Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a “single-tube”

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.