Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361
Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361
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DOI：
10.1128/aem.00433-14
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发表时间：
2014-03
影响因子：
4.4
通讯作者：
Gaoyan Wang;D. Manns;J. Churey;R. Worobo
中科院分区：
文献类型：
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作者：
Gaoyan Wang;D. Manns;J. Churey;R. Worobo
苏云金素H是由苏云金芽孢杆菌SF361产生的抗菌肽。苏云金素H的31个氨基酸形成了一个由四对非常独特的硫-α-碳硫醚键维持的发夹结构。苏云红素H的产生依赖于一个包含10个开放阅读框的假定基因簇。该基因簇包括3个串联结构基因（thnA1、thnA2和thnA3），编码3个相同的40个氨基酸的苏云金素H前肽，以及其他7个被认为负责前肽加工、调控、修饰、输出和自身免疫的基因。利用新型表达载体pGW133转化苏云红素H缺陷宿主，构建了一个同源的苏云红素H表达系统。该寄主命名为B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3，通过从天然的苏云金素H产生菌染色体上删除三个串联结构基因构建而成。将苏云红蛋白H的天然启动子thnA1和Cry蛋白终止子克隆到大肠杆菌b中，构建了苏云红蛋白H表达载体pGW133。苏云金菌穿梭载体pHT315。产生了33种不同的pGW133变体，每种变体含有thnA1基因的不同点突变，并分别转化为苏云金芽孢杆菌SF361 ΔthnA1 ΔthnA2 ΔthnA3。这些位点导向突变体在硫醚键形成位置上含有单个自由基或保守氨基酸取代，或在所有其他非丙氨酸氨基酸上含有一个自由基取代。随后比较了携带不同pGW133变异体的苏云金芽孢杆菌SF361 ΔthnA1 ΔthnA2 ΔthnA3对三种不同指示菌株的细菌素活性。
ABSTRACT Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared.