The role of protein :protein interactions in regulating flagellar assembly

Abstract

The number of flagella in Salmonella enterica serovar Typhimurium cells is closely related with the activity of FlhD4C2, a transcriptional regulator that controls the synthesis of a flagellum. In multiflagelated cells of S. enterica, FlhD4C2 activity is negatively controlled by the Type 3 Secretion chaperone, FliT. FliT interacts with the flagellar filament cap protein FliD. FliD is thus indicated as an anti-regulator in the same aspect describing FlgM inhibition of σ28 activity. Protein interactions between FliT, FliD and FlhD4C2 were explored in detail. Two independent studies, Native gel electrophoresis and gel filtration, revealed the stoichiometry of FliT:FliD interaction to be 1 to 1. In addition, Bacterial Two Hybrid (B2H) analysis showed FliT interacting with FliD In vivo. FliT was also shown to interact with FlhD4C2 by destroying the FlhD4C2 complex. FlhD4C2 activity reduced in the presence of FliT but not all FlhD4C2 activity was inhibited. Biochemical analysis of the interaction between FlhD4C2 and DNA showed that FlhD4C2 bound to DNA was insensitive to FliT regulation. Using a FliT variant, FliT94, that had the last α-helix deleted, helped show that the binding site of FliT to both FliD and FlhD4C2 possibly overlap. The negative regulation of FliT is proposed to be a flagellar specific regulatory mechanism that effectively decreases FlhD4C2 activity to a minimum level rather than inhibiting all flagellar production. FliD counteracts FliT activity to increase FlhD4C2 activity by forming the FliT:FliD complex. Importantly, FlhD4C2 bound to DNA is insensitive to FliT regulation and permits a low number of cells in a population to build a low number of flagella, even during the action of FliT. This study highlighted the importance of including the regulatory protein:DNA interaction in the working model of the regulatory circuit. This model unveils a mechanism to allow bacterial cells to remain prepared to respond quickly and efficiently to the decision to control the activity of flagellation to multiple signals generated by the external environment, internal cues and flagellar specific feedback mechanisms.