Organisation and mechanism of bacterial twin arginine translocases

The bacterial Tat pathway facilitates the translocation of pre-folded proteins over the cytoplasmic membrane. In Gram-negative bacteria, TatA, TatB and TatC (each an integral membrane protein) are the essential components. Most of our understanding of Tat function in bacteria has come from studies on Escherichia coli, a Gramnegative bacterium. Gram-positive bacteria have Tat systems that are composed of just a single TatA and TatC protein. The absence of TatB suggests a different organisation and translocation mechanism to the Tat systems of Gram-negative bacteria. Here the Tat pathway of Bacillus subtilis, a Gram-positive bacterium, was analysed in detail for the first time revealing important structural differences to the E. coli Tat pathway. Complementation experiments reveal the Tat pathway of B. subtilis is active in E. coli, pointing to functional conservation between Gram-negative and Gram-positive bacteria. The complexes formed by TatA and TatC in B. subtilis were investigated. TatA and TatC form a tight complex that is significantly smaller than its E. coli TatABC counterpart, possibly reflecting the presence of a different number of TatA and/or TatC units within this complex. TatA in B. subtilis like in E. coli also forms homooligomeric complexes separately from TatC. Unlike E. coli TatA complexes that vary enormously in size, the TatA complexes of Gram-positive bacteria are small and homogeneous in nature, suggesting an entirely different translocation mechanism involving a single defined translocon rather than a spectrum of size variants as proposed for E. coli. The TatA proteins from Gram-positive bacteria may be bifunctional and perform the roles of E. coli TatA and TatB. Here the first direct evidence to support this hypothesis is presented and domains important for both TatA and TatB roles identified. Finally a soluble population of TatA identified in B. subtilis was analysed and evidence is presented that suggests it maybe mis-localised