B. thuringiensis

Treatment Code Mean % Corrected larval mortality of P. operculella

after exposing to artificial UV irradiation for Persistence (hours)

0 hr 4hr 8hr 12hr 16hr 20hr 24hr

B. thuringiensis var. kurstaki

HD-73

Parent strain 88 ±5.20 46 ±5.20 33 ±6.93 28 ±5.20 16 ±6.93 9 ±6.35 4 ±0.58 4.225 hrs. ≈ 1.6 days

The selected EMS-mutants

B.t.EMS-M1 92±6.35 85±4.04 73±7.51 59±6.93 52±7.51 47±6.93 40±5.77 18.186 hrs. ≈ 6.8 days

B.t.EMS-M3 93 ±4.62 85 ±6.93 76 ±8.08 63 ±8.08 59 ±8.08 50 ±5.77 46 ±5.20 22.810 hrs. ≈ 8.6 days

EMS= Ethyl Methane Sulphonate * An exposure time of 4 hours in the solar simulator is equivalent to ≈ 1.5 days in the field (McGuire et al., 1996).

Pigment production, extraction and isolation: Mutant isolates had the ability to produce a dark brown pigment that obtained in Bp media containing L-

tyrosine after 4-day-old culture, only weak production of pigment was detected in the parent strain while B.t.EMS-M3 mutant isolate gave the darkest brown color among isolates. (Figure 3- A and B).

(A) (B)

Fig. 3. The parent strain B. thuringiensis var. kurstaki (HD-73)

(on the right side) and the EMS-mutant isolate B.t.EMS-M3 (on the left side) producing pigment in liquid Bp medium (A) and on plated BP medium (B),

after incubation for 4 days at 28ºC The growth rate of tested B. thuringiensis isolates was detected by measuring the light absorption of their

culture broth (BP media at pH 8) at the wavelength of 600nm, results was shown in Figure (4) which indicates that the maximum growth rate was obtained with the parent strain. The pigment production rate of tested B. thuringiensis isolates was detected by measuring the light absorption of their culture supernatant at the wavelength of 540 nm, results was shown in Figure (4) which indicates that the maximum pigment production rate was obtained with the mutant isolate B.t.EMS-M3. After the extraction and isolation of the pigment by acidic precipitation, the amount of the produced pigment from each isolate was weigh and shown in Figure (4). The maximal production of the pigment weight by B.t.EMS-M3 mutant isolate was 0.41 mg/ml of the fermented culture.

Pigment characteristics: The pigment produced by the parent strain B. thuringiensis var. kurstaki (HD-73) and their selected mutant

isolates were not easily soluble in water and insoluble in organic solvents such as ethanol, ether and chloroform. But there were easily soluble in alkaline solvent, especially in heated ones. A dark brown precipitate was produced when FeCl3 was added to the pigment solution. Thus this pigment was expected to be melanin pigment.



Fig. 4. The cell growth rate as O.D. in the culture broth at 600 nm; the pigment production rate in the supernatant of the culture broth at 540 nm and the weight of pigment powder extracted from the tested B. t. isolates after grown

in 100 ml of the fermented cultures (BP media) after incubation at 28ºC on a shaker at 200 rpm for 4 days. *The cell growth rate and the pigment production rate of the tested

B. t. isolates in BP media pH 8, O.D. = Optical Density. ** The tested Parent strain (B. t.), and the selected mutant isolate (B.t.EMS-M3).****Pigment powder (in mg). *** The tested B.t. isolates are parent strain B. thuringiensis var. kurstaki (HD-73), and their selected mutant

isolates (B.t.EMS-M1) and (B.t.EMS-M3). Absorbance spectra of the pigment extracted from B. thuringiensis isolates: The UV and visible absorption spectra of the pigment was obtained by spectrophotometer (Beckman

DU640, Beckman, Coulter, Inc., Fullerton, CA, U.S.A.) scanning at 200-700 nm, Table (3) and Figures (5-A, B, C, D). Synthetic melanin (Sigma Co.) used as standard melanin; its absorbance to UV at wavelength range of 200-700 nm was measured. The absorbance values of the standard melanin were 2.055 and 0.134 at wavelengths of 226.5 and 602 nm, respectively. This means that the maximum absorbance at wavelength of 226.5 nm. Table (3) indicate that, the pigments extracted from the parent strain B. thuringiensis var. kurstaki (HD-73) and the selected mutant isolates (B.t.EMS-M1) and (B.t.EMS-M3) showed a variation with respect to their absorbance values for different wavelengths, but all extracted pigments absorbed wavelengths between 226.5 and 230.5 nm, which represent the absorbance value of standard melanin. The absorbance values of the pigment extracted from the parent strain B. thuringiensis var. kurstaki (HD-73) and their selected mutants (B.t.EMS-M1) and (B.t.EMS-M3) were 1.976, 1.072 and 2.615 at wavelengths of 227, 293 and 227.5 nm, respectively. The only extracted pigment which gave the maximum value of absorbance (2.615) was extracted from the mutant isolate B.t.EMS-M3 at wavelength of 227.5 nm that was similar to standard melanin which gave absorbance value about 2.055 at wavelength of 226.5 nm, Table (3) and Figures (5-A, B, C, D).

