Inspired by Tiago’s 2016 study that mapped the atomic structure of the conjugative F-pilus1, I was “infected” (pun intended) with fascination in this peculiar nanomachine. Since I joined his lab, my idea of bacterial conjugation – a process whereby two bacteria exchange DNA horizontally through a conjugative pilus - changed drastically. During early years of molecular biology, F. Jacob and E. Wollman developed the “Interrupted Mating Experiment”, whereby bacterial conjugation was prematurely aborted by agitation at different timepoints, resulting in a partial transfer of DNA2. This technique allowed for generating first genomic maps of the E.coli chromosome, but resulted in a wide-spread assumption that conjugation is a fragile process, prone to agitation of the environment.
It was therefore incredibly puzzling why the F-plasmids (which encode the conjugative F-pili) are so vastly widespread in gastrointestinal tracts of humans and animals – which are undeniably turbulent environments3. There must be something that distinguishes the F-pilus from other conjugative pili, that allows it to thrive in such tempestuous environments. This question formulated the exciting project that aimed to assess the dynamics of bacterial connections created by the F-pilus, and ultimately understand the biomechanical potential of this polymer. Now, we are delighted to share our unexpected but impactful results in the paper "The F-pilus biomechanical adaptability accelerates conjugative dissemination of antimicrobial resistance and biofilm formation".
Hostile territory - the perfect territory
Since the first discovery of bacterial conjugation in 19464, the F-pilus has become the paradigm for studying this phenomenon. Nowadays, it is well-established that the F-pilus is a peculiar example of conjugative pili as it can operate in liquid environments due to its flexible nature and thin morphology. These properties distinguish it from thick and rigid pili, which are capable of conjugating exclusively on solid surfaces 5. Following this idea, we tested how exactly the F-pilus operates in turbulent environments and compared conjugation efficiencies between liquid-steady and liquid-agitated conditions (Fig. 1A). It became clear that the conjugation efficiency was not decreased by agitation (Fig. 1B). In fact, it was significantly enhanced. Moreover, even the high-speed vortexing was not enough to disrupt interbacterial connections. It seems that once the F-pilus recognises a mating opportunity, the two bacteria remain fixed together, and DNA transfer proceeds successfully despite agitation of the environment.
We hypothesized that such stable connections could form ignition points for biofilm expansion. By referring to the pioneering study by Jean-Marc Ghigo in 2001, which first documented the involvement of the F-pili in biofilm formation6 – we decided to test differences in biofilm formation across the previously described steady versus agitated conditions. The results were consistent with conjugation experiments – biofilm mass was visibly greater after agitation (Fig. 1C). Furthermore, we visualised formed biofilms with electron microscopy, which revealed a tightly packed network of bacteria connected by F-pili (Fig. 1D). Operation of the F-pili in agitated environments clearly accelerated expansion of a protective biofilm.
Not-so-fragile sex machine
Having observed this worrying trend, we were under the obligation to understand in detail the adaptations of the F-pilus which allow for such high efficiency of conjugation and biofilm formation. Magnus Andersson’s lab expertise on measuring mechanical properties of polymers was exactly what allowed us to document biomechanical properties of the F-pilus. We displayed - with a biophysical precision - how stretchy, bendy, and sturdy the F-pilus is. By a setup where an immobilised bacterium is pulled by its F-pilus with atomic tweezers (Fig. 2A), we observed a spring-like behaviour of the F-pilus, comparable to that of the highly flexible type IV pili. After multiple rounds of stretching and relaxing, there was no visible exhaustion of the filament - meaning that the F-pilus has adapted to continuous dynamic movements, important for its operation in turbulent environments (Fig. 2B). Notably, even the highest pulling forces in our setup were unable to – even partially - dislocate the F-pili from the surface of the bacterium, underlining its robustness
Role of phospholipids revealed
An interesting property of the F-pilus structure is the presence of phospholipid moieties threaded between F-pilin subunits. These are conserved among conjugative pili – including in those of Archea7 - however their role remained unknown – mainly because it is impossible to remove them in vivo and study the change in F-pilus behaviour. One appealing hypothesis is that presence of phospholipids stabilises the molecular architecture of conjugative pili, therefore increasing mechanical (and potentially thermochemical) robustness. We resolved to steered molecular dynamics simulations (sMD) conducted by Joseph Baker's lab. The freedom of an in silico setting allowed us to simulate a phospholipid-lacking F-pilus – a structure unobtainable in nature. Our pulling simulations show clearly its structural inferiority compared to the native F-pilus, which was significantly more robust and capable to withstand pulling (see movie below). We therefore first describe the role of phospholipid molecules in conjugative pili as a molecular glue that helps keep the pilus structure together. In nature, this is an extremely valuable adaptation that directly affects conjugation efficiency and biofilm formation.
Molecular properties, populational threat
With this paper we aim to bring more attention to the mechanisms how antibiotic resistance genes spread among bacteria, and what adaptations accelerate this process. Collectively, our work is also a great example of how cooperation between different fields of science can lead to great development in our understanding of complex biological processes on multiple levels. With molecular detail we documented the elastic and highly dynamic properties of the F-pilus, which augmented our microbiological experiments that revealed how F-pili thrive in turbulent environments. All of these answered a clinically relevant issue that affects humans on a global scale – that is – why the F-plasmids are so widespread in human and animal guts.
1. Costa, T. R. D. et al. Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex. Cell 166, 1436-1444.e1410 (2016).
2. Jacob, F. & Wollman, E. L. Sexuality and the genetics of bacteria (1961).
3. Rozwandowicz, M. et al. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. Journal of Antimicrobial Chemotherapy 73, 1121–1137 (2018).
4. Lederberg, J. & Tatum, E. L. Gene Recombination in Escherichia Coli. Nature 158, 558-558 (1946).
5. Bradley, D. E. Morphological and serological relationships of conjugative pili. Plasmid 4, 155-169 (1980).
6. Ghigo, J.-M. Natural conjugative plasmids induce bacterial biofilm development. Nature 412, 442-445 (2001).
7. Beltran, Leticia C et al. Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery. Nature communications vol. 14,1 666. 2023