Periodic Reporting for period 1 - CMDNAUP (Composition and Mechanism of the DNA-uptake Pilus of Vibrio cholerae)
Reporting period: 2016-09-01 to 2018-08-31
Horizontal gene transfer (HGT), the ability to acquire novel genes from other organisms, can facilitate rapid bacterial evolution including the spread of genes encoding antibiotic resistance and virulence factors. One widely used form of HGT is natural competence for transformation, which allows bacteria to take up DNA from the environment and maintain it in a heritable state. In the Gram-negative bacterium Vibrio cholerae, which is an important human pathogen that causes the pandemic disease Cholera, natural competence is activated during growth on chitinous surfaces, which are ubiquitous in the aquatic environment. To transport DNA into the cell, competent bacteria use type IV pili, a widespread and versatile class of cell surface polymers. Recent work from our laboratory established that V. cholerae produces a bona fide DNA-uptake pilus composed of the major pilin subunit PilA, and that DNA-uptake requires the combined action of this pilus and a periplasmic DNA-binding protein called ComEA. These results were consistent with the long-standing model in which the pilus retracts to bring DNA into the periplasm. However, although well supported by genetic evidence this process had never been visualised in action. Therefore the main objective of this action was to visualise the DNA-uptake pilus in live cells and investigate its dynamics.
In this work we developed new tools to visualise the DNA-uptake pilus using a cysteine labelling approach. Using this method we show that DNA-uptake pili are highly dynamic and that they retract prior to DNA-uptake. On average competent cells produce 1-2 pili per min per cell and within this timeframe about 2/3 cells produce a pilus. Moreover, we confirmed that these dynamics are dependent on the retraction ATPase, PilT. Indeed, in the absence of PilT, cells produce multiple static pili. Unexpectedly, these hyper-piliated cells have the ability to auto-aggregate via direct pilus-pilus interaction, and in liquid culture form macroscopic aggregates that rapidly sediment. In pandemic strains, which all produce an identical PilA, the aggregation phenotype is conserved. However, we discovered that extensive strain-to-strain variability in PilA, present in environmental strains, controls the ability of pili to interact but does not affect their ability to mediate transformation. Remarkably, the variability between different PilA subunits creates highly specific interactions that allow pili to distinguish between one another and thus, specifically interact with pili composed of the same PilA subunit. Finally, we went on to show that during growth on chitinous surfaces cells exist within large interconnecting networks of pili, suggesting that these interactions likely function during surface colonisation.
The demonstration here that DNA-uptake pili are dynamic provides direct evidence for the long-standing retraction hypothesis of how type IV pili function in natural transformation. Moreover, the cysteine labelling approach we have used will likely be useful in studying other aspects of DNA-uptake pilus assembly, such as the contribution of various accessory proteins and how its assembly dynamics are regulated. The most important discovery of this work, however, is that sequence variability between the major subunit of a type IV pilus enables pili to distinguish between one another. Indeed, the ability of pili to ‘recognise’ one another immediately suggests that it could function as a mechanism for bacterial kin recognition. The results generated by this action will therefore likely be of interest not only to researchers interested in natural transformation and type IV pili but also to the wider community and especially researchers interested in how bacteria physically sense and interact with their surroundings.