Assessment of toxic potential of local Jordanian Bacillus thuringiensis strains on Drosophila melanogaster and Culex sp. (Diptera).

AIMS: To assess the toxic potential of different local Jordanian Bacillus thuringiensis isolates on larvae of Drosophila melanogaster and Culex sp. METHODS AND RESULTS: Scanning electron microscopy revealed the presence of spherical, bi-pyramidal, and bi-pyramidal and cuboidal parasporal bodies produced by the toxic isolates. Spherical inclusions dominated. The toxicity of the isolates to the two insects, determined using 24-well plates or vials, indicated that the 50% lethal concentration (LC50) of the bacterial suspension for D. melanogaster and Culex sp. larvae varied from 4.60 to 8.65, and from 5.30 to 6.74, respectively. CONCLUSION: Comparison of the LC50 values of isolate 82 with those of the reference strain B. t. israelensis showed that this isolate has a higher toxicity potential. SIGNIFICANCE AND IMPACT OF THE STUDY: Some local Jordanian B. thuringiensis isolates exhibit toxic potential that could be used to control some important pests, and could replace chemical pesticides.

Identification of geosmin as a volatile metabolite of Anabaena sp.

A cyanobacterial isolate recovered from Lake Ogletree, the principal drinking water supply for the city of Auburn, Alabama (USA) was identified as Anabaena sp. The isolate was shown to produce the most common off-flavor compound, geosmin.

Environmental and nutritional factors affecting geosmin synthesis by Anabaena sp.

A cyanobacterium isolated from a source-water reservoir during a spring odor and taste episode and identified as Anabaena sp. consistently produced geosmin during laboratory culture on modified BG-11 liquid medium. Maximal geosmin/biomass occurred at 20 degrees C and a light intensity of 17 microE/m2/s; geosmin/chla values directly correlated with increasing light intensity (r2 = 0.95, P < 0.01). It was concluded that at 20 degrees C, increasing light intensity favors less chla synthesis and higher geosmin synthesis; at 17 microE/m2/s, increasing temperature stimulates chla production (to 25 degrees C) while repressing geosmin synthesis (above 20 degrees C). Nutritional factors promoting biomass, chla, and geosmin synthesis by Anabaena sp. were also investigated. For cultures grown at 17 microE/m2/s and 20 degrees C for 20 days, both ammonium-N and nitrate-N generally enhanced the growth of Anabaena sp. Nitrate-N promoted more chla production (r2 = 0.99) than ammonium-N. Geosmin synthesis was directly correlated with ammonium-N concentrations (r2 = 0.89), with low nitrate-N (123.5 micrograms/l) favoring maximal geosmin production (2.8 micrograms/l). Increasing nitrate-N concentrations promoted a three-fold increase in chla content with geosmin synthesis decreased by two-fold. Geosmin/mg biomass was directly related to ammonium-N concentration; high nitrate-N levels suppressed geosmin production. No geosmin was detected at or below 118 micrograms phosphate-phosphorus/l. Geosmin, dry weight biomass, and chla production were correlated with increasing phosphorus (P) concentration (r2 = 0.76, 0.96 and 0.98, respectively). No geosmin was detected when copper was present in growth media at or above 6.92 micrograms Cu2+/l (CuSO4.5H2O). Dry weight biomass and chla production were negatively correlated with Cu2+ ion concentrations.

Diversity of Bacillus thuringiensis in different habitats of northern Jordan.

A survey of Bacillus thuringiensis was conducted for 17 locations in Northern Jordan representing 12 different habitats. Eighty isolates were identified as B. thuringiensis in the majority of the tested samples. Results showed that soils contaminated with the slaughterhouses waste materials had the highest content of spore-forming bacteria [(4.05-2.2) 10(7) CFU/g)] and B. thuringiensis [(4.05-7.9) 10(7) CFU/g)] with a (5.5%-14.9%) and (5.2%-7.7%) of the total viable bacterial count, respectively. These bacteria were more abundant in soils contaminated with such animal by-products.

Bacterial degradation of hydrocarbons as evidenced by respirometric analysis.

