Surface attachment is the crucial first step for a single cell transitions from a planktonic to a surface
associated state, which can lead to the development of multicellular communities called biofilms.
Microbes extensively use pili for initial surface attachment. Pili are filamentous appendages that
extend into the extracellular environment and can attach to a wide range of surfaces. This Thesis
contributes to the understanding of how pili work and how bacteria transition from a planktonic to
a surface bound life style. This will aid future development in creating new ways to prevent bacterial
attachment and biofilm formation and thereby avoid the necessity for the removal of fully developed
biofilms which often requires harsh physical and chemical treatments which can be impractical in a
biomedical context.
We used single cell studies, microfluidic methods and quantitative computational analysis to study
in detail the mechanism of pili-mediated attachment in Caulobacter crescentus and Pseudomonas
aeruginosa. In C. crescentus we confirm the recently described ability of pili to retract, which was
previously considered not possible for this type of pili. We characterized this functionality in greater
detail and our results highlight the importance of pili in reorienting cells and bringing the cell body
closer to surfaces, whereby cells can promote long term attachment by secreting a glue-like substance
called holdfast. We also investigated the role of the second messenger c-di-GMP during pilimediated cell attachment and biofilm formation. We show a novel role for c-di-GMP in directly
regulating elongation and retraction of pili in C. crescentus and P. aeruginosa. In P aeruginosa a novel
c-di-GMP effector, FimW, regulates surface attachment and walking behaviour, and how its
asymmetric distribution drives surface colonization. In C. crescentus we show that c-di-GMP
positively regulates attachment. We manipulated a key component of the secretion machinery, HfsK,
and show that c-di-GMP not only regulates the timing of holdfast synthesis, but also its cohesion
and adhesion properties. Lastly, we report a novel protein, PdeL, which is both a phosphodiesterase
and a transcriptional factor that regulates the expression of biofilm related genes in Escherichia coli.
In the appendixes we describe in detail the process for creating microfluidic devices, extensively
used in the studies described in this thesis. Moreover, we include a manual for the use of WHISIT, a
custom-made software program for the analysis of bacterial fluorescent signals in an automated and
quantitative approach
