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

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences


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.

Technical Summary

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.


10 25 50

publication icon
Frain KM (2017) The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity. in Biochimica et biophysica acta. Molecular cell research

Description The Tat system has been extensively studied in E. coli but the Gram positive systems show interesting differences that are porly understood. We characterised TatAC system from Bacillus subtilis and showed that the systems can still function when expressed in an E. coli background. We characterised the TatAC and TatA subcomplexes in great detail and showed that the properties of the complexes provided new insights into the mechanism by which this system transports large, fully folded proteins across the bacterial plasma membrane.
Exploitation Route Expression of the B subtilis Tat system in an E. coli tat null mutant yielded a strain with important properties: the system exports proteins to the periplasm but a leay outer membrane phenotype leads to release of the product into the culture medium. This system has been patented and is being investigated together will collaborating biotech companies
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Patent awarded, and this new platform is currently being tested by industrial groups
First Year Of Impact 2013
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

Title New E. coli strains 
Description We have developed new strains of E. coli that export proteins to the periplasm by a novel pathway 
Type Of Material Cell line 
Year Produced 2010 
Provided To Others? Yes  
Impact Several groups are testing these strains with a view to using them for production production on analytical or production scale 
Description Open day presentations 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Hosted open day visits to my lab in majority of UCAS days - 2 groups of UCAS applicants + parents per open day, at least 10 open days per year

Increased understanding of biotech projects at Kent
Year(s) Of Engagement Activity 2007,2009,2011,2012,2013,2014,2015,2016
Description School visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk sparked questions and discussion

Not applicable
Year(s) Of Engagement Activity 2010,2011,2015