• Applied Biological Chemistry
• /
• 제51권4호
• /
• pp.263-268
• /
• 2008

Seventy-one Bacillus cereus strains (12 references and 59 food isolates) were analyzed for the occurrence of five different enterotoxin genes (nheABC, hblCDA, entFM, cytK, and bceT) and one emetic toxin cereulide synthetase gene (ces) by PCR (polymerase chain reaction). PCR analysis revealed eight toxigenic patterns in all B. cereus strains tested; they all carried both entFM and nheABC. The presence of hblCDA, cytK, and bceT varied according to the enterotoxin-producing strains, among which hblCDA was the least frequently detected in the food-isolated strains. Only five B. cereus strains harbored ces, associated with the emetic type of food poisoning; however, these strains were devoid of hblCDA, cytK, and bceT.

• BMB Reports
• /
• 제46권3호
• /
• pp.175-180
• /
• 2013

Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin becomes inactive when isoleucine at position 150 was replaced by alanine. To investigate the functional role of this position, Ile150 was substituted with Leu, Phe, Glu and Lys. All mutant proteins were produced at high level, solubilized in carbonate buffer and yielded protease activated product similar to those of the wild type. Intrinsic fluorescence spectra analysis suggested that these mutants retain similar folding to the wild type. However, mosquito larvicidal and hemolytic activities dramatically decreased for the I150K and were completely abolished for I150A and I150F mutants. Membrane binding and oligomerization assays demonstrated that only I150E and I150L could bind and form oligomers on lipid membrane similar to that of the wild type. Our results suggest that amino acid at position 150 plays an important role during membrane binding and oligomerization of Cyt2Aa2 toxin.

• BMB Reports
• /
• 제41권11호
• /
• pp.820-825
• /
• 2008

Bacillus thuringiensis Cyt2Aa toxin is a mosquito-larvicidal and cytolytic $\delta$-endotoxin, which is synthesized as a protoxin and forms crystalline inclusions within the cell. These inclusions are solubilized under alkaline conditions and are activated by proteases within the larval gut. In order to assess the functions of the N-and C-terminal regions of the protoxin, several N- and C-terminal truncated forms of Cyt2Aa were constructed. It was determined that amino acid removal at the N-terminal, which disrupts the $\beta$1 structure, might critically influence toxin production and inclusion formation. The deletion of 22 amino acids from the C-terminus reduced the production and solubility of the toxin. However, the removal of more than 22 amino acids from the C-terminus or the addition of a bulky group to this region could result in the inability of the protein to adopt the proper folding. These findings directly demonstrated the critical roles of N- and C-terminal amino acids on the production and folding of the B. thuringiensis cytolytic $\delta$-endotoxin.

• BMB Reports
• /
• 제43권6호
• /
• pp.427-431
• /
• 2010

Cyt2Aa2 is a mosquito-larvicidal protein produced as a 29 kDa crystalline protoxin from Bacillus thuringiensis subsp. darmstadiensis. To become an active toxin, proteolytic processing is required to remove amino acids from its N- and C-termini. This study aims to investigate the functional role of amino acid residues on the N-terminal ${\beta}1$ and C-terminal ${\alpha}F$ of Cyt2Aa2 protoxin. Mutant protoxins were constructed, characterized and compared to the wild type Cyt2Aa2. Protein expression data and SDS-PAGE analysis revealed that substitution at leucine-33 (L33) of ${\beta}1$ has a critical effect on dimer formation and structural stability against proteases. In addition, amino acids N230 and I233-F237 around the C-terminus ${\alpha}F$ demonstrated a crucial role in protecting the protoxin from proteolytic digestion. These results suggested that ${\beta}1$ and ${\alpha}F$ on the Nand C-terminal ends of Cyt2Aa2 protoxin play an important role in the molecular interaction and in maintaining the structural stability of the protoxin.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제2권2호
• /
• pp.185-189
• /
• 2001

Wild type Bacilus thuringiensis subsp. israelensis and B. sphaericus toxins have been used separately as active in ingredients for bacterial insecticides to control mosquito larvae due to their comparable toxicity to chemical insecticides. Cry11B, recently cloned from B. thuringiensis subsp. jegathesan, shows higher toxicity against three major species of mosquito larvae than Cry11A, one of the major component of B. thuringiensis subsp. israelensis inclusion body. To determine whether the combination of cry11B and B. sphaericus binary toxins is as toxic as B. thuringiensis subsp. israelensis parental strain, cry11B and B. sphaericus binary toxins genes were co-expressed as an operon using cytlA promoters/STAB-SD hybrid expression system in B. thuringiensis subsp. israelensis acrystalliferous strain 4Q7. However, unexpectedly, B. sphaericus binary toxins were barely produced, whereas relatively large amount of Cry11B was produced. When this strain was grown in four different media, NB+G and Peptonized Milk produced more toxin proteins and spores per unit of media than GYS and G-Tris. Toxicity of this strain against fourth instar Culex quinquefasciatus was ranged from of 8.3 to 45.7 ng/ml, with NB+G culture being the highest, and GYS culture was the lowest.