(A) (B) (C) (D)

Fig. 5. The absorption rate of melanin extracted from isolate to UV-light at 0.01 % concentration using spectrophotometer at the wavelength range of 200-700 nm. (A) synthetic melanin (Sigma comp.), (B) Extraction from the parent strain B. thuringiensis var. kurstaki (HD-73, (C) Extraction from the mutant isolate (B.t.EMS-M1),

(D) Extraction from the mutant isolate (B.t.EMS-M3)

0.14

0.018 0.015

0.5

0.41

0.7

36.88

41.241.48

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Parent strain B.t.EMS-M1 B.t.EMS-M3

strains

O.D

. at 6

00 n

m &

O.D

. at 5

40 n

m

34

35

36

37

38

39

40

41

42

Pigm

ent w

ight

(mg)

O.D. at 600 nm O.D. at 540 nm Pigment weight (mg)



Table 3. The absorbance values of the pigment that extracted from the tested B. thuringiensis isolates to UV-light at 0.01% concentration using spectrophotometer

at the wavelength range of 200-700 nm

Tested pigment Peak picks Wavelength (nm) Corresponding absorbance values

Synthetic melanin 602, 226.5 0.134, 2.055

Parent strain 605.5, 243.5, 227 0.026, 1.490, 1.976 B.t.EMS-M1 602.5, 293, 248.5, 200.5 0.040, 1.072, 2.828, 0.921 B.t.EMS-M3 602, 249.50, 227.5 0.045, 2.275, 2.615

*The tested parent strain B. thuringiensis var. kurstaki (HD-73) and the selected mutant isolates. Plasmid panding patterns of some B. thuringiensis isolates: Figure (6) illustrates that the plasmid content of the control B. thuringiensis var. kurstaki (HD-73) strain

harboring 6 plasmids lanes (1&5) and also, 1 and 4 plasmids lane (3) and lane (4) of its selected mutants (B.t.EMS-M1) and (B.t.EMS-M3), respectively. On the other hand, the plasmid of mutant isolate (B.t.EMS-M1) was cured by EMS, whereas lost two plasmids of its indigenous plasmids and contain only one plasmid lane (8). These results indicate that the exposing to either UV light or EMS success in cured some of plasmids in the parent strain and concluded that either UV light or EMS have some mechanisms in plasmid curing. This result represents that the mutagen agents may play a role in plasmid replication (Figure 6).

Fig. 6. Plasmid profile of the tested B. thuringiensis isolates. Lanes 1&5, DNA marker, DNA ladder ranged from 1000 to 100 bp.; lane 2, parent strain B. thuringiensis var. kurstaki (HD-73);

lane 3, the selected mutant (B.t.EMS-M1) and lane 4, the selected mutant (B.t.EMS-M3 Crystal protein pattern of some B. thuringiensis isolates: In this study, the parent strain B. thuringiensis var. kurstaki (HD-73) strain and their mutant isolates

(B.t.EMS-M1 and B.t.EMS-M3) were compared depending on their crystal protein patterns. After the extraction of the crystal proteins from each tested strain, the analysis of them was performed using 12% SDS polyacrylamid gel. The electrophoretic patterns of the tested B.t. isolates were illustrated in Figure (7), which reveals bands with different molecular weights. The polymorphic bands at molecular weights; 168, 145, 40, 35 and 27 KDa were lost from the mutants B.t.EMS-M1 and B.t.EMS-M3.

Crystal protein profile shown in Table (4) was generated depending on the analysis of SDS-PAGE crystal protein banding pattern data of the parent strain and their mutant isolates. The mutant isolates (B.t.EMS-M1 and B.t.EMS-M3) and their parent strain B. thuringiensis var. kurstaki (HD-73) crystal proteins were compared.

