Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.DOI: http://dx.doi.org/10.7554/eLife.07380.001

Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ('wide' and 'narrow'), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.

The ESCRT machinery, comprising of multiple proteins and subcomplexes, is crucial for membrane remodelling in eukaryotic cells, in processes that include ubiquitin-mediated multivesicular body formation, membrane repair, cytokinetic abscission, and virus exit from host cells. This ESCRT system appears to have simpler, ancient origins, since many archaeal species possess homologues of ESCRT-III and Vps4, the components that execute the final membrane scission reaction, where they have been shown to play roles in cytokinesis, extracellular vesicle formation and viral egress. Remarkably, metagenome assemblies of Asgard archaea, the closest known living relatives of eukaryotes, were recently shown to encode homologues of the entire cascade involved in ubiquitin-mediated membrane remodelling, including ubiquitin itself, components of the ESCRT-I and ESCRT-II subcomplexes, and ESCRT-III and Vps4. Here, we explore the phylogeny, structure, and biochemistry of Asgard homologues of the ESCRT machinery and the associated ubiquitylation system. We provide evidence for the ESCRT-I and ESCRT-II subcomplexes being involved in ubiquitin-directed recruitment of ESCRT-III, as it is in eukaryotes. Taken together, our analyses suggest a pre-eukaryotic origin for the ubiquitin-coupled ESCRT system and a likely path of ESCRT evolution via a series of gene duplication and diversification events.

Background:Secretins are key components of complex DNA and protein transport machineries. Results: An unusual secretin ␣␣␤␣␤␤␣ fold was identified as a ring-building motif essential for piliation but not for transformation. Conclusion: Type IV pilus structures are not essential for transformation in T. thermophilus. Significance: This is the first report of a ring-building domain of a unique secretin complex in T. thermophilus.

Natural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species. Thermus thermophilus HB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whether T. thermophilus has any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that in T. thermophilus, T4P and natural transformation are linked but distinct systems.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin-like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS-CoV-2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultrastable scaffold.

The thermophilic bacterium Thermus thermophilus HB27 is known for its highly efficient natural transformation system, which has become a model system to study the structure and function of DNA transporter in thermophilic bacteria. The DNA transporter is functionally linked to type IV pili (T4P), which are essential for twitching motility and adhesion to solid surfaces. However, the pilus structures themselves are dispensable for natural transformation. Here, we report that the cellular mRNA levels of the major structural subunit of the T4P, PilA4, are regulated by environmental factors. Growth of T. thermophilus in minimal medium or low temperature (55 °C) leads to a significant increase in pilA4 transcripts. In contrast, the transcript levels of the minor pilin pilA1 as well as other T4P genes are nearly unaffected. The elevated pilA4 mRNA levels are accompanied by an increase in piliation of the cells but not by elevated natural transformation frequencies. Hyperpiliation leads to increased adhesion to plastic surfaces. The increased cell-surface interactions are suggested to represent an adaptive response to temperature stress and may be advantageous for survival of T. thermophilus.

Natural transformation systems and type IV pili are linked in many naturally competent bacteria. In the Gram-negative bacterium Thermus thermophilus, a leading model organism for studies of DNA transporters in thermophilic bacteria, seven competence proteins play a dual role in both systems, whereas two competence genes, comEA and comEC, are suggested to represent unique DNA translocator proteins. Here we show that the T. thermophilus ComEA protein binds dsDNA and is anchored in the inner membrane. comEA is co-transcribed with the flanking comEC gene, and transcription of this operon is upregulated by nutrient limitation and low temperature. To our surprise, a comEC mutant was impaired in piliation. We followed this observation and uncovered that the impaired piliation of the comEC mutant is due to a transcriptional downregulation of pilA4 and the pilN both playing a dual role in piliation and natural competence. Moreover, the comEC mutation resulted in a dramatic decrease in mRNA levels of the pseudopilin gene pilA1, which is unique for the DNA transporter. We conclude that ComEC modulates transcriptional regulation of type IV pili and DNA translocator components thereby mediating a response to extracellular parameters.