Institute of Biochemistry and Biophysics, Polish Academy of Sciences (IBB PAS), Pawińskiego 5a, 02-106, Warsaw, Poland.
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
Sci Rep. 2018 Oct 25;8(1):15790. doi: 10.1038/s41598-018-34087-2.
Mannose phosphotransferase system (Man-PTS) serves as a receptor for several bacteriocins in sensitive bacterial cells, namely subclass IIa bacteriocins (pediocin-like; pediocins) and subclass IId ones - lactococcin A (LcnA), lactococcin B (LcnB) and garvicin Q (GarQ). Here, to identify the receptor for three other narrow-spectrum subclass IId bacteriocins - garvicins A, B and C (GarA-C) Lactococcus garvieae mutants resistant to bacteriocins were generated and sequenced to look for mutations responsible for resistance. Spontaneous mutants had their whole genome sequenced while in mutants obtained by integration of pGhost9::ISS1 regions flanking the integration site were sequenced. For both types of mutants mutations were found in genes encoding Man-PTS components IIC and IID indicating that Man-PTS likely serves as the receptor for these bacteriocins as well. This was subsequently confirmed by deletion of the man-PTS operon in the bacteriocin-sensitive L. garvieae IBB3403, which resulted in resistant cells, and by heterologous expression of appropriate man-PTS genes in the resistant Lactococcus lactis strains, which resulted in sensitive cells. GarA, GarB, GarC and other Man-PTS-targeting bacteriocins differ in the amino acid sequence and activity spectrum, suggesting that they interact with the receptor through distinct binding patterns. Comparative analyses and genetic studies identified a previously unrecognized extracellular loop of Man-PTS subunit IID (γ+) implicated in the L. garvieae sensitivity to the bacteriocins studied here. Additionally, individual amino acids localized mostly in the sugar channel-forming transmembrane parts of subunit IIC or in the extracellular parts of IID likely involved in the interaction with each bacteriocin were specified. Finally, template-based 3D models of Man-PTS subunits IIC and IID were built to allow a deeper insight into the Man-PTS structure and functioning.
磷酸甘露糖转移酶系统（Man-PTS）作为敏感细菌细胞中几种细菌素的受体，即 IIa 亚类细菌素（类肠毒素；肠毒素）和 IId 亚类 - 乳球菌素 A（LcnA）、乳球菌素 B（LcnB）和 garvicin Q（GarQ）。在这里，为了鉴定另外三种窄谱 IId 细菌素 - garvicin A、B 和 C（GarA-C）的受体，我们生成了对细菌素有抗性的乳球菌 garvieae 突变体并对其进行测序以寻找负责抗性的突变。自发突变体的整个基因组被测序，而通过整合侧翼整合位点的 pGhost9::ISS1 区域获得的突变体则被测序。对于这两种类型的突变体，在编码 Man-PTS 成分 IIC 和 IID 的基因中发现了突变，这表明 Man-PTS 可能也是这些细菌素的受体。随后，通过在细菌素敏感的 L. garvieae IBB3403 中删除 man-PTS 操纵子，得到了抗性细胞，以及通过在抗性乳球菌 lactis 菌株中异源表达适当的 man-PTS 基因，得到了敏感细胞，这一结果得到了验证。GarA、GarB、GarC 和其他 Man-PTS 靶向细菌素在氨基酸序列和活性谱上有所不同，表明它们通过不同的结合模式与受体相互作用。比较分析和遗传研究鉴定了 Man-PTS 亚基 IID（γ+）的一个以前未被识别的细胞外环，该环与本研究中研究的细菌素对 L. garvieae 的敏感性有关。此外，还指定了位于亚基 IIC 的糖通道形成跨膜部分或 IID 的细胞外部分的单个氨基酸，这些氨基酸可能与每种细菌素的相互作用有关。最后，基于模板的 Man-PTS 亚基 IIC 和 IID 的 3D 模型被构建，以允许更深入地了解 Man-PTS 的结构和功能。
