Assembly and mechanism of bacterial twin-arginine translocation systems
Baglieri, J. (2012) Assembly and mechanism of bacterial twin-arginine translocation systems. PhD thesis, University of Warwick.
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The bacterial twin-arginine translocation (Tat) pathway is able to export pre-folded
cofactor containing proteins across the cytoplasmic membrane. Tat substrates bear
cleavable N-terminal signal peptides that are characterized by the presence of a
critical and highly conserved twin-arginine motif which lends the Tat pathway its
In Escherichia coli and many other Gram-negative bacteria, three integral membrane
proteins: TatA, TatB and TatC are essential for Tat-dependent translocation. In
contrast Bacillus subtilis possesses a simpler TatAC system which lacks the TatB
component. In E. coli the TatA protein assembles into homo-oligomeric complexes
that vary considerably in size. The TatA proteins found in B. subtilis do not exhibit
the same degree of heterogeneity and this suggested mechanistic differences between
the Tat pathways of Gram-negative and Gram-positive bacteria. How the Tat system
works is still poorly understood, and the work presented in this thesis sought to gain
insights into the assembly and mechanism of E. coli and B. subtilis Tat pathways.
This work focused on the study of two previously uncharacterized components: the
E. coli TatA paralog TatE subunit and B. subtilis TatAc subunit.
In this thesis the purification and characterization of E. coli TatE complexes is
reported. Using analytical gel filtration chromatography, blue-native gelelectrophoresis
(BN-PAGE) and single-particle analysis of purified TatE complexes,
it was found that the TatE complexes are more discrete than the highly
heterogeneous TatA complexes. This finding, together with the ability of TatE to
support the translocation of the 90-kDa TorA protein, suggested alternative
translocation models in which single TatE complexes do not contribute the bulk of
the translocation channel, similar to the B. subtilis model.
In addition, co-purification and BN-PAGE experiments demonstrated for the first
time that TatE interacts with TatA to form TatAE mixed complexes in the
membrane, and reveals a completely novel form of Tat complex that might be
A soluble population of TatE was also identified in E. coli cell extracts, and phase
separation experiments using Triton X-114 suggested it may be mis-localized.
In a separate set of studies, the ability of the B. subtilis TatAc protein to form active
translocases in combination with the B. subtilis TatCd or TatCy proteins was
investigated for the first time. The TatAcCd and TatAcCy mixed translocases were
able to translocate several E. coli Tat substrates including, TorA, AmiA and AmiC.
Finally BN-PAGE and gel filtration chromatography showed that the TatAcCd and
TatAcCy complexes were significantly smaller than the previously described E. coli
TatABC substrate-binding complex.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||Translocation (Genetics), Escherichia coli -- Physiology, Bacillus subtilis -- Physiology|
|Official Date:||July 2012|
|Institution:||University of Warwick|
|Theses Department:||School of Life Sciences|
|Supervisor(s)/Advisor:||Robinson, Colin, 1958-|
|Sponsors:||Seventh Framework Programme (European Commission) (FP7/2007-2013)|
|Extent:||xvii, 167,  leaves : ill.|
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