Proteome- and Metabolome Research, Faculty of Biology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
Jungbunzlauer Austria AG, Pernhofen 1, 2064 Wulzeshofen, Austria.
J Proteomics. 2022 Apr 15;257:104513. doi: 10.1016/j.jprot.2022.104513. Epub 2022 Feb 8.
Xanthomonas is a phytopathogenic bacterium and of industrial interest due to its capability to produce xanthan, used as a thickener and emulsifier in the food and non-food industry. Until now, proteome analyses of Xcc lacking a detailed view on the proteins involved in xanthan biosynthesis. The proteins involved in the biosynthesis of this polysaccharide are located near, in or at the cell membrane. This study aims to establish a robust and rapid protocol for a comprehensive proteome analysis of Xcc strains, without the need to isolate different cell fractions. Therefore, a method for the analysis of the whole cell proteome was compared to the isolation of specific fractions regarding the total number of identified proteins, the overlap, and the differences between the approaches. The whole cell proteome analysis with extended peptide separation methods resulted in more than 3254 identified proteins covering 73.1% of the whole proteome. The protocol was used to study xanthan production in a label-free quantification approach. Expression profiles of 8 Gum proteins were compared between the stationary and logarithmic growth phase. Differential expression levels within the operon structure indicate a complex regulatory mechanism for xanthan biosynthesis. Data are available via ProteomeXchange with identifier PXD027261. SIGNIFICANCE: Bacteria are metabolite factories with a wide variety of natural products. Thus, proteome analyses play a crucial role to understand the biological processes within a cell behind the biosynthesis of those metabolites. Proteins involved in the biosynthesis of secreted products are often organised on, in or around the membrane allowing metabolite channelling. Experiments targeting those biosynthesis pathways on protein level often require the analysis of multiple cell fractions like cytosolic, inner, and outer membrane. This is time consuming and demands different protocols. The protocol presented here is a rapid and robust solution to study biosynthetic pathways of biological or biotechnological interest in a single approach on protein level, where gene products are partitioned across multiple cell fractions. The use of a single method also simplifies the comparison of different experiments, for example, production vs. nonproduction conditions.
黄单胞菌是一种植物病原菌，由于其能够生产黄原胶而具有工业价值，黄原胶被用作食品和非食品工业中的增稠剂和乳化剂。到目前为止，对缺乏详细的黄单胞菌生物合成 xanthan 相关蛋白视图的 Xcc 进行了蛋白质组分析。该多糖生物合成涉及的蛋白质位于细胞膜附近、内部或表面。本研究旨在建立一种快速而稳健的 Xcc 菌株全细胞蛋白质组分析方法，而无需分离不同的细胞组分。因此，比较了全细胞蛋白质组分析与特定组分分离方法，以比较鉴定蛋白的总数、重叠度以及两种方法的差异。采用扩展肽分离方法进行全细胞蛋白质组分析，鉴定到的蛋白超过 3254 种，覆盖整个蛋白质组的 73.1%。该方案用于在无标记定量方法中研究 xanthan 的生产。在静止和对数生长期比较了 8 种 Gum 蛋白的表达谱。操纵子结构内的差异表达水平表明 xanthan 生物合成的复杂调控机制。数据通过 ProteomeXchange 以标识符 PXD027261 提供。意义：细菌是具有多种天然产物的代谢工厂。因此，蛋白质组分析在理解细胞内生物合成这些代谢物背后的生物学过程中起着至关重要的作用。分泌产物生物合成涉及的蛋白通常位于膜上、内部或周围，允许代谢物通道化。针对那些生物合成途径的蛋白水平实验通常需要分析多个细胞组分，如细胞质、内膜和外膜。这既耗时又需要不同的方案。本文提出的方案是一种快速而稳健的解决方案，可在单个蛋白水平上研究生物或生物技术感兴趣的生物合成途径，其中基因产物分配到多个细胞组分中。单一方法的使用还简化了不同实验的比较，例如，生产与非生产条件的比较。
