An important toxin‐antitoxin (TA) system hok/sok, encoded by R1 plasmid of Escherichia coli, is involved in the post segregation killing of cells that have lost the plasmid. The lethal properties of hok protein have been utilized for the environmental containment of microbes and the development of potential vaccine candidates. This study aimed to demonstrate the potent anti‐microbial property of a 19 amino acid (AA) long N‐terminal fragment of hok peptide. This was accomplished by designing a conditional suicide system based on hok gene expression cloned in an anhydrotetracycline (aTc) inducible vector – pASK75. Heat shock and electroporation were utilized for the transformation of Escherichia coli and Vibrio cholerae cells, respectively. The minimal induction concentration (MI_dC) of aTc, determined by analyzing the expression of green fluorescent protein cloned separately into pASK75 vector, was 30 ng/mL. As hok gene was synthesized de novo (using recombinant polymerase chain reaction) in our study, various random sized hok fragments were generated (as a result of the error‐prone nature of Taq polymerase). The smallest hok fragment able to bring about effective antimicrobial killing was a 19 AA long N‐terminal fragment of hok having the wild type sequence, except for the carboxy terminus AA residue. The MI_dC of aTc in our experiments was 6‐fold lower than previously reported, making our bacterial clones suitable for use in mammalian systems as potential vaccine candidates. Based on our experiments, we hypothesize the 19 AA long N‐terminal fragment of hok peptide to be the smallest possible hok fragment sufficient to bring about effective antimicrobial killing.

中文翻译：

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将hok基因克隆到脱水四环素诱导型pASK75载体中显示出hok肽的19个氨基酸长的N端片段的有效抗菌作用。

由大肠杆菌R1质粒编码的一种重要的毒素-抗毒素（TA）系统hok / sok参与了丢失该质粒的细胞的分离后杀死。hok蛋白的致命特性已被用于微生物的环境控制和潜在疫苗候选物的开发。这项研究旨在证明hok肽的19个氨基酸（AA）长N端片段的有效抗菌特性。这是通过基于克隆在无水四环素（aTc）诱导型载体– pASK75中的hok基因表达设计一个条件自杀系统来完成的。利用热休克和电穿孔法转化大肠杆菌和霍乱弧菌单元格。通过分析分别克隆到pASK75载体中的绿色荧光蛋白的表达确定的aTc的最低诱导浓度（MI _d C）为30 ng / mL。由于在我们的研究中hok基因是从头合成的（使用重组聚合酶链反应），因此产生了各种随机大小的hok片段（由于Taq聚合酶容易出错的性质）。能够有效杀死细菌的最小的hok片段是hok的19 AA长N端片段，除了羧基末端的AA残基外，它具有野生型序列。该MI _d在我们的实验中，aTC的C值比以前报道的低6倍，这使我们的细菌克隆适合作为潜在的疫苗候选物在哺乳动物系统中使用。根据我们的实验，我们假设hok肽的19个AA长N端片段是可能足以引起有效抗菌杀伤作用的最小的hok片段。