Rapid Assessment of Water Toxicity by Plasmonic Nanomechanical Sensing.

The ability to rapidly and accurately detect water toxicity is crucial for monitoring water quality and assessing toxic risk, but such detection remains a great challenge. Here, we present a plasmonic nanomechanical sensing (PNMS) system for the rapid assessment of water toxicity. This technique is based on the plasmonic sensing of the nanomechanical movement of single bacterial cells, which could be inhibited upon exposure to potential toxicants. By correlating the amplitude of nanomechanical movement with bacterial activity, we detected a variety of toxic substances in water. The direct readout of bacterial activity via PNMS allowed for a high sensitivity to toxicants in water, thereby enabling us to evaluate the acute toxicological effect of chemical compounds rapidly. The PNMS method is promising for online alerts of water quality safety and for assessing chemical hazards. We anticipate that PNMS is also suitable for a wide range of other applications, including bacterial detection and high-throughput screening of antibacterial materials.

Microencapsulation of an indigenous isolate of Bacillus thuringiensis by spray drying.

In this study, microencapsulation by spray drying was performed to protect spores and crystals of an indigenous isolate of Bacillus thuringiensis Se13 from environmental stress. The effects of wall material, inlet temperature, and outlet temperature on microencapsulation of Bt-Se13 were investigated using Taguchi's orthogonal array. The most suitable wall material determined as maltodextrin DE10. The optimum inlet and outlet temperatures of spray drier were determined as 160 °C and 70 °C, respectively. The number of viable spores, mean particle size, wetting time, percentage of suspensibility and moisture content of the product produced under optimum conditions were determined as 8.1 × 1011 cfu g-1, 13.462 µm, 25.22 s, 77.66% and 7.29%, respectively. As a result of efficiency studies on Spodoptera exigua in the laboratory conditions, the LC50 was determined as 1.6 × 104 cfu mL-1. Microencapsulated Bt-Se13 based bio-pesticide may be registered for the control of S. exigua and can be tested against other lepidopterans which share the same environment.

Specific Cytotoxic Effects of Parasporal Crystal Proteins Isolated from Native Saudi Arabian Bacillus thuringiensis Strains against Cervical Cancer Cells.

Currently, global efforts are being intensified towards the discovery of local Bacillus thuringiensis (Bt) isolates with unique anticancer properties. Parasporins (PS) are a group of Bt non-insecticidal crystal proteins with potential and specific in vitro anticancer activity. However, despite the significant therapeutic potential of PS-producing Bt strains, our current knowledge on the effects of these proteins is limited. Hence, the main objective of this study was to screen Bt-derived parasporal toxins for cytotoxic activities against colon (HT-29) and cervical (HeLa) cancerous cell lines. Nine non-larvicidal and non-hemolytic Bt strains, native to Saudi Arabia, were employed for the isolation of their parasporal toxins. 16S rDNA sequencing revealed a 99.5% similarity with a reference Bt strain. While PCR screening results indicated the absence of selected Cry (Cry4A, Cry4B, Cry10 and Cry11), Cyt (Cyt1 and Cyt2) and PS (PS2, PS3 and PS4) genes, it concluded presence of the PS1 gene. SDS-PAGE analysis revealed that proteolytically-cleavaged PS protein profiles exhibit patterns resembling those observed with PS1Aa1, with major bands at 56 kDa and 17 kDa (Bt7), and 41 kDa and 16 kDa (Bt5). Solubilized and trypsinized PS proteins from all Bt strains exhibited a marked and dose-dependent cytotoxicity against HeLa cancerous cells but not against HT-29 cells. IC50 values ranged from 3.2 (Bt1) to 14.2 (Bt6) with an average of 6.8 µg/mL. The observed cytotoxicity of PS proteins against HeLa cells was specific as it was not evident against normal uterus smooth muscle cells. RT-qPCR analysis revealed the overexpression of caspase 3 and caspase 9 by 3.7, and 4.2 folds, respectively, indicative of the engagement of intrinsic pathway of apoptosis. To the best of our knowledge, this is the first report exploring and exploiting the versatile repertoire of Saudi Arabian environmental niches for the isolation of native and possibly novel Saudi Bt strains with unique and specific anticancer activity. In conclusion, native Saudi Bt-derived PS proteins might have a potential to join the arsenal of natural anticancer drugs.

An acridine orange spore germination fluorescence microscopy versus spectral paradox.

The metachromatic fluorophore acridine orange (AO) has demonstrated green fluorescent staining of dormant Bacillus spores and orange to red staining of transcriptionally active vegetative cells when used in the mid-micoMolar range. Despite the microscopic observation of numerous bright orange to red fluorescent vegetative cells following germination induction, no clear spectral emission peaks > 590 nm have ever been reported for spectrofluorometric analysis involving AO in conjunction with spore germination. This microscopy versus spectrofluorometry paradox is documented in the present report and hypotheses are put forth to explain the very weak spectral changes in the red region which do not appear to correlate with the abundant orange-red fluorescence of nascent vegetative cells seen through the fluorescence microscope.

