SummaryThe widespread role of pili as colonization factors in pathogens has long been recognized in Gramnegative bacteria and more recently in Gram-positive bacteria, making the study of these hair-like filaments a perennial hot topic for research. No other pili are found in as many or as diverse bacteria as type IV pili. This is likely a consequence of their ancient origin and unique ability to promote multiple and strikingly different phenotypes such as attachment to surfaces, aggregation, uptake of DNA during transformation, motility, etc. Two decades of investigations in several model species have shed some light on the structure of these filaments and the molecular basis of some of the properties they confer. Moreover, recent discoveries have led to a better knowledge of the genetic basis and molecular mechanisms of type IV pili biogenesis. This brings us a few steps closer to understanding how these filaments are produced, but leaves us wondering whether (as in the famous motto that inspired the title) out of the many models studied will emerge one unifying mechanism.

A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39°C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 10 6 mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.

Prokaryotes have engineered sophisticated surface nanomachines that have allowed them to colonize Earth and thrive even in extreme environments. Filamentous machineries composed of type IV pilins, which are associated with an amazing array of properties ranging from motility to electric conductance, are arguably the most widespread since distinctive proteins dedicated to their biogenesis are found in most known species of prokaryotes. Several decades of investigations, starting with type IV pili and then a variety of related systems both in bacteria and archaea, have outlined common molecular and structural bases for these nanomachines. Using type IV pili as a paradigm, we will highlight in this review common aspects and key biological differences of this group of filamentous structures.

SummaryAlthough type IV pili (Tfp) are among the commonest virulence factors in bacteria, their biogenesis by complex machineries of 12-15 proteins, and thereby their function remains poorly understood. Interestingly, some of these proteins were reported to merely antagonize the retraction of the fibres powered by PilT, because piliation could be restored in their absence by a mutation in the pilT gene. The recent identification of the 15 Pil proteins dedicated to Tfp biogenesis in Neisseria meningitidis offered us the unprecedented possibility to define their exact contribution in this process. We therefore systematically introduced a pilT mutation into the corresponding non-piliated mutants and characterized them for the rescue of Tfp and Tfp-mediated virulence phenotypes. We found that in addition to the pilin, the main constituent of Tfp, only six Pil proteins were required for the actual assembly of the fibres, because apparently normal fibres were restored in the remaining mutants. Restored fibres were surface-exposed, except in the pilQ/T mutant in which they were trapped in the periplasm, suggesting that the PilQ secretin was the sole Pil component necessary for their emergence on the surface. Importantly, although in most mutants the restored Tfp were not functional, the pilG/T and pilH/T mutants could form bacterial aggregates and adhere to human cells efficiently, suggesting that Tfp stabilization and functional maturation are two discrete steps. These findings have numerous implications for understanding Tfp biogenesis/function and provide a useful groundwork for the characterization of the precise function of each Pil protein in this process.