Characterization of Amylolysin, a Novel Lantibiotic from Bacillus amyloliquefaciens GA1

BackgroundLantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity. Methodology/FindingsBacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact invitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus. Conclusion/SignificanceApart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens.

背景：羊毛硫抗生素（lantibiotics）是一类热稳定肽类，以含有硫醚氨基酸羊毛硫氨酸（lanthionine）和甲基羊毛硫氨酸（methyllanthionine）为核心特征。它们可抑制革兰氏阳性菌的生长，包括单核细胞增生李斯特菌（Listeria monocytogenes）、金黄色葡萄球菌（Staphylococcus aureus）以及蜡样芽孢杆菌（Bacillus cereus）——这些均为食源性疾病或医院内感染的致病菌。羊毛硫抗生素的生物合成机制由基因簇编码，该基因簇包含编码前羊毛硫抗生素肽的结构基因，以及参与前羊毛硫抗生素修饰、调控、转运与免疫的其他功能基因。

方法与结果：研究发现解淀粉芽孢杆菌（Bacillus amyloliquefaciens）GA1可产生一种抗菌肽，命名为解淀粉溶素（amylolysin），其对包括耐甲氧西林金黄色葡萄球菌在内的一系列革兰氏阳性菌具有抑制活性。基因组表征分析鉴定得到一个假定的羊毛硫抗生素基因簇，该基因簇包含结构基因amlA、参与修饰的基因amlM、转运基因amlT、调控基因amlKR以及免疫基因amlFE。敲除amlA会导致该抗菌活性完全丧失，由此证实所鉴定的基因簇与解淀粉溶素的合成直接相关。对纯化后的解淀粉溶素进行基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF）和液相色谱-质谱（LC-MS）分析，结果表明该肽为一种此前未被报道过的新型羊毛硫抗生素。解淀粉溶素在体外可与脂质II（lipid II，即跨细胞质膜的肽聚糖单体载体）特异性结合，且其拥有独特的修饰基因，这提示该肽属于B类羊毛硫抗生素。解淀粉溶素的免疫机制似乎仅由AmlF和AmlE两种蛋白介导，这在芽孢杆菌属中较为罕见。

结论与意义：除解淀粉芽孢杆菌Y2菌株和HIL Y-85,544728菌株产生的枯草菌素（mersacidin）外，关于该物种合成B类羊毛硫抗生素的报道十分稀少。本研究对解淀粉芽孢杆菌中另一类B类羊毛硫抗生素代表物进行了完整的遗传与结构表征。