Asano S I, Nukumizu Y, Bando H, Iizuka T, Yamamoto T
Faculty of Agriculture, Hokkaido University, Japan.
Appl Environ Microbiol. 1997 Mar;63(3):1054-7. doi: 10.1128/aem.63.3.1054-1057.1997.
A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.
从蜡样芽孢杆菌FM1中克隆了一个新的肠毒素基因，并测定了其核苷酸序列。此前，已从同一蜡样芽孢杆菌菌株中分离出一种在高等动物中引起典型肠毒素症状的45 kDa蛋白（K. Shinagawa，第181 - 193页，载于A. E. Pohland等人编著的《食品和饲料中的微生物毒素》，1990年），但尚未有关于该肠毒素基因克隆的报道。在本研究中，制备了针对纯化肠毒素的特异性抗体，并用于筛选以λgt11载体构建的蜡样芽孢杆菌FM1基因组文库。发现一个免疫阳性克隆包含完整的蛋白质编码区以及一些5'和3'侧翼区域。克隆基因推导的氨基酸序列表明，该蛋白富含β结构，并且包含一些不寻常的序列，如连续的Asn残基。为了从苏云金芽孢杆菌中克隆肠毒素基因，根据蜡样芽孢杆菌基因的核苷酸序列合成了两条PCR引物。这些引物旨在扩增完整的蛋白质编码区。用苏云金芽孢杆菌亚种sotto和苏云金芽孢杆菌亚种israelensis菌株的DNA制剂进行PCR，成功扩增出一段大小与蜡样芽孢杆菌肠毒素蛋白质编码区几乎相同的DNA片段。扩增的DNA片段的核苷酸序列表明，这些苏云金芽孢杆菌菌株含有一个与蜡样芽孢杆菌肠毒素基因非常相似的肠毒素基因。在一个反应中用四对引物对另外的苏云金芽孢杆菌菌株进行进一步的PCR筛选，结果显示一些其他的苏云金芽孢杆菌菌株含有类肠毒素基因。
