Novel twin-arginine translocase (Tat) complexes from Gram-positive bacteria

Bacteria export many essential proteins across the plasma membrane that surrounds the cell, and the export mechanisms have attracted a great deal of interest. One reason for this interest is the complexity of the processes involved: the plasma membrane is designed to be tightly sealed and the transport of a large protein molecule is an inherently difficult process. Another factor is that these transport mechanisms represent excellent potential targets for novel anti-microbial compounds, since they are surface-exposed and there are no homologues in mammals. One protein export system of particular interest is the twin-arginine translocation (Tat) system, because this exports fully-folded proteins across this membrane by an unknown mechanism. Previous studies have focused on Gram-negative bacteria (which have 2 cell membranes) but we have recently found that the Tat system of the Gram-positive Bacillus subtilis has features that are very different in fundamental respects (yet the system still functions when expressed in a Gram-negative cell, indicating a common translocation mechanism). The aim of the work is to characterise the two Tat complexes of this organism, and determine how they come together to transport proteins. We will study the composition of the substrate-binding TatAC complex, study the structure and function of the uniquely simple TatA complex, and generate high-resolution structural data through a combination of electron microscopy and X-ray crystallography.

The Tat system transports folded proteins across the bacterial plasma membrane and plant thylakoid membrane, using a mechanism that is unique but very poorly understood. Studies on Gram-negative bacteria have shown the existence of a TatABC substrate-binding complex and a separate TatA complex that may form the translocation channel. The latter is remarkably heterogeneous, and this has been proposed to provide flexibility of pore size. Gram-positive bacteria lack a tatB gene, suggesting the presence of different Tat systems, but almost nothing has been published about the Tat systems in these organisms. We have carried out the first study of Tat complexes from a Gram-positive organism (Bacillus subtilis), after expression of the B. subtilis tatAC genes in an Escherichia coli tat mutant. The results reveal major surprises in the form of a much smaller core TatAC complex and single form of TatA complex. We propose to carry out a combined structural/mechanistic study on these complexes, in which we will: (i). Test the hypothesis that TatA variability is linked to size of translocation pore required in vivo. The uniquely homogeneous TatA complex represents a key tool for this purpose. (ii). Characterise the substrate-binding TatAC in terms of subunit stoichiometry and subunit function. (iii). Carry out single-particle electron microscopy to determine the domain organisation of the TatAC and TatA complexes. (iv). Embark on a major effort to crystallise the TatAC complex from B. subtilis or other candidate Gram-positive bacteria.