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Marine microbes support global carbon cycling by sequestration and metabolizing of marine carbon. Understanding how these microbes use unique motility modalities to navigate the physicochemical environment of the ocean is crucial to understanding microbial carbon metabolism. Motility in several marine Rhodobacter strains exhibit dendritic motility, but underlying genetic mechanisms remain poorly characterized. To lay groundwork for future study of genetic mechanisms for dendritic motility in novel Rhodobacter strains HOT5_B8 and HOT5_C3, we use timelapse microscopy to qualitatively and quantitatively characterize patterns in dendrite formation. Preliminary results determine that dendritic motility is faster than non-dendritic motility in HOT5_B8 and HOT5_C3. Further, key differences in HOT5_B8 and HOT5_C3 behaviors are used as evidence to posit putative density-dependent mechanisms in the formation and behaviors of dendrites. / 2023-09-10T00:00:00Z

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Motility and chemotaxis have been implicated in the process of biofilm formation in a wide range of species. Using a combination of microscopy and image analysis, genetics, microbiology and biochemistry, the initial approach of Rhodobacter sphaeroides cells to a solid surface has been characterised. Interestingly, these data suggest that for R. sphaeroides alterations in motility and swimming behaviour may result in differences in biofilm formation simply by changing the number of cells which reach the surface. This is in contrast to a few other well-studied species where the motility apparatus, the flagellum, has been shown to play an active role in surface sensing and the transition to biofilm growth. Tracking swimming cells and measuring surface attachment revealed that changes in motility affect the ability of cells to attach to a surface, with non-motile cells attaching least and mutants with frequent stops attaching less than smooth swimming cells with few stops. Tracking attaching cells and classifying their method of attachment revealed that flagellar tethering is not essential for R. sphaeroides attachment. Competition assays with fluorescently labelled strains showed that the initial imbalance between motile and non-motile cells remains as microcolonies develop over 48 hours,and the proportion of non-motile cells remains fairly constant. Development on a surface over 48 hours was similar for motile and non-motile strains, including aflagellate strains, once attached. Using parameters calculated by tracking swimming cells to calculate the effective diffusion coefficient in a simple model of cell movement suggested that motion alone could explain the differences in attachment without assuming different cell properties. In particular, aflagellate strains might be hindered from surface attachment by their reduced motility alone. This is interesting since some other bacterial species use the flagellum as a surface sensor.

This thesis investigates bacterial motility from the mechanism permitting individual selfpropulsion to the complex collective flocking motility in Escherichia coli and Bacillus subtilis cells. Understanding bacterial swimming has intrigued scientists for decades and recently there has been a growing interest in collective swimming behaviour. The first part of this thesis reviews the characteristics of E. coli and B. subtilis cells subsequently describing the governing physics and constraints of self-propulsion in the low Reynolds regime. The second part of this thesis presents three self-contained experimental sections, examining individual swimming in non-conventional body shaped cells and subsequently focusing on concentrated bacterial swimming in normal cells. We first investigated motility in mutant spherical E. coli cells KJB24 motivated by simulations, which often model bacteria as self-propelled spheres. Somewhat unexpectedly these spherical cells do not exhibit runs and tumbles but diffuse slower than expected. As an introduction to working with microbiology and to familiarise with microbiology techniques we investigated why these spherical cells do not swim. Secondly we investigated how cellular motility varies as a function of body length by inhibiting cell division in wild-type E. coli with cephalexin; which remained motile despite body elongation. Fluorescent flagella visualization provided evidence of multiple bundle formations along the lateral walls as a mechanism to sustain motility. The average swimming velocity, body and flagella rotation rates, the number of flagella and number of flagella bundles were extracted experimentally as a function of length. The extracted experimental parameters for normal sized cells were consistent with Purcell’s model. We explored simple adaptations and scaling of this model to describe motility for filamentous cells, which agrees with experimental values. The main focus is on collective behaviour of B. subtilis by examining the onset from individual swimming to collective motility using time-lapse microscopy. Results demonstrated a smooth transition where cells self-organize into domains expanding rapidly by recruiting cells. We present advancements in B. subtilis fluorescent flagella staining which revealed unexpected multiple flagella bundle arrangements during runs, contradictory to general conjectures. Novel visualisation of flagella filaments during reversal events is presented in both E. coli and B. subtilis cells, providing experimental evidence for complex flagella ‘flipping’. Cellular reversal is hypothesized as a mechanism for quorum polarity facilitating collective swimming. We present novel flagella imaging in the setting of collective behaviour showing evidence to support quorum polarity. Subsequently we extracted the run length distributions of cells as a function of concentration, yielding a decreasing trend with increasing concentration. Using particle tracking we quantitatively extracted the mean squared displacement of swimming cells versus passive tracers at different concentrations during collective swimming, these novel results are discussed in respect to recent simulations. These presented experiments provide new insights into collective behaviour improving current understanding of this phenomenon.

Suspensions of the aerobic bacteria {\it Bacilus subtilis} develop patterns and flows from the interplay of motility, chemotaxis and buoyancy.In sessile drops, such bioconvectively driven flows carry plumes down the slanted meniscus and concentrate cells at the drop edge, while in pendant drops such self-concentration occurs at the bottom.These dynamics are explained quantitatively by a mathematical model consisting of oxygen diffusion and consumption, chemotaxis, and viscous fluid dynamics.Concentrated regions in both geometries comprise nearly close-packed populations, forming the collective ``Zooming BioNematic'' (ZBN) phase.This state exhibits large-scale orientational coherence, analogous to the molecular alignment of nematic liquid crystals, coupled with remarkable spatial and temporal correlations of velocity and vorticity, as measured by both novel and standard applications of particle imaging velocimetry.To probe mechanisms leading to this phase, response of individual cells to steric stress was explored, finding that they can reverse swimming direction at spatial constrictions without turning the cell body.The consequences of this propensity to flip the flagella are quantified, showing that "forwards" and "backwards" motion are dynamically and morphologically indistinguishable.Finally, experiments and mathematical modeling show that complex flows driven by previously unknown bipolar flagellar arrangements are induced when {\it B. subtilis} are confined in a thin layer of fluid, between asymmetric boundaries.The resulting driven flow circulates around the cell body ranging over several cell diameters, in contrast to the more localized flows surrounding free swimmers.This discovery extends our knowledge of the dynamic geometry of bacteria and their flagella, and reveals new mechanisms for motility-associated molecular transport and inter-cellular communication.

Understanding dispersal is a central aim of evolutionary ecology. Theoretical analyses of dispersal have been crucial in identifying key variables which contribute to its evolution and maintenance, but the supporting empirical data remains elusive. Microbes offer a powerful model system on which ecological and evolutionary theory can be experimentally tested with controlled and replicated experiments, and with the convenient malleability of selective pressures and bacterial genomics. Pseudomonas aeruginosa is an ubiquitous, opportunistic pathogen that is able to induce acute or chronic infections in a broad array of hosts. As well as in vivo environments, P. aeruginosa can be found in a range of ecological habitats, from solid to aqueous, and as such requires a variety of dispersal mechanisms (including swimming, gliding on a surfactant and ‘crawling’) for effective colonisation and infectivity. In this thesis, I present a collection of papers which outline empirical ecological and evolutