Xanthomonas campestris pv. campestris (Xcc) are Gram negative phytopathogens listed under the top five important phytopathogenic bacteria. They infect Brassicaceae and cause increasingly crop failure in agricultural. Additionally, the strain is of industrial interest due to its capability of producing the secondary metabolite xanthan which also plays an important role in phytopathology. In our study we worked with the wild type strain Xcc B100, of which the whole genome sequence was revealed in 2008 and refined 2017. Moreover, extracellular proteins as well as first analysis of the phospho proteome have been investigated. Till now a comprehensive proteome analysis of Xcc B100 is missing.
Proteome isolation was carried out using different isolation and nLC MS protocols to identify the largest possible number of proteins. With trifluoroethanol-separation the cytosolic proteome was isolated. Therefore, also a reversed phase fractionation was done. Isolation of the exoproteins was done with FASP-preparation. Additionally, the periplasmatic and membrane proteome was isolated. All samples were prepared in five biological replicates and measured on a nanoLC system coupled to a QExactive Plus. Data analysis was done with Proteome Discoverer TM.
In total more than 120 nLC MS measurements were done leading to a total number of 1,442,553 PSMs which can be associated 3,077 when minimal 2 unique peptides were used for identification. This presents 69% of whole putative proteome of Xcc B100. In this list many proteins located in clusters involved in virulence, like hrp or DFS could be identified.
With 3,077 detected proteins and therefore 69 % of the whole putative protein repertoire of Xcc B100, this work represents the most comprehensive proteome analysis in this strain to date. The identification of a variety of different proteins known to be involved in virulence could give a great groundwork for further analysis.