Pili: the microbes' Swiss army knifes

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

Allosteric control of type III secretion systems by the second message cyclic-di-GMP

Cyclic di-GMP (cdG) is a ubiquitous second messenger in bacteria, regulating transcriptional and post-transcriptional processes and allosterically controlling protein function. While the mechanisms of cdG metabolism are well understood, the downstream targets of this molecule are poorly characterised. To understand the role of cdG signalling in plant-associated species, cdG-capture compound pull-down experiments were performed to identify potential binding proteins in Pseudomonas fluorescens. One of the top targets identified was the flagella export AAA+ ATPase FliI, which was shown to bind specifically and tightly to cdG. FliI-cdG interaction was demonstrated for diverse bacterial FliI homologs. Excitingly, high-affinity binding was observed for the type-III secretion system (T3SS) homolog, HrcN and the type-VI ATPase, ClpB2. A combination of techniques was used to predict the FliI cdG binding site at the interface between two FliI subunits. Although the addition of cdG inhibits the ATPase activity of both FliI and HrcN in vitro, this occurs at a non-physiological cdG concentration suggesting that this does not represent the in vivo role of binding. However, when cdG concentrations are artificially increased, the export of flagellin subunits is significantly reduced, suggesting a link between cdG binding and protein export. Changes in the in vitro multimerization state of the protein were also observed upon the addition of cdG. As part of this study, novel and highly specific tools for nucleotide-protein interactions were developed and existing biochemistry techniques were optimised. These assays were employed to characterise cdG binding to four more proteins, which were identified as cdG binders. The results generated in this study broaden the existing knowledge about cdG binding protein diversity. The identification of FliI as a cdG binder suggests a novel cdG-dependent control mechanism for the function of bacterial export pathways including the flagellum and the T3SS, through allosteric interaction with export ATPase proteins

The Effects of Retinol during In Vivo and In Vitro Oocyte Maturation and Embryonic Development.

Previous studies demonstrated that retinol administration to ewes, followed by natural service, resulted in embryos with improved competence to develop in vitro. In vivo studies with sheep and in vitro experiments with bovine embryos, were designed to evaluate the effects of retinol and to understand its mechanism(s) of action. The primary objective of the first experiment was to analyze ovine oocyte metabolism, and to assess the effects of retinol on this process. Sheep oocytes were matured in vitro over a 24 hour period in the presence of different radiolabeled substrates. Results revealed that oxidative metabolism measured by glutamine showed no significant differences over all time periods. Pyruvate oxidation was highest early in maturation and then decreased. Glycolysis was highest at the middle time period. Differences in metabolism between oocytes from retinol-treated ewes and those from control ewes were not detected. Next, varying concentrations of retinol were added either during in vitro maturation (IVM) or in vitro culture (IVC) of bovine oocyte and embryos, respectively. Our results demonstrated that 5mM retinol supplementation during IVM tended to improve embryonic development measured by the rate of blastocyst development. This concentration proved even more beneficial if the control blastocyst rate was below 20%. Furthermore, 10mM retinol appeared detrimental during IVC but not during IVM. In the third experiment, we evaluated the effects of 5mM retinol and 100mM cysteamine on bovine oocyte glutathione content. In addition, we investigated the combinatorial effects of retinol and cysteamine on in vitro bovine embryonic development. We did not observe an increase in glutathione levels in bovine oocytes treated with retinol. However, in the presence of cysteamine bovine oocytes exhibited an increase in glutathione content. Retinol and cysteamine treatment during IVM and IVC increased bovine blastocyst development, which may indicate that retinol is increasing the utilization or uptake of cysteine from the medium. Next, we evaluated the role of exogenous retinol supplementation to superovulated ewes on the glutathione content of mature oocytes collected from the oviducts. Our results did not reveal differences in glutathione content of oocytes from retinol-treated ewes versus those from control ewes. Antioxidant transcripts encoding for Mn-SOD, Cu-Zn SOD, GS, and GSTp, were detected in ovine oocytes matured in vivo. However, retinol did not appear to impact the expression levels of these transcripts