Insights into assembly of the type IVa pilus machine in Myxococcus xanthus
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Abstract
Type IVa pili (T4aP) are widely distributed and highly versatile bacterial cell surface structures
that function in motility, adhesion, biofilm formation and virulence. Key to their function is their
ability to undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning
T4aP machine. Assembly of the T4aP machine in Myxococcus xanthus follows an outside-in
parthway, starting with the incorporation of the PilQ secretin in the OM, which then recruits the
periplasmic, inner membrane and cytoplasmic components. Additionally, a complex composed
of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip
mediating adhesion. Here we focuse on the assembly of the bipolarly localized T4aP machine
in the rod-shaped bacterium M. xanthus.
The genome of M. xanthus encodes for three sets of minor pilins and PilY1. Here, we find that
one of these minor pilin and PilY1 gene clusters includes a noncanonical cytochrome c, which
we named TfcP. While TfcP has an unusually low redox potential that makes a function in
respiration unlikely, it is conditionally essential for T4aP dependent motility by promoting the
accumulation of PilY1.1 in the presence of low calcium concentrations. We suggest that TfcP
extends the range of calcium concentration at which PilY1.1 can function, making its function
more robust against environmental fluctuations.
Next, we focused on the assembly of new T4aP machines in the new poles of cells after cell
division. We demonstrate that PilQ starts to be recruited to the nascent poles during
cytokinesis, but mostly is recruited to the new poles after completion of cytokinesis. This
recruitment depends on the peptidoglycan-binding AMIN domains of PilQ and we propose that
this mechanism is general for AMIN domain containing secretins. Additionally, PilQ transiently
recruits the pilotin Tgl to the nascent and new poles, which then induces the multimerization
of PilQ in the outer membrane. We suggest that the transient interaction between PilQ and Tgl
is mediated by the unfolded β-lip of PilQ, the domain that is integrated into the outer membrane.
In addition, we uncover that the diguanylate cyclase DmxA is important for the symmetric
assembly of the T4aP machine at the new cell poles after cytokinesis. In the absence of DmxA,
cells exhibit a misregulated cell polarity and a very heterogeneous polar localization of the
T4aP machine, which leads to an increased reversal frequency. DmxA is recruited to the
division site by components of the divisiome shortly before the completion of cytokinesis and
rapidly elevates the cellular c-di-GMP level. We suggest that this burst of c-di-GMP regulates
the symmetric incorporation of polar landmarks at the new cell poles in both daughter cells.
Lastly, we establish a detailed protocol for the application of miniTurboID-based proximity
labeling in M. xanthus. We apply this protocol to the polarity regulator MglA, and utilize
proximity labeling to compare the conditional interactome of MglA.
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Dates
Created: 2023Issued: 2024-03-20Updated: 2024-03-20
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
DDC-Numbers
570
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Herfurth, Marco André (0000-0003-4291-0283): Insights into assembly of the type IVa pilus machine in Myxococcus xanthus. : Philipps-Universität Marburg 2024-03-20. DOI: https://doi.org/10.17192/z2023.0678.