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Architecture of the type IVa pilus machine.

ABSTRACT: Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo-electron tomography of intact Myxococcus xanthus cells to visualize type IVa pili and the protein machine that assembles and retracts them (the type IVa pilus machine, or T4PM) in situ, in both the piliated and nonpiliated states, at a resolution of 3 to 4 nanometers. We found that T4PM comprises an outer membrane pore, four interconnected ring structures in the periplasm and cytoplasm, a cytoplasmic disc and dome, and a periplasmic stem. By systematically imaging mutants lacking defined T4PM proteins or with individual proteins fused to tags, we mapped the locations of all 10 T4PM core components and the minor pilins, thereby providing insights into pilus assembly, structure, and function.

SUBMITTER: Chang YW

PROVIDER: S-EPMC5929464 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Architecture of the type IVa pilus machine.

Chang Yi-Wei YW Rettberg Lee A LA Treuner-Lange Anke A Iwasa Janet J Søgaard-Andersen Lotte L Jensen Grant J GJ

Science (New York, N.Y.) 20160310 6278

Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo-electron tomography of intact Myxococcus xanthus cells to visualize type IVa pili and the protein machine that assembles and retracts them (the type IVa pilus machine, or T4PM) in situ, in both the piliated and nonpiliated states, at a resolution of 3 to 4 nanometers. We found that T4PM comprises an outer membrane pore, fou ...[more]

PMID: 26965631

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Project description:Type IVa pili (T4aP) are important for bacterial motility, adhesion, biofilm formation, and virulence. This versatility is based on their cycles of extension, adhesion, and retraction. The conserved T4aP machine (T4aPM) drives these cycles; however, the piliation pattern varies between species. To understand how these patterns are established, we focused on the T4aPM in Myxococcus xanthus that assembles following an outside-in pathway, starting with the polar incorporation of the PilQ secretin forming a multimeric T4aP conduit in the outer membrane. We demonstrate that PilQ recruitment to the nascent poles initiates during cytokinesis, but most are recruited to the new poles in the daughters after the completion of cytokinesis. This recruitment depends on the peptidoglycan-binding AMIN domains in PilQ. Moreover, the pilotin Tgl stimulates PilQ multimerization in the outer membrane, is transiently recruited to the nascent and new poles in a PilQ-dependent manner, and dissociates after the completion of secretin assembly. Altogether, our data support a model whereby PilQ polar recruitment and multimerization occur in two steps: the PilQ AMIN domains bind septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates secretin multimerization in the outer membrane. Using computational analyses, we provide evidence for a conserved mechanism of T4aPM pilotins whereby the pilotin transiently interacts with the unfolded β-lip of the secretin monomer, i.e., the region that eventually inserts into the outer membrane. Finally, we suggest that the presence/absence of AMIN domain(s) in T4aPM secretins contributes to the different T4aPM localization patterns across bacteria. IMPORTANCE Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria.

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Architecture of the type IVa pilus machine.

Publications

Architecture of the type IVa pilus machine.

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