The microbial biodegradability of mineral oil and other hydrocarbons, namely hexane, decane and tetradecane was determined using the Warburg constant volume respirometer. Results of oxygen uptake indicated that hexane and tetradecane were more degradable than mineral oil and decane. Rhodococcus erythropolis and Erwinia cancerogena showed the highest (0.866) and lowest (0.115) oxygen quotient (Qo2) values, respectively, when exposed to mineral oil. Staphylococcus warneri and Enterobacter cloacae showed the highest (2.895) and (2.816) Qo2 values, respectively, when exposed to hexane; whereas E. cloacae and E. cancerogena showed the lowest Qo2 values (1.289 and 1.824), respectively. Both R. erythropolis and E. cloacae had the highest Qo2 values (2.859 and 2.289), respectively, when exposed to tetradecane. More oxygen was consumed by R. erythropolis than the other bacterial cultures when exposed to all hydrocarbons. In contrast, less oxygen was taken by E. cancerogena than the other bacterial cultures when exposed to all hydrocarbons, except for hexane.

Isolation, identification and analysis of antibacterial activity of soil streptomycetes isolates from north Jordan.

A total of 90 different Streptomyces isolates were recovered from 36 soil samples and assessed for their antibacterial activity. Nine isolates were identified by the absence of an aerial mycelium. The rest were grouped into six colour series, namely grey, white, yellow, green, red and polymorphic colours (pink, orange or violet) with total numbers of 29, 18, 14, 8, 3 and 9, respectively. The isolates (68%) showed a reverse side culture pigmentation, 30% produced melanin and 25% produced other soluble pigments. Isolates (48%) were characterized by flexuous spore chains, 21% with spiral and 10% for each of the rectus and retinaculum apertum arrangement. The antibiotic activity against a wide range of bacteria was exhibited by 54% of the isolates which were effective against Bacillus subtilis (57%), Staphylococcus aureus (47%), Escherichia coli (24%), Klebsiella spp (16%), and Shigella spp (12%). The lowest activity (8%) was exhibited against Pseudomonas spp and Salmonella spp. The antibacterial activity of the isolates was divided into four groups according to the diameter of the inhibition zone produced. Groups 3 and 4 with larger inhibition zones indicated their potential as a possible source of novel antibiotics.

Characterization and analysis of antibiotic activity of some aquatic actinomycetes.

Nine different isolates of aquatic actinomycetes identified as Streptomyces spp. were studied for their morphological and cultural characteristics. One of these isolates (C4-S, Streptomyces violaceusniger) was extensively studied for its inhibitory effect against a wide range of Gram-positive, Gram-negative bacteria, Mycobacterium vaccae ATCC 29678, Candida albicans and several food associated filamentous fungi and yeasts. Most of these were characterized by flexous sporophore morphology and their inability to produce cultural pigments. Bioassay results indicated that S. violaceusniger of 10 days culture age was highly active against Gram-positive cocci and bacilli with an inhibition zone of 16-25 mm, and slightly active against M. vaccae ATCC 29678 with an inhibition zone of 5-10 mm. The inhibitory effect was slight against Escherichia coli, Aspergillus niger 1 and C. albicans with an inhibition zone of 8-10 mm for each of them. There was no inhibitory effect of S. violaceusniger against other Gram-negative bacteria, filamentous fungi and yeast. The nature of the active molecule produced by S. violaceusniger showed a maximum absorption in the UV region at 210-260 nm.

Effect of glutathione L-cystein and L-djenkolic acid in the synthesis and mutagenicity of azide metabolite in Bacillus subtilis ATCC 6633 strain.

The Bacillus subtilis ATCC 6633 strain synthesizes a mutagenic metabolite from sodium azide and O-acetylserine. Mutagenicity of azide was decreased in growth media containing 10(-4) M glutathione, L-cysteine or L-djenkolic acid whereas dithiothritol (DTT) added at the same concentration did not reduce the mutagenicity of azide. Likewise, glutathione, L-cysteine, L-djenkolic acid, and DTT were found to have no effect in reducing the mutagenicity of the in vitro produced metabolite using bacterial cell-free extract. These results suggest that O-acetyl-serine sulfhydrylase catalyzes the reaction of azide and O-acetylserine to form a mutagenic metabolite, which is ninhydrin positive and migrates in TLC to an Rf value similar to that of azidoalanine in both acidic and basic solvent systems.

