Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16286-90. doi: 10.1073/pnas.1008368107. Epub 2010 Aug 30.
During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC(50) to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.
在细菌自噬过程中，一个分化的亚群从其遗传上相同的兄弟姐妹中收获营养物质，以允许在营养有限的条件下继续生长。本研究采用假设驱动的成像质谱（IMS）来鉴定枯草芽孢杆菌自噬系统中活性代谢物，该系统中，产芽孢细胞裂解非产芽孢的兄弟姐妹。分别鉴定出与提议的自噬因子孢子杀伤因子（SKF）和孢子延迟蛋白（SDP）产物序列匹配的两个候选分子，确定了最终产物的结构。SKF 是一种 26 个氨基酸（aa）的环状肽，经过翻译后修饰，一个二硫键和一个半胱氨酸硫醚桥接到一个甲硫氨酸的α-位，这是一种以前在生物学中未描述过的翻译后修饰。SDP 是一个 42 个残基的肽，具有一个二硫键。在固体培养基上的点测试中，过量表达的 SKF 和 SDP 具有杀菌作用，SDP 的杀菌效果更高。然而，只有纯化的 SDP 会影响液体培养基中的枯草芽孢杆菌细胞，在荧光显微镜和生长测定中。具体而言，SDP 以浓度依赖的方式延迟生长，导致细胞通透性增加，最终导致细胞裂解，并伴有膜管和球体的产生。同样，SDP 但不是 SKF 能够抑制金黄色葡萄球菌和表皮葡萄球菌的生长，其 IC50 与万古霉素相当。这项研究，通过鉴定 SKF 和 SDP 的结构，突出了 IMS 在微生物群落代谢交换研究中的优势，也表明 IMS 是发现新型生物活性分子的一种很有前途的方法。
