ABSTRACT
Bacterial ï¬agella are extracellular ï¬laments that drive swimming in bacteria. During motor assembly, ï¬agellins are transported unfolded through the central channel in the ï¬agellum to the growing tip. Here, we applied in vivo ï¬uorescent imaging to monitor in real time the Vibrio alginolyticus polar ï¬agella growth. The ï¬agellar growth rate is found to be highly length-dependent. Initially, the ï¬agellum grows at a constant rate (50 nm/min) when shorter than 1500 nm. The growth rate decays sharply when the ï¬agellum grows longer, which decreases to ~9 nm/min at 7500 nm. We modeled ï¬agellin transport inside the channel as a one-dimensional diffusive process with an injection force at its base. When the ï¬agellum is short, its growth rate is determined by the loading speed at the base. Only when the ï¬agellum grows longer does diffusion of ï¬agellin become the rate-limiting step, dramatically reducing the growth rate. Our results shed new light on the dynamic building process of this complex extracellular structure.
Subject(s)
Flagella/physiology , Intravital Microscopy , Optical Imaging , Organelle Biogenesis , Vibrio alginolyticus/physiology , Flagellin/metabolism , Kinetics , Protein TransportABSTRACT
The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.