Expression and activation control of the T6SS4 machinery in Yersinia pseudotuberculosis

The type VI secretion system (T6SS) is a contact dependent protein delivery system of Gram-negative bacteria to intoxicate prokaryotic or eukaryotic cells. The enteropathogen Yersinia pseudotuberculosis (Ypstb) encodes four complete clusters (T6SS1-4) with potentially different functions controlled by intricate, independent regulatory pathways. Under laboratory conditions, only T6SS4 is mildly expressed which makes thorough analysis of each system difficult. The recent identification of the T6SS4 activator RovC allows a more detailed investigation of the regulation of this cluster.

Flow cytometry was used to analyse T6SS4 expression on a single cell level. We observed heterogenous expression of T6SS4 (T6SS4+ and T6SS4- subpopulations) within a Ypstb population, which strongly depends on temperature and growth phase. Heterogeneous expression is not only limited to T6SS4, as also rovC is heterogeneously expressed, indicating an unknown additional transcriptional regulator upstream of rovC. Although all T6SS4 genes are under the control of the same promoter, qRT-PCR revealed different amounts of gene transcripts within the cluster, suggesting additional post-transcriptional pathways. However, expression of T6SS4 compounds alone does not result in a firing event. Analysis with fluorescence microscopy could show that several additional triggers such as high cell density and osmotic pressure are required to activate the T6SS4.

Our preliminary data show a tight regulation on transcriptional, post-transcriptional and post-translational levels, that differ from already known pathways in other organisms. However, as little is known about the T6SSs of Ypstb so far, further research is required to understand their function and the purpose of this heterogeneous expression.