转座子Tn917诱变的炭疽杆菌芽孢形成缺陷株的筛选

Screening the spo Mutations of Bacillus anthracis by Tn917-Mediated Insertional Mutagenesis

目的:诱导转座子Tn917随机插入炭疽杆菌染色体,产生在不同位点突变的突变体库,从中筛选芽孢形成缺陷型突变株。方法:用含转座子Tn917的质粒pLTV3转化炭疽杆菌,以低浓度红霉素诱导转座因发生转座,产生大量的突变株。进而用氯化三苯基四氮唑染色法和复红美蓝染色法从突变体库中筛选芽孢形成缺陷株;用Southern杂交法对芽孢形成缺陷株进行验证。结果:对2000个突变体进行了筛选,共得到6株芽孢形成缺陷株,在LB培养基中培养5d后,镜下仍未见有芽孢形成,呈现明显的芽孢形成缺陷特征。Southern杂交表明野生株无杂交带,突变株均有且只有1条杂交带,且杂交带的位置不尽相同。结论:转座子Tn917可以单拷贝随机诱变炭疽杆菌野生株,产生在不同位点突变的突变株。

Objective： To generate mutant pools of Bacillus anthracis A16R by transposon Tn917-mediated insertional mutagenesis and screen the spo mutations. Methods： B.anthracis was transformed with temperature-sensitive plasmid pLTV3 that harbors Tn917. The culture of one transformant was diluted with fresh medium and incubated with erythromycin of low concentration in order to induce the transposition of Tn917. The mutant pools of B.anthracis was generated. The spo mutations was screened by use of triphenyl tetrazolium chloride （TTC） and fuchsin basic-methylene blue stained. And Southern hybridization was used to confirm the spo mutations. Results： Six spo mutants were screened from 2 000 Tn917 insertion mutants and they could not sporulation when growing in LB medium for five days. The probe hybridized to one EcoR Ⅰ fragment of each transposant, and each hybridization fragment had different molecular size. The probe did not hybridize to the wild B. anthracis DNA. Conclusions： Transposon Tng17 mutagenesis results in single chromosome insertions with apparent randomness in B.anthracis.