Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype

The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Within its biosynthetic gene cluster, the immunity gene pepI, providing producer self-protection, is localized upstream of the structural gene pepA. Pep5 production and the immunity phenotype have been found to be tightly coupled (M. Reis, M. Eschbach-Bludau, M. I. Iglesias-Wind, T. Kupke, and H.-G. Sahl, Appl. Environ. Microbiol. 60:2876–2883, 1994). To study this phenomenon, we analyzed pepA and pepI transcription and translation and constructed a number of strains containing various fragments of the gene cluster and expressing different levels of immunity. Complementation of a pepA-expressing strain with pepI in trans did not result in phenotypic immunity or production of PepI. On the other hand, neither pepA nor its product was found to be involved in immunity, since suppression of the translation of the pepA mRNA by mutation of the ATG start codon did not reduce the level of immunity. Moreover, homologous and heterologous expression of pepI from a xylose-inducible promoter resulted in significant Pep5 insensitivity. Most important for expression of the immunity phenotype was the stability of pepI transcripts, which in the wild-type strain, is achieved by an inverted repeat with a free energy of −56.9 kJ/mol, localized downstream of pepA. We performed site-directed mutagenesis to study the functional role of PepI and constructed F13D PepI, I17R PepI, and PepI 1-65; all mutants showed reduced levels of immunity. Western blot analysis indicated that F13D PepI and PepI 1-65 were not produced correctly or were partially degraded, while I17R PepI apparently was less efficient in providing self-protection than the wild-type PepI.

羊毛硫抗生素（lantibiotic）Pep5由表皮葡萄球菌（Staphylococcus epidermidis）5菌株产生。在其生物合成基因簇中，赋予产生菌自身抗性的免疫基因pepI，定位于结构基因pepA的上游区域。Pep5的合成与免疫表型紧密偶联（M. Reis、M. Eschbach-Bludau、M. I. Iglesias-Wind、T. Kupke与H.-G. Sahl，Appl. Environ. Microbiol. 60:2876–2883, 1994）。为研究该现象，我们对pepA与pepI的转录及翻译过程进行了分析，并构建了一系列携带该基因簇不同片段、且免疫表达水平各异的工程菌株。将pepI以反式形式互补至表达pepA的菌株中，并未获得免疫表型或检测到PepI的表达产物。另一方面，pepA及其编码产物均未参与免疫过程：通过突变pepA的ATG起始密码子以抑制其mRNA的翻译，并未降低菌株的免疫水平。此外，从木糖诱导型启动子进行pepI的同源与异源表达，可使菌体显著获得Pep5抗性。免疫表型的表达最关键的影响因素为pepI转录本的稳定性；在野生型菌株中，该稳定性由定位于pepA下游、自由能为−56.9 kJ/mol的反向重复序列所介导。我们通过定点诱变研究了PepI的功能作用，并构建了F13D PepI、I17R PepI以及PepI 1-65三种突变体，所有突变体的免疫水平均出现下降。蛋白质印迹（Western blot）分析结果显示，F13D PepI与PepI 1-65无法正常合成或发生了部分降解，而I17R PepI在为菌株提供自身抗性方面的效率显然低于野生型PepI。