Fig. 7. SDS- PAGE -profile of water soluble crystal proteins of the tested B. thuringiensis isolates. The numbers at the right indicate wide molecular weight marker proteins (in kilo-Daltons) (Pharmacia). Lane 1, mutant (B.t.EMS-M3); Lane2, mutant

(B.t.EMS-M1); Lane 3, parent strain and Lane 4, Protein markers



Table 4. Densitometric analysis of water soluble protein bands (SDS-PAGE) for the tested B. thuringiensis isolates, representing band number and molecular weight (MW), where (+)

means presence and (-) means absence of band

Band No. Protein band (kDa)

the tested B. thuringiensis isolates B.t.WT-EMS-M3 B.t.WT-EMS-M1 Control (B.t. WT)

1 168 - - + 2 145 - - + 3 137 + - - 4 136 - - + 5 127 - + - 6 118 + - - 7 67 - + - 8 66 + - - 9 59 - + - 10 58 + - - 11 47 - + - 12 45 - - + 13 43 - + - 14 40 - - + 15 39 + - - 16 35 - - + 17 34 - + - 18 33 + - - 19 27 - - + 20 25 - + - 21 24 + - - 22 23 - + - 23 20 + - - 24 19 - + - 25 17 + + - 26 15 + - - 27 14 - + -

Data were obtained by integrating the peaks from scanning densitometry of Comassie-blue stained gels by using a gel documentation system image analysis gel works ID advanced software (Gel Biometral, Germany). Heterogeneous high molecular-size material frequently appears as a smear above the 127 and 137 kDa band and may accumulate at the stacking gel-separating gel interface.

4. DISCUSSION The effectiveness of pest biological control could be increased by the production of genetically improved

microorganisms such as B. thuringiensis [43]. Effective performance requires either improvement of the environment to favour the biocontrol agent or genetic improvement of the agent. Genetic improvement of any organism is referring to development of one or more of its desired characters through genetic techniques such as mutation. Mutation is an inheritable change in the base sequence of the DNA of an organism, or a permanent change of the genetic material, either in a single gene or in the numbers or structures of the chromosomes through an operation called mutagenesis [44].

Melanin can protect microorganisms from UV because it can eliminate free radicals by acting as a free trap. Melanin is a natural product, easily biodadable in nature, so it will not pollute the surroundings, making it a perfect photoprotective agent [35]. In this study, a method to obtain a pigment-producing mutant of B. thuringiensis var. kurstaki (HD-73) was described through utilize Ethyl Methane Sulphonate (EMS) as a chemical mutagen [28]. Two B. thuringiensis mutant isolates were selected depending on their persistence ability against UV-irradiation and the highly insecticidal activity against 1st instar of P. operculella larvae to characterize the dark-brown pigment. The higher toxicity of the mutant isolates against the previously mentioned different pests more than its parent may explain the production of melanin which is reported to be an excellent UV protective agent [16]. The pigment was extracted from the culture broth of the selected mutant isolates gave the darkest brown color in comparing to the control strain [16, 35]. Produced melanin was variable increase than that of control strain; this variation may be due to the variety of the used microorganism [15]. The resistance of the obtained mutant isolates to UV could be due to the presence of melanin in the spore. Whereas, enhanced UV stability of insecticidal activity of these mutant isolates, as compared to the parent strain when exposed to UV irradiation, could be either due to adsorption of melanin in the crystal proteins or incorporation of melanin in the crystal at the time of synthesis coat [27]. Practically all B. thuringiensis strains harbor a set of self-replicating, extra-chromosomal DNA molecules or plasmids, which vary in number and size in the different strains [45, 46]. Every B. thuringiensis strain can have a variable number of plasmids responsible for the synthesis of different endotoxins; plasmids can bear several, usually identical, toxin genes [47-49]. Hence, plasmid behavior is of considerable significance for insecticidal activity [50, 51]. The SDS-



PAGE patterns of the reference strains showed different polymorphic bands with different molecular weights. The mutant isolates have bands more than their parent strain; this may be due to the genetic alterations that happened to the mutant isolates due to the mutation by EMS [52]. Fundamentally the tested B. thuringiensis strains; the wild type B. t. var. kurstaki (HD-73) strain which represent the control, its two mutant isolates (B.t.WT-EMS-M1 and B.t.WT-EMS-M3), showed observable differences in the crystal protein banding patterns and their the equal bands volume, based on their genetic back ground and / or the effect of the mutagenic treatment by EMS on the plasmids numbers and / or genes content that finally affect on the gene expression whereas expressed in the equal bands volumes. The results showed lost of some bands in the selected mutants than the parent strain may allow changing the composition of the cell envelop Performs to increase melanin secretion. Parton, [53] concluded that the progressive loss of lipopolysaccharide components which occurs from the S chemotype through various degrees of roughness to Re is accompanied by a change in the envelope protein composition, apparently between Rb and Rc.

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