20161202-Stubenrauch-Thesis.pdf (46.15 MB)
Download fileThe assembly of fimbrial ushers, inverse autotransporters and LptDE into bacterial outer membranes
thesis
posted on 2016-12-07, 22:33 authored by Christopher James StubenrauchThe
translocation and assembly module (TAM) is capable of assembling outer membrane
proteins, but its function has remained enigmatic in that: (i) only the
AIDA-like autotransporter family had been shown to require the TAM, and (ii)
outer membrane protein biogenesis is generally considered to be catalysed only
by the β-barrel assembly machinery (BAM) complex. To assess whether the TAM
plays a broader role in β-barrel assembly, I developed a pulse chase assay to
characterise the specific contributions of the TAM and the BAM complex for a
range of outer membrane protein substrates.
Working on the hypothesis that the TAM assembles more complicated outer membrane proteins that diverge from the classical β-barrel architecture, five candidate TAM substrates were analysed for their requirement of the TAM: FimD (a fimbrial usher), intimin (an inverse autotransporter), TolC, PhoE, and LptDE. While the PhoE data was inconclusive, the four remaining candidate substrates were found to have varying requirements for the TAM. Furthermore, six related fimbrial ushers and a second inverse autotransporter were also confirmed to require the TAM for efficient biogenesis.
FimD contains an unusual extracellular loop that is readily proteolytically degraded on addition of exogenous proteinase K. Because distinct proteolytic fragmentation "fingerprints" were generated depending on the presence or absence of the TAM, this provided a tool to dissect the contribution of the TAM toward FimD biogenesis. The TAM was subsequently found to initiate β-barrel assembly from the C-terminal end of the substrate, and while the BAM complex was also capable of assembling FimD, it was significantly less efficient because it initiated β-barrel assembly from a central region of the protein. This data was interpreted to mean that the BAM complex is subsidiary to the TAM during fimbrial usher biogenesis.
In contrast, although assembly of the LptDE complex was also shown to be assisted by the TAM, considering both the BAM and LptDE complexes are essential (but the TAM is not), the TAM likely plays a minor role in LptDE assembly that is subsidiary to the BAM complex. As such, the hypothesis that only the BAM complex is necessary for β-barrel biogenesis should be extended to include the contribution that TAM makes - depending on the substrate - as either the major or minor outer membrane protein insertase.
Working on the hypothesis that the TAM assembles more complicated outer membrane proteins that diverge from the classical β-barrel architecture, five candidate TAM substrates were analysed for their requirement of the TAM: FimD (a fimbrial usher), intimin (an inverse autotransporter), TolC, PhoE, and LptDE. While the PhoE data was inconclusive, the four remaining candidate substrates were found to have varying requirements for the TAM. Furthermore, six related fimbrial ushers and a second inverse autotransporter were also confirmed to require the TAM for efficient biogenesis.
FimD contains an unusual extracellular loop that is readily proteolytically degraded on addition of exogenous proteinase K. Because distinct proteolytic fragmentation "fingerprints" were generated depending on the presence or absence of the TAM, this provided a tool to dissect the contribution of the TAM toward FimD biogenesis. The TAM was subsequently found to initiate β-barrel assembly from the C-terminal end of the substrate, and while the BAM complex was also capable of assembling FimD, it was significantly less efficient because it initiated β-barrel assembly from a central region of the protein. This data was interpreted to mean that the BAM complex is subsidiary to the TAM during fimbrial usher biogenesis.
In contrast, although assembly of the LptDE complex was also shown to be assisted by the TAM, considering both the BAM and LptDE complexes are essential (but the TAM is not), the TAM likely plays a minor role in LptDE assembly that is subsidiary to the BAM complex. As such, the hypothesis that only the BAM complex is necessary for β-barrel biogenesis should be extended to include the contribution that TAM makes - depending on the substrate - as either the major or minor outer membrane protein insertase.
