Function of the type-III secretion system ATPase complex in protein export of the bacterial flagellum

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The bacterial flagellum and the evolutionarily related injectisome of many Gram-negative pathogens utilize a conserved type-III secretion system (T3SS) for the export of substrate proteins. The T3SS consists of the export gate embedded in the basal body and a soluble ATPase complex (FliH, FliI and FliJ in case of the flagellum). An unique feature of T3SS is its ability to switch substrate selectivity from early-type to late-type substrates that is instrumental for proper flagellum assembly. The ATPase complex presumably contributes to targeting of chaperone-substrate complexes and unfolding of substrate proteins, yet, the T3SS retains some degree of function also in ATPase-deficient mutants. Further, it recently has been shown that the second messenger cyclic di-GMP (cdG) binds to the ATPase FliI, suggesting a possible novel role of the second messenger during flagellar assembly. Accordingly, the physiological roles of cdG binding and ATP hydrolysis via the ATPase FliI during the T3SS protein secretion process remain elusive.Based on preliminary results, we hypothesize that cdG binding modulates the oligomerization state and therefore ATPase activity of FliI, which in turn regulates the substrate specificity switch of the flagellar T3SS from early-type to late-type substrates. In this project, we aim to clarify the role of ATP hydrolysis and cdG binding to the ATPase complex during the protein secretion process. Specifically, we want to investigate: (i) how cdG binding modulates the ATPase activity of FliI and thereby regulates the substrate specificity; (ii) if and how FliI contributes to secretion substrate unfolding and chaperone-substrate release and; (iii) how the ATPase function and cdG-binding modulates the sub-cellular localization and oligomerization of FliI.The various questions will be addressed in a comparative approach by analyzing protein secretion, ATPase oligomerization and sub-cellular localization of wildtype FliI and mutants deficient in ATP hydrolysis and/or putative cdG binding using a combination of genetics and biochemical approaches, as well as single-molecule/superresolution microscopy techniques.In summary, we expect that these experiments will establish the physiological relevance of ATP hydrolysis and the putative cdG-binding for the function of FliI and investigate several potential mechanisms how cdG binding might regulate protein export via T3SS.