Reactance and resistance: main properties to follow the cell differentiation process in Bacillus thuringiensis by dielectric spectroscopy in real time.

During growth, Bacillus thuringiensis presents three phases: exponential phase (EP), transition state (TS), and sporulation phase (SP). In order to form a dormant spore and to synthesize delta-endotoxins during SP, bacteria must undergo a cellular differentiation process initiated during the TS. Dielectric spectroscopy is a technique that can be utilized for continuous and in situ monitoring of the cellular state. In order to study on-line cell behavior in B. thuringiensis cultures, we conducted a number of batch cultures under different conditions, by scanning 200 frequencies from 42 Hz to 5 MHz and applying fixed current and voltage of 20 mA and 5 V DC, respectively. The resulting signals included Impedance (Z), Angle phase (Deg), Voltage (V), Current (I), Conductance (G), Reactance (X), and Resistance (R). Individual raw data relating to observed dielectric property profiles were correlated with the different growth phases established using data from cellular growth, cry1Ac gene expression, and free spores obtained with conventional techniques and fermentation parameters. Based on these correlations, frequencies of 0.1, 0.5, and 1.225 MHz were selected for the purpose of measuring dielectric properties in independent batch cultures, at a fixed frequency. X and R manifest more propitious behavior in relation to EP, TS, SP, and spore release, due to particular changes in their signals. Interestingly, these profiles underwent pronounced changes during EP and TS that were not noticed when using conventional methods, but were indicative of the beginning of the B. thuringiensis cell differentiation process.

The Use of Polystyrene Beads to Prepare Arrayed Samples of Bacillus thuringiensis for Microscopic Examination.

A common activity in the global search for useful Cry toxins is the microscopic screening of bacterial colonies for the presence of Bacillus thuringiensis. High-throughput screens require that aliquots from large numbers of colonies be arrayed on a microscopic slide. However, precisely placing a small amount of bacteria on a slide, and at a density that is useful for microscopic examination, is both difficult to achieve and time consuming. Herein we share a simple technique that utilizes a hooked wand and small polystyrene beads to quickly collect, and uniformly apply, aliquots of bacterial colonies onto gridded microscope slides in a manner optimal for viewing. If desired, libraries of examined bacteria can simultaneously be generated by discharging the beads into indexed multiwell plates. This simple and inexpensive method is robust, suitable for both light and phase contrast microscopy, and has been also used successfully to screen randomly mutated bacteria for phenotypic changes.

Activity of the Bacillus thuringiensis NprR-NprX cell-cell communication system is co-ordinated to the physiological stage through a complex transcriptional regulation.

NprR is a quorum sensor of the RNPP family found in bacteria of the Bacillus cereus group. In association with its cognate peptide NprX, NprR controls the expression of genes essential for survival and sporulation of Bacillus thuringiensis during its necrotrophic development in insects. Here, we report that the nprR-nprX genes are not autoregulated and are co-transcribed from a σ(A) -dependent promoter (PA ) located upstream from nprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY represses nprR-nprX transcription during the exponential growth phase and the quorum sensor PlcR activates nprR-nprX transcription at the onset of stationary phase. We show that nprX is also transcribed independently of nprR from two promoters, PH and PE , dependent on the sporulation-specific sigma factors, σ(H) and σ(E) respectively. Both promoters ensure nprX transcription during late stationary phase while transcription from PA has decreased. These results show that the activity of the NprR-NprX quorum sensing system is tightly co-ordinated to the physiological stage throughout the developmental process of the Bacillus.

Characterization of one novel cry8 gene from Bacillus thuringiensis strain Q52-7.

Bacillus thuringiensis (Bt) is the most widely used insecticidal microbe due to its specific toxicity and safe use with respect to animals and the environment. In this study, we isolated Bt strain Q52-7 from a soil sample collected in the Qian Shan District, Liao Ning Province, China. We observed that the Q52-7 strain produced spherical crystals. The Bt Q52-7 strain had high toxicity against Asian Cockchafer (Holotrichia parallela), exhibiting an LC50 of 3.80 × 10(9) cfu/g, but is not toxic for Anomala corpulenta Motschulsky and Holotrichia oblita. Using general cry8 primers, we amplified a 1.3 kb fragment with the polymerase chain reaction. Specific primers were designed for the amplified fragment to clone the full-length coding region. A novel gene, cry8Na1, had 69 % sequence similarity with cry8Ca1. cry8Na1 gene was successfully expressed in the HD-73(-) acrystalliferous mutant of Bt subsp. Kurstaki HD-73. Bioassays demonstrated that the Cry8Na1 protein is highly toxic for the H. parallela, with a 50 % lethal concentration of 8.18 × 10(10) colony forming units per gram.

