Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA.
Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, USA.
J Bacteriol. 2019 Feb 25;201(6). doi: 10.1128/JB.00662-18. Print 2019 Mar 15.
Bacterial endospores produced by and species can remain dormant and highly resistant to environmental insults for long periods, but they can also rapidly germinate in response to a nutrient-rich environment. Multiple proteins involved in sensing and responding to nutrient germinants, initiating solute and water transport, and accomplishing spore wall degradation are associated with the membrane surrounding the spore core. In order to more fully catalog proteins that may be involved in spore germination, as well as to identify protein changes taking place during germination, unbiased proteomic analyses of membrane preparations isolated from dormant and germinated spores of and were undertaken. Membrane-associated proteins were fractionated by SDS-PAGE, gel slices were trypsin digested, and extracted peptides were fractionated by liquid chromatography and analyzed by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. More than 500 proteins were identified from each preparation. Bioinformatic methods were used to characterize proteins with regard to membrane association, cellular function, and conservation across species. Numerous proteins not previously known to be spore associated, 6 in and 68 in , were identified. Relative quantitation based on spectral counting indicated that the majority of spore membrane proteins decrease in abundance during the first 20 min of germination. The spore membranes contained several proteins thought to be involved in the transport of metal ions, a process that plays a major role in spore formation and germination. Analyses of mutant strains lacking these transport proteins implicated YloB in the accumulation of calcium within the developing forespore. Bacterial endospores can remain dormant and highly resistant to environmental insults for long periods but can also rapidly germinate in response to a nutrient-rich environment. The persistence and subsequent germination of spores contribute to their colonization of new environments and to the spread of certain diseases. Proteins of and were identified that are associated with the spore membrane, a position that can allow them to contribute to germination. A set of identified proteins that are predicted to carry out ion transport were examined for their contributions to spore formation, stability, and germination. Greater knowledge of spore formation and germination can contribute to the development of better decontamination strategies.
和 物种产生的细菌芽孢可以在很长一段时间内处于休眠状态，并对环境刺激具有高度抗性，但它们也可以迅速在营养丰富的环境中发芽。参与感应和响应营养发芽剂、启动溶质和水运输以及完成孢子壁降解的多种蛋白质与包围孢子核心的膜相关联。为了更全面地编目可能参与孢子发芽的蛋白质，并确定发芽过程中发生的蛋白质变化，对来自 和 休眠和发芽孢子的膜制剂进行了无偏蛋白质组学分析。通过 SDS-PAGE 分离膜相关蛋白，凝胶切片用胰蛋白酶消化，提取的肽通过液相色谱分离，并用基质辅助激光解吸电离串联飞行时间质谱分析。从每个制剂中鉴定出超过 500 种蛋白质。使用生物信息学方法对与膜结合、细胞功能和跨物种保守性相关的蛋白质进行了特征描述。鉴定出许多以前未知的与孢子相关的蛋白质， 中有 6 种， 中有 68 种。基于光谱计数的相对定量表明，大多数孢子膜蛋白在发芽的前 20 分钟内丰度降低。孢子膜含有几种被认为参与金属离子运输的蛋白质，这一过程在孢子形成和发芽中起着主要作用。缺乏这些运输蛋白的突变体分析表明，YloB 参与了发育中的前孢子内钙的积累。细菌芽孢可以在很长一段时间内处于休眠状态，并对环境刺激具有高度抗性，但也可以在营养丰富的环境中迅速发芽。孢子的持久性和随后的发芽有助于它们在新环境中的定植，并有助于某些疾病的传播。鉴定出与孢子膜相关的 和 蛋白，它们的位置可以使它们有助于发芽。一组被预测进行离子转运的鉴定蛋白被检查其对孢子形成、稳定性和发芽的贡献。对孢子形成和发芽的更多了解可以有助于开发更好的消毒策略。