• 미생물학회지
• /
• 제45권4호
• /
• pp.425-429
• /
• 2009

Bacillus cereus 설사독소 전사조절유전자인 plcR 및 papR의 특이적 프라이머 쌍을 사용한 PCR로 설사 독소유전자 함유 B. cereus 균의 검출 가능성을 조사하였다. 적어도 한 종류 이상의 독소유전자들(hblACD, nheABC, cytK)을 함유한 96균주의 B. cereus를 대상으로 plcR-papR 유전자에 특이적인 프라이머들을 이용하여 PCR을 한 결과 모두 양성을 나타냈다. 그러나 독소 유전자를 함유하지 않은 48균주의 Bacillus들로 같은 분석을 한 결과 모두 음성을 보였다. PCR에 의한 plcR-papR 유전자 검출법은 설사독소 유전자를 함유한 B. cereus를 확인하는데 간편하면서도 정확한 결과를 제공하였다.

• Journal of Microbiology and Biotechnology
• /
• 제19권8호
• /
• pp.749-759
• /
• 2009

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%). SDS-PAGE 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.

• Journal of Microbiology and Biotechnology
• /
• 제25권12호
• /
• pp.2043-2048
• /
• 2015

Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the non-hemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

• 한국수산과학회지
• /
• 제53권6호
• /
• pp.870-877
• /
• 2020

Twenty-three Bacillus cereus strain isolated from commercial jeotgal were investigated for 11 toxin genes and susceptibility to 25 different antimicrobials. The hemolytic enterotoxins hblA, hblC, and hblD were detected in 13.0%, and non-hemolytic enterotoxins nheA, nheB, and nheC were detected in 26.1%, 100%, and 100% of the isolates, respectively. The positive rates of cytK, entFM, becT, hlyII, and ces were 73.9%, 60.9%, 26.1%, 8.7%, and 0.0%, respectively. According to the disk diffusion susceptibility test, all of the strains studied were resistant to cefuroxime, followed by cefoxitin (78.3%), oxacillin (78.3%), ampicillin (69.6%), penicillin G (69.6%), and amoxicillin (65.2%). However, all the strains were susceptible to 11 other antimicrobials, including amikacin, chloramphenicol, and ciprofloxacin. The average minimum inhibitory concentrations of amoxicillin, ampicillin, and cefuroxime against B. cereus were 462.9, 235.0, and 135.0 ㎍/mL, respectively. These results highlight the need for sanitizing commercial jeotgal, and provide evidence to help reduce the risk of jeotgal contamination by antimicrobial-resistant bacteria.

• 한국식품위생안전성학회지
• /
• 제28권2호
• /
• pp.138-145
• /
• 2013

어린이집 유아들이 공용으로 사용하는 수건의 미생물 오염도와 분리된 식중독 미생물의 장독소 유전자 및 장독소 생산 특성을 분석하여 어린이집 유아 사용 수건의 위생안전성을 확보하고자 어린이가 손 씻은 후 공용으로 사용하는 수건 22개 (사용 전 7개, 사용 중 15개)를 연구대상으로 하였다. 일반세균수는 평균 6.2 log CFU/100 $cm^2$로 검출되었고 진균수는 평균 4.1 log CFU/100 $cm^2$로 검출되었으며 대장균군은 사용 전 수건의 경우 7건 중 4건 (57.1%), 사용 중인 수건의 경우 15건 모두에서 (100%) 검출되었다. 어린이 사용 수건의 미생물 오염도가 높게 나타나 수건의 위생적인 살균 세탁 및 상대습도가 낮은 곳에 보관하는 등의 보관 방법 개선이 필요한 것으로 판단되었다. Salmonella spp.의 경우 실험에 사용된 모든 수건에서 검출되지 않았으나, Staph. aureus는 22건 중 5건 (22.7%), B. cereus는 11건 (50.0%)에서 검출되어 B. cereus 오염이 가장 심각한 것으로 나타났다. Staph. aureus 장독소 유전자와 독소 단백질은 모두 불검출 되었으나 분리 동정된 11균주의 B. cereus 장독소 유전자 실험 결과 hblC, hblD, hblA 유전자가 각각 72.7, 72.7, 54.5% 검출되고 nheA, nheB, nheC 유전자가 각각 81.8, 72.7, 54.5% 검출되었으며 cytK, entFM 장독소 유전자는 각각 45.5, 90.0% 검출되었다. B. cereus 장독소 실험결과 HBL 장독소는 11균주 중 8균주 (72.7%), NHE 장독소는 5균주 (45.5%) 검출되었고 HBL과 NHE 장독소 중 하나 이상을 생산하는 B. cereus 균주는 10균주 (90.9%)로 나타났다. 수건에 오염된 B. cereus 균주의 교차오염에 따른 식중독 위험성이 상존하는 것으로 나타나 개인별 수건 또는 종이 타올 사용을 고려하여야 할 것으로 판단되었다.