Does changing the predicted dynamics of a phospholipase C alter activity and membrane binding?

The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro(245) and Pro(254), are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro(245) disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro(245) variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro(254) appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding.

Molecular characterization and genetic diversity of insecticidal crystal protein genes in native Bacillus thuringiensis isolates.

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.

Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India.

Bacillus thuringiensis (Bt) strains were isolated from 94 samples from different geographical regions. Novel types of crystalline inclusion bodies were observed from some of the isolates. Crystalline inclusions of bipyramidal, spherical and cuboidal morphology were found produced by most of the isolates. Isolate GS12 showed crystal on one side of spore while isolate GM108 formed crystals on both termini of spore. Isolate GN31 produced large sized bipyramidal crystals. SDS-PAGE analysis of the spore crystal suspension showed major protein bands in the range of 29 and 140 kDa. Two new serovars of Bt viz. GS4 and GN24 having H3abce and H3ab serotype respectively were isolated. Toxicity comparable to the reference strain Bacillus thuringiensis subs. kurstaki (Btk) HD-1 was observed for the isolates GM20, GM17 and MP3 against larvae of Helicoverpa armigera. Some of the isolates harboring cry genes like cry1Ac and cry2 did not show any toxicity towards H. armigera while most of the isolates were harboring cry1, cry1Ac and cry2 gene.

Germinant generation from δ-endotoxin of Bacillus thuringiensis strain 1.1.

The novel finding of this study is that the δ-endotoxin present in the spore coat of Bacillus thuringiensis strain 1.1 (Bt1.1), plays a central role in spore germination by generation of germinant via its ß-glucosidase activity and is based on the following: (i) the crystals of Bt1.1 consist of the 140 kDa δ-endotoxin which exhibits ß-glucosidase enzymatic activity. Besides crystals, δ-endotoxin is also located in the spore coat and at this site displays ß-glucosidase activity, resulting in glucose production; (ii) glucose is an efficient germinant of both Bt1.1 and acrystalliferous Bt4.1 strain; (iii) substrates of ß-glucosidase can activate the germination of Bt1.1 spores, but not those of the acrystalliferous Bt4.1 sister strain that do not contain the 140 kDa δ-endotoxin; (iv) Reduction or enhancement of enzymatic activity of δ-endotoxin, results in retardation or acceleration of germination and outgrowth, respectively. Bt1.1 cells secrete a 60 kDa polypeptide which displays ß-glucosidase activity as indicated by zymogram analysis and which is immunologically related to the 140 kDa δ-endotoxin.

Optimization of spray drying process for Bacillus thuringiensis fermented wastewater and wastewater sludge.

Response surface methodology was used to optimize spray drying process for producing biopesticide powders of Bacillus thuringiensis by using fermented broth of starch industry wastewater and wastewater sludge. Analysis of variance was carried out using number of viable spores in the powder as dependent variable. The determination coefficients of models were 92 and 94% for fermented broth of starch industry wastewater and wastewater sludge, respectively. Under the optimal conditions of the operational parameters of spray drying, the numbers of viable spores were 2.2 × 10(8) and 1.3 × 10(8) CFU/mg in the dry powders for starch industry wastewater and wastewater sludge respectively, with a loss of viable spores of 18 and 13% when compared with their respective fermented broths. The entomotoxicity (measured by the bioassay method) of the powders obtained under optimal conditions showed a loss of 28 and 18% when compared with the fermented broth of starch industry wastewater and wastewater sludge, respectively. The optimized results of spray drying were used for field application calculations. The volume of fermented broth required to produce powder formulated product when compared with the volume required for liquid formulation product in order to treat 1 ha of balsam fir was less and offered several advantages.

Characterization of ISBth4, a functional new IS231 variant from Bacillus thuringiensis MEX312.

