Sushida Hirotoshi, Ishibashi Naoki, Zendo Takeshi, Wilaipun Pongtep, Leelawatcharamas Vichien, Nakayama Jiro, Sonomoto Kenji
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Department of Fishery Products, Faculty of Fisheries, Kasetsart University, 50 Paholyothin Rd., Chatuchak, Bangkok 10900, Thailand.
J Biosci Bioeng. 2018 Jul;126(1):23-29. doi: 10.1016/j.jbiosc.2018.01.015. Epub 2018 Feb 13.
EnkT is a novel ATP-binding cassette (ABC) transporter responsible for secretion of four bacteriocins, enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z), produced by Enterococcus faecium NKR-5-3. It is generally recognized that the secretion of a bacteriocin requires a dedicated ABC transporter, although molecular mechanisms of this secretion are yet to be revealed. In order to characterize the unique ability of EnkT to secrete multiple bacteriocins, the role of N-terminal leader peptides of bacteriocin precursors was evaluated using Ent53C precursor as a model. The 18-amino acid leader peptide of Ent53C (Lc) was modified by site-directed mutagenesis to generate various point mutations, truncations, or extensions, and substitutions with other leader peptides. The impact of these Lc mutations on Ent53C secretion was evaluated using a quantitative antimicrobial activity assay. We observed that Ent53C production increased with Ala substitution of the highly conserved C-terminal double glycine residues that are recognized as the cleavage site. In contrast, Ent53C antimicrobial activity decreased, with decrease in the length of the putative α-helix-forming region of Lc. Furthermore, EnkT recognized and transported Ent53C of the transformants possessing heterologous leader peptides of enterocin A, pediocin PA-1, brochocins A and B, and lactococcins Qα and Qβ. These results indicated that EnkT shows significant tolerance towards the sequence and length of leader peptides, to secrete multiple bacteriocins. This further demonstrates the functional diversity of bacteriocin ABC transporters and the importance of leader peptides as their recognition motif.
EnkT是一种新型的ATP结合盒（ABC）转运蛋白，负责分泌由粪肠球菌NKR-5-3产生的四种细菌素，即肠球菌素NKR-5-3A、C、D和Z（Ent53A、C、D和Z）。虽然细菌素分泌的分子机制尚未明确，但一般认为细菌素的分泌需要一种专门的ABC转运蛋白。为了表征EnkT分泌多种细菌素的独特能力，以Ent53C前体为模型评估了细菌素前体N端前导肽的作用。通过定点诱变对Ent53C的18个氨基酸前导肽（Lc）进行修饰，以产生各种点突变、截短或延伸，并替换为其他前导肽。使用定量抗菌活性测定法评估这些Lc突变对Ent53C分泌的影响。我们观察到，用丙氨酸取代被认为是切割位点的高度保守的C端双甘氨酸残基后，Ent53C的产量增加。相反，随着Lc假定的α螺旋形成区域长度的减少，Ent53C的抗菌活性降低。此外，EnkT识别并转运了具有肠球菌素A、片球菌素PA-1、布氏菌素A和B以及乳球菌素Qα和Qβ异源前导肽的转化体的Ent53C。这些结果表明，EnkT对前导肽的序列和长度具有显著的耐受性，能够分泌多种细菌素。这进一步证明了细菌素ABC转运蛋白的功能多样性以及前导肽作为其识别基序的重要性。
