A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results

Rampersad, Joanne; Ammons, David

doi:10.1186/1471-2180-5-52

Cited by 45 publications

(12 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After binding, the toxin molecules go through oligomerization and membrane insertion that cause pore formation, and lead to eventual cell-cytolysis [5–7]. The formation of ICPs is the only phenotypic character that differentiates the two taxonomically closely related species, Bt and Bacillus cereus [8, 9], which have been proposed to be considered as a single species [10]. Hofte & Whitely [11] were the first to describe four Cry and two Cyt δ-endotoxins genes on the basis of insecticidal activity.…”

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.)

et al. 2016

View full text Add to dashboard Cite

BackgroundWorldwide, mosquito vectors are transmitting several etiological agents of important human diseases, including malaria, causing millions of deaths every year. In Saudi Arabia, as elsewhere, vector-control is based mostly on chemical insecticides which may be toxic and cause environmental deprivation. Here, to support the development of bio-pesticide alternatives, a study was conducted to identify native Bacillus thuringiensis (Bt) isolates with improved toxicity against the malaria vector, Anopheles gambiae (s.l.).MethodsSixty-eight Bt isolates were obtained from 300 soil and other samples collected from 16 sites across Saudi Arabia. Bt identification was based on morphological characteristics of colonies, shape of parasporal crystals and biochemical profiles. After characterization of their mosquitocidal activity, larvicidal strains were described through 16S ribosomal DNA gene sequencing, cry, cyt and chi genes PCR-amplification profiles, and SDS-PAGE protein analyses.ResultsSpherical Bt crystals were predominant amongst the 68 isolates (34%), while irregular, bi-pyramidal and spore-attached crystals were found in 32, 13 and 21% of strains, respectively. LC50 and LC90 bioassays showed that 23/68 isolates were larvicidal, with distinct biochemical activity profiles compared to non-larvicidal Bt strains. Eight larvicidal strains showed larvicidal activity up to 3.4-fold higher (LC50 range: 3.90–7.40 μg/ml) than the reference Bti-H14 strain (LC50 = 13.33 μg/ml). Of these, 6 strains had cry and cyt gene profiles similar to Bti-H14 (cry4Aa, cry4Ba, cry10, cry11, cyt1Aa, cyt1Ab, cyt2Aa). The seventh strain (Bt63) displaying the highest larvicidal activity (LC50 = 3.90 μg/ml) missed the cry4Aa and cyt1Ab genes and had SDS-PAGE protein profiles and spore/crystal sizes distinct from Bti-H14. The eight strain (Bt55) with LC50 of 4.11μg/ml had cry and cyt gene profiles similar to Bti-H14 but gave a chi gene PCR product size of 2027bp. No strains harbouring cry2, cry17 + 27, cry24 + 40, cry25, cry29, cry30, or cyt2Ba were detected.ConclusionThis study represents the first report of several Saudi indigenous Bt strains with significantly higher larvicidal efficacy against An. gambiae than the reference Bti-H14 strain. The very high toxicity of the Bt63 strain, combined with distinct cry and cyt genes and SDS-PAGE-protein profiles makes it a promising candidate for future applications in mosquito bio-control.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1922-6) contains supplementary material, which is available to authorized users.

show abstract

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.)

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Building on this previous work, we undertook a study to identify genes responsible for crystal localization in a novel filamentous strain of B. thuringiensis called Bt2-56 ( Fig. 1A and A=) (6,14).Inactivation of gene cry256 causes loss of crystals from the exosporium. Genes in Bt2-56 were mutated by random transposon insertion using plasmid pMarA (7).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

A Novel Bacillus thuringiensis Cry-Like Protein from a Rare Filamentous Strain Promotes Crystal Localization within the Exosporium