Genetic determinants of active antibiotic-producing soil streptomycetes.

Plasmids or covalently closed circular (CCC)-DNA molecules are abundant in the genus Streptomyces, and have been suggested to be involved in the genetic control of the production of many antibiotics in these organisms. In this study, 21 active antibiotic-producing Streptomyces isolates were screened for their plasmid content by an alkaline lysis method which revealed the presence of a small plasmid DNA in the positive control Streptomyces lividans ATCC 35287, containing pIJ702 plasmid (5.65 kb in size). However, no low molecular weight plasmids were observed in the tested antibiotic-producing Streptomyces strains suggesting that antibiotic production in these strains is likely chromosomally encoded DNA. Treatment of 2 Streptomyces strains with 10 mM ethidium bromide (EB) resulted in the failure to produce aerial mycelia and antibiotic activity.

Detection of genetic polymorphism by RAPD-PCR among isolates of Bacillus thuringiensis.

Sixteen isolates of Bacillus thuringiensis recovered from different Jordanian habitats were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the molecular level. Total genomic DNA from each isolate and three reference strains were amplified using 10-mer primers. Electrophoretic analysis of the amplification products revealed the incidence of polymorphism among the isolates. Pair-wise comparisons of polymorphic products were used to construct a dendrogram applying the cluster analysis. Fifteen of the isolates were all in one major cluster which was divided into six small groups. Such analysis showed some regional variation among the isolates, but did not indicate a clearly defined habitat locational pattern of the DNA polymorphism.

Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates.

Random amplified polymorphic DNA (RAPD) was used for identification and assessment of genetic diversity between isolates of Streptomyces from soil. Genomic DNA from 18 Streptomyces isolates and 2 reference strains were amplified using four different 10-mer primers. Different DNA fingerprinting patterns were obtained for all the isolates. Electrophoretic and cluster analysis of the amplification products revealed incidence of polymorphism among the isolates and none of them was identical to the reference strains although there were some common amplification bands. Two highly divergent groups were determined among the isolates. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.

Antibacterial activity of Orobanche cernua extract.

The effect of the parasitic plant Orobanche cernua extract was evaluated on 5 different Gram-positive and Gram-negative bacteria. Dilution experiments show that the minimum inhibitory concentration (MIC; microgram/ml) of the extract was as follows: Staphylococcus aureus-1526.5; Streptococcus sp.-3125; Streptococcus pyogenes-25,000 and both for Bacillus cereus and Escherichia coli-50,000 micrograms/ml. Data indicated that the MIC of 3125 micrograms/ml is equal to cefotaxime (30 micrograms) and tobromycin (10 micrograms) activity against Streptococcus sp. and S. aureus, respectively; 25,000 and 50,000 micrograms/ml is equal to cefotaxime (30 micrograms) activity against S. pyogenes and B. cereus, respectively, and 50,000 micrograms/ml is equal to nalidixic acid (30 micrograms) activity against E. coli. It can be concluded that the O. cernua extract displays remarkable activity against some Gram-positive bacteria such as Streptococcus and Staphylococcus.

Degradation of geosmin-like compounds by selected species of gram-positive bacteria.

Biodegradation of geosmin produced by Streptomyces halstedii (A-1 strain) was investigated using seven Gram-positive bacteria. Bacterial action on the A-1 extract was followed at 15, 45 and 120 min. Bacillus cereus ssp. thuringiensis HD-1, B. cereus 3711 and B. cereus ssp. mycoides 4379 showed no effect. However, Arthrobacter atrocyaneus, Arth. globiformis, Chlorophenolicus N-1053 and Rhodococcus maris caused a colour change from blue to yellow (positive reaction). These data suggest that geosmin can be degraded by specific species of Gram-positive bacteria. Biodegradation of geosmin, which causes bad odour in drinking water, can be used as a new technology in water treatment.