This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteranspecific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.
Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of killing. In this study a truncated form of cry1Ab gene derived from Bacillus thuringinsis (Bt) subsp. aegypti isolate Bt7 was engineered into the genome of the baculovirus Autographa californica multiple nuclearpolyhedrosis wild type virus, in place of the polyhedrin gene by using homologous recombination in Spodoptera frugiperda (Sf) cells between a transfer vector carrying the Bt gene and the wild type virus linearized DNA. Recombinant wild type virus containing the cry1Ab gene was detected as blue occlusion-negative plaques in monolayers of Sf cells grown in the presence of X-Gal. In Sf cells infected with plaque-purified recombinant virus, the cry1Ab gene was expressed to yield a protein of approximately 82-kDa, as determined by immunoblot analysis. The toxicity of the recombinant virus expressing the insecticidal crystal protein (ICP) was compared to that of the wild-type virus. Infected-cell extract was toxic to cotton leaf worm Spodoptera littoralis second instar larvae and the estimated LC50 was 1.7 μg/ml for the recombinant virus compared with that of wild-type virus which was 10 μg/ml.
Significance and Impact of the Study: Insecticidal activity of Bacillus thuringiensis subsp. aegypti was determined, and its vegetative insecticidal protein was subjected to FPLC for protein purification. This work contributes to improve understanding the different toxins secreted during vegetative growth of Bt. Moreover, the N-terminal amino acid sequences of 88-kDa protein was only 92% identical to that of vip3A, and for 44 kDa was 92% identical with Cry35a, suggesting that we might have identified a new genes. Finally, we have proven these proteins to be novel insecticidal agents that may complement the use of known insecticidal proteins derived from Bacillus. Abstract Bacillus thuringiensis subsp. aegypti C18 is an Egyptian isolate, obtained from dead pink bollworm larvae. Insecticidal active proteins against different insect were purified from BtaC18 strain during vegetative states. Both the bacterial pellet and cell-free supernatant obtained during vegetative growth had insecticidal activity against black cutworm (BCW). Bioassays revealed that the pellet after 48 h of growth is more potent and toxic against BCW. The toxin in the pellet was active at very high temperatures but lost toxicity after boiling or autoclaving. Proteins extracted from the BtaC18 pellet were further purified by ammonium sulfate precipitation, and the 40% fraction was then subjected to fast protein liquid chromatography (FPLC). Seven major protein peaks were detected after FPLC (Pi-a, b, c, d, e, f and g). Pic protein fraction was active against BCW with an estimated LC 50 = 26 ng cm À2 , Pid protein killed 50% of European corn borer (ECB) at 46 ng cm À2 , and Pif showed insecticidal activity against western corn root worm (WCRW) with estimated LC 50 was 94 ng cm À2 . Based on the significant and high toxicity of Pic against BCW and Pif against WCRW, the 88-and 44-kDa proteins were further characterized by N-terminal amino acid sequencing.
The threat of heavy metals pollution to public health and wildlife has led to an increased interest in developing systems that can remove or neutralize its toxic effects in industrial effluents and municipal wastewater. Tolerance to a range of heavy metal ions was determined for bacteria which had been isolated from wastewater collected from Makkah city, Saudi Arabia. Isolates were tolerant to cupper, cadmium, zinc, and cobalt although the levels of tolerance to the different concentrations of metal ions were specific for each isolate. One isolate was able to tolerate all four metal ions tested; phenotypic and genotypic investigation revealed that isolate (S7) resembled similarities with Pseudomonas aeruginosa. The results of this study showed the potential applicability of the isolated heavy metal-tolerant strain Pseudomonas aeruginosa (S7) in the treatment of heavy metal containing solutions. Further studies on the genomic structure of isolate (S7) are required to investigate its capabilities to remove/reduce heavy metals in contaminated microcosms. Methods Isolation of bacteria Water samples were taken from industrial wastewater ponds found in Makkah City, Saudi Arabia. The samples were collected in sterile screw-capped bottles containing 0.1% sodium thiosulfate to prevent bacterial oxidation. The collected samples were preserved in an ice box and transported to laboratory for direct bacteriological examination. Plate dilution method was employed for bacterial isolation [9] using LB agar supplemented with 0.5 mM of Cu(NO 3) 2 , CdCl 2 , Zn(NO 3) 2 and Co(NO 3) 2 respectively. An aliquot of 50 µl of each dilution was spread on the surface of LB agar plates; three replicates of each dilution were prepared. Inoculated plates were incubated at 30-35ºC for 24-48 h. The O.D. readings of each culture were taken at 0, 24, 48 and 72 h. The results were recorded based on three trials for each experiment. Identification tests Random collection of different colonies with various morphological characteristics was selected from LB agar plates. All unknown colonies were subjected to microscopic, biochemical and molecular identification as follows: Microscopic examination: Gram staining technique was carried out as described by Reddy et al. [9]. Biochemical characterization: Biochemical testes included: Oxidase, Catalase tests, Fermentation of Sugars, Urease test, IMVIC Test, aesculine hydrolysis, gelatin hydrolysis were carried out according to Reddy et al. [9]. J o u rn al of M ic ro b ia l & Bioc h e m ic a l Te chno lo g y
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