Ammons¹,

Reyna²,

Granados³

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

dMutation of a novel cry-like gene (cry256) from Bacillus thuringiensis resulted in a protein crystal, normally located within the spore's exosporium, being found predominately outside the exosporium. The cry256 gene codes for a 3-domain Cry-like protein that does not correspond to any of the known Cry protein holotypes. During sporulation, the bacterium Bacillus thuringiensis produces a protein crystal comprised of one or more members of the Cry family of protein toxins. The crystal is normally located outside the exosporium, which surrounds the spore (1); however, relatively few strains of B. thuringiensis have been shown to localize their crystals within the exosporium (2, 3). With the aim of understanding how some crystals are localized within the exosporium, Debro et al. showed that in B. thuringiensis subsp. finitimus, the genes necessary for localizing crystals within the exosporium where located on two plasmids (4). Zhu et al. later identified these genes (5) and showed that not all B. thuringiensis organisms used these genes to localize crystals within their exosporium. Building on this previous work, we undertook a study to identify genes responsible for crystal localization in a novel filamentous strain of B. thuringiensis called Bt2-56 ( Fig. 1A and A=) (6,14).Inactivation of gene cry256 causes loss of crystals from the exosporium. Genes in Bt2-56 were mutated by random transposon insertion using plasmid pMarA (7). Mutants with crystals located outside the exosporium were identified by viewing stained preparations of sporulated mutants (8). Approximately 84% of the crystals from a mutant called M1-11 were observed outside the exosporium (Fig. 1B and B=).Inverse PCR using primers left-tn1 and oIPCR2a (Table 1) was used to amplify M1-11 genomic DNA flanking the transposable element, indicating that transposons had integrated into both a histidine kinase gene and a cry-like gene that we call cry256. The DNA sequence of both strands of the entire cry256 gene and its immediate flanking regions was then determined by "walking" the gene, using primers designed from previously sequenced DNA regions (GenBank accession number JQ670887). The DNA sequence of cry256 from Bt2-56 (the unmutated parent strain of M1-11) was also determined, using an Illumina GAIIx sequencer at the University of Houston's IMD Sequencing Center. The 36-bp sequencing reads were assembled using Velvet (9) and visualized with the software program Tablet (10). DNA translations were performed using a Web-based DNA-to-protein tool (11; the Web application utilized is located at http://insilico.ehu.es /translate/). The transposon was found to have inserted into a region that separated the last 49 amino acids of the protein encoded by cry256 from the rest of protein (see file S1 in the supplemental material [the insertion site highlighted in red at position 1528]). Aside from the transposon, the DNA sequence of cry256 in mutant M1-11 was identical to that obtained for the wild-type cry256 gene in Bt2-56.Since the cause-and-effect relationship ...

show abstract

“…In order to ascertain the ability of native Bt isolates to demonstrate appreciable activity against second instar larvae (6 h after moulting) of H. armigera , "quick screen" bioassays were performed (Rampersad and Ammons 2005). "Surface contamination" method involving higher doses (1mg of total protein mL -1 of culture supernatant) of the actively growing cultures (18 h) smeared onto young chick pea leaves in triplicate were employed.…”

Section: R a F Tmentioning

confidence: 99%

Molecular and insecticidal characterization of Vip3A protein producingBacillus thuringiensisstrains toxic againstHelicoverpa armigera(Lepidoptera: Noctuidae)

et al. 2016

View full text Add to dashboard Cite

Vegetative insecticidal proteins (Vip) represent the second generation of insecticidal proteins produced by Bacillus thuringiensis (Bt) during the vegetative growth stage of growth. Bt-based biopesticides are recognized as viable alternatives to chemical insecticides; the latter cause environmental pollution and lead to the emergence of pest resistance. To perform a systematic study of vip genes encoding toxic proteins, a total of 30 soil samples were collected from diverse locations of Kashmir valley, India, and characterized by molecular and analytical methods. Eighty-six colonies showing Bacillus-like morphology were selected. Scanning electron microscopy observations confirmed the presence of different crystal shapes, and PCR analysis of insecticidal genes revealed a predominance of the lepidopteran-specific vip3 (43.18%) gene followed by coleopteran-specific vip1 (22.72%) and vip2 (15.90%) genes in the isolates tested. Multi-alignment of the deduced amino acid sequences revealed that vip3 sequences were highly conserved, whereas vip1 and vip2 showed adequate differences in amino acid sequences compared with already reported sequences. Screening for toxicity against Helicoverpa armigera larvae was performed using partially purified soluble fractions containing Vip3A protein. The mortality levels observed ranged between 70% and 96.6% in the isolates. The LC50 values of 2 of the native isolates, JK37 and JK88, against H. armigera were found to be on par with that of Bt subsp. kurstaki HD1, suggesting that these isolates could be developed as effective biopesticides against H. armigera.

show abstract

A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results

Cited by 45 publications

References 31 publications

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.)

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.)

A Novel Bacillus thuringiensis Cry-Like Protein from a Rare Filamentous Strain Promotes Crystal Localization within the Exosporium

Molecular and insecticidal characterization of Vip3A protein producingBacillus thuringiensisstrains toxic againstHelicoverpa armigera(Lepidoptera: Noctuidae)

Contact Info

Product

Resources

About