A new insertion variant of IS231 family, designated ISBth4, was identified from Bacillus thuringiensis MEX312. ISBth4 is 2046bp in length and is delimited by two 17bp inverted repeats (IR) with one mismatch, flanked by two perfect 11bp direct repeats (DR). ISBth4 contains two open reading frames (ORFs), ORF1 and ORF2, which encode 72 and 473 amino acids, respectively. Multiple sequence alignments revealed that the potential transposase of ISBth4 contained five conserved domains N1, N2, N3, C1 and C2 that are similar to other IS231 elements; and the typical catalytic triad D(N2)-70-D(N3)-150-E(C1) and Y(2)R(3)E(6)K motifs as hallmarks of IS4 elements. Comparison of the amino acids of the potential ISBth4 transposase with those from other publicly available B. cereus group IS231 elements revealed a close similarity with ISBce7 (94% identity), ISBce5 (90%), IS231Y (89%) and ISBce8 (86%), and lower similarity to IS231N (49%), IS231M (48%) and ISBce12 (40%). Phylogenetic analysis of the evolutionary relationships between ISBth4 and the other IS231 elements showed that ISBth4 is more closely related to the IS231 sequences isolated from B. cereus strains than those from B. thuringiensis strains. In vivo transposition activity of ISBth4 was discovered in a mutant B18 from a MEX312 background, indicating that it is a functional insertion sequence in its B. thuringiensis natural host.

Diversity of Bacillus thuringiensis strains isolated from citrus orchards in spain and evaluation of their insecticidal activity against Ceratitis capitata.

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDSPAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

High-energy ball milling treatment of soybean for Bacillus thuringiensis culture media.

Soybean meal has been intensively used as a substrate in culture media for several microorganisms. However, the fermentable sugar containing the soybean needs to be released from the solid matrix through different processes. Against this backdrop, the present study explores the use of high-energy ball milling as a one-step treatment method for expedited production of fermentable sugars of textured soybean. The best result is observed after only 5 min of milling, obtaining 34.1 times more fermentable sugars than untreated textured soybean, and 2.5 times more than commercially used soybean meal. Notably, the textured soybean ball-milled has been used as a substrate for Bacillus thuringiensis var. kurstaki HD-73 fermentation. The cell and spore production is also compared with a standard Rowe media. The maximum cell concentration obtained in the entire fermentation process using ball-milled textured soybean media is found to be higher than the concentration obtained using the standard Rowe media. In addition, it is observed that there is a direct correlation between maximum cell production and reducing sugar concentration generated by the high-energy ball milling treatment. No fermentation inhibitors or by-products are generated during the physical treatment.

Negative cross-resistance between structurally different Bacillus thuringiensis toxins may favor resistance management of soybean looper in transgenic Bt cultivars.

High adoption rates of single-gene Bacillus thuringiensis (Bt) Cry1Ac soybean impose selection pressure for resistance in the soybean looper, Chrysodeixis includens, a major defoliator in soybean and cotton crops. To anticipate and characterize resistance profiles that can evolve, soybean looper larvae collected from field crops in Brazil in 2013 were selected for resistance to Cry1Ac. Using two methods of selection viz., chronic exposure to Cry1Ac cotton leaves and the seven-day larval exposure to purified Cry1Ac on the artificial diet, 31 and 127-fold resistance was obtained in 11 and 6 generations of selection, respectively. The resistance trait had realized heritability of 0.66 and 0.72, respectively, indicating that most of the phenotypic variation in Cry1Ac susceptibility of the soybean looper larvae was due to additive genetic variation. The Cry1Ac-selected populations showed positive cross-resistance to Cry1Ab (6.7-8.7 fold), likely because these Bt toxins have a very similar molecular structure. Importantly, the Cry1Ac-selected populations became more susceptible to Cry2Aa and Cry1Fa, showing negative cross-resistance (up to 6-fold, P < 0.05). These results indicate that Cry1Ac, Cry1Fa, and Cry2A are compatible in a multi-toxin approach to minimize the risk of rapid adaptation of the soybean looper to Bt toxins.

New strategy for isolating novel nematicidal crystal protein genes from Bacillus thuringiensis strain YBT-1518.

We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes--cry55Aa1, cry6Aa2, and cry5Ba2--were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

Quantitation and differentiation of bioparticles based on the measurements of light-scattering signals with a common spectrofluorometer.

By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, Delta lambda = 0), we obtained synchronous light scattering (SLS) signals that related to Rayleigh and Mie scatterings. It was found that the SLS signals could be applied for quantitation and differentiation of model bioparticles such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium. In PBS buffer, these model bioparticles could form colloidal suspensions or dispersions of sizes ranging from hundreds of nanometers to tens of micrometers, giving SLS signals with the intensity being proportional to the amount of bioparticles in the range from 1.7 x 10 (5) to 1.7 x 10 (9) CFU/mL. A further finding is that polarized synchronous light scattering (PSLS) signals of I 0 degrees -30 degrees against I 0 degrees -0 degrees , which could be obtained by introducing polarizing sheets accessory of the spectrofluorometer, and the derivative synchronous light scattering (DrSLS) signals, which could be obtained directly with the extension function of the spectrofluorometer, offer differentiation information of bioparticles connected with their size, shape, refractive indexes, and inner structure. Refractive indexes of spherical bacteria were then calculated based on light scattering signals.