Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product
Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product
复制标题
针对羊毛硫抗生素 Pep5 的生产者免疫：免疫基因 pepI 的鉴定及其基因产物的定位和功能分析
DOI：
10.1128/aem.60.8.2876-2883.1994
复制
发表时间：
1994
影响因子：
4.4
通讯作者：
H. Sahl
中科院分区：
文献类型：
--
作者：
M. Reis;M. Eschbach;M. I. IGLESIAS;T. Kupke;H. Sahl
羊毛硫抗生素Pep 5由表皮葡萄球菌5产生。Pep 5生产和生产者免疫与20-kb质粒pED 503相关。将含有Pep 5结构基因pepA的pED 503的1.3kb Kpn Ⅰ片段亚克隆到大肠杆菌-葡萄球菌穿梭载体pCU 1中，并将重组质粒pMR 2转移到Pep 5和免疫阴性突变株S. epidermidis 5 Pep 5-（缺乏pED 503）。该克隆不产生活性Pep 5，但显示出与野生型菌株相同程度的对Pep 5的不敏感性。1.3 kb Kpn I片段的测序和突变体的分析表明，Pep 5免疫中涉及两个基因，即结构基因pepA本身和pepI，pepA上游的一个短的开放阅读框。为了鉴定69个氨基酸的pepI基因产物，我们构建了一个E. coli麦芽糖结合蛋白-PepI融合克隆。通过用抗麦芽糖结合蛋白-PepI抗血清进行免疫印迹，在野生型菌株和免疫突变体（携带质粒pMR 2和pMR 11）的可溶性和膜级分中检测到免疫肽PepI。携带pepI无pepA或pepI有不完全pepA的菌株不具有免疫力，也不产生pepI。用盐和EDTA洗涤膜减少了这部分中PepI的量，用Triton X-100处理几乎完全除去了肽。此外，PepI被添加到植物稳定的原生质体中的蛋白酶水解。这表明PepI松散地附着在细胞质膜的外侧。脯氨酸摄取和流出实验免疫和nonimmune菌株也表明，PepI可以在膜网站。
The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Pep5 production and producer immunity are associated with the 20-kb plasmid pED503. A 1.3-kb KpnI fragment of pED503, containing the Pep5 structural gene pepA, was subcloned into the Escherichia coli-Staphylococcus shuttle vector pCU1, and the recombinant plasmid pMR2 was transferred to the Pep5- and immunity-negative mutant S. epidermidis 5 Pep5- (devoid of pED503). This clone did not produce active Pep5 but showed the same degree of insensitivity towards Pep5 as did the wild-type strain. Sequencing of the 1.3-kb KpnI-fragment and analysis of mutants demonstrated the involvement of two genes in Pep5 immunity, the structural gene pepA itself and pepI, a short open reading frame upstream of pepA. To identify the 69-amino-acid pepI gene product, we constructed an E. coli maltose-binding protein-PepI fusion clone. The immunity peptide PepI was detected in the soluble and membrane fractions of the wild-type strain and the immune mutants (harboring the plasmids pMR2 and pMR11) by immunoblotting with anti-maltose-binding protein-PepI antiserum. Strains harboring either pepI without pepA or pepI with incomplete pepA were not immune and did not produce PepI. Washing the membrane with salts and EDTA reduced the amount of PepI in this fraction, and treatment with Triton X-100 almost completely removed the peptide. Furthermore, PepI was hydrolyzed by proteases added to osmotically stabilized protoplasts. This suggests that PepI is loosely attached to the outside of the cytoplasmic membrane. Proline uptake and efflux experiments with immune and nonimmune strains also indicated that PepI may act at the membrane site.