Defining the molecular basis of dynamic localization of type VI secretion system assembly in "Pseudomonas aeruginosa"

Interbacterial competition is a highly resource-intensive and physiologically complex undertaking. Consequently, gene regulation and the functions of large macromolecular complexes are often driven by environmental signals. One particularly crucial complex for inter-species competition in Gramnegative bacteria is the type VI secretion system (T6SS), commonly used to deliver toxins across bacterial membranes into other cells. The expression of type VI secretion system (T6SS) assemblies varies across species and is commonly regulated on a transcriptional and post-translational level. Studies in different model organisms have found a variety of phenotypes associated with T6SS assembly.
Pseudomonas aeruginosa, encodes three separate T6SS on distinct loci. The main anti-bacterial T6SS, H1-T6SS, is tightly regulated on a post-translational level and is assembled in response to outer membrane damage, producing a characteristic “tit-for-tat” phenotype wherein adjacent cells appear to be dueling with their T6SS. This response was previously shown to depend on a threonine phosphorylation pathway (TPP) consisting of a Thr kinase/phosphatase pair (PpkA/PppA) as well as the proteins TagQ/TagR/TagS and TagT, which act upstream of kinase activation. TagQ is an outer membrane (OM) lipoprotein that sequesters TagR to the OM, while TagR has been shown to promote PpkA activity. TagST forms an ATP-binding cassette (ABC)-transporter like complex that has some ATPase activity in vitro but does not seem to affect the localization of either TagQ or TagR. In particular, it remains unknown if any of these proteins interact directly or form complexes either before or during signal transduction. Moreover, the mechanism by which a signal is passed on to PpkA has remained elusive.
In this thesis, I used a combination of biochemical, structural and microscopy approaches to describe a mechanism for activation of H1-T6SS assembly by membrane damage. I set out first to characterize the function of TagQ, which I found to form a 1:2 complex with TagR in vivo using coimmunoprecipitation (Co-IP) approaches valdiated by mass spectrometry (MS) and mass photometry. Analysis of TagR by mass photometry revealed that it can form a dimer in the absence of TagQ but only at supraphysiological concentrations. Subsequently, I found that TagQR complex formation is induced upon membrane damage using several different methods of inducing damage, validated by live cell microscopy. Following up on previously reported roles of TagR in stimulating PpkA activity, I wondered if TagQ could potentially be involved as well through its binding to TagR. Strikingly, I was able to detect a supercomplex formed by TagQR binding directly to PpkA using Co-IP and in vitro pulldowns. Using in silico modeling approaches supported by live cell imaging of mutants, I was able to show a potential mechanism for PpkA dimerization by the TagQR complex. Similarly, I was able to shed light on the role of TagS/T by studying its own interactions by in vitro and in vivo approaches. Finally, I was curious to see whether PpkA would have any phosphorylation targets that could produce the gene regulation effects seen in other envelope stress responses. Using MS and Co-IP approaches I found that the TagQRST-PpkA pathway is exclusive to H1-T6SS activation, making it a truly unique membrane damage response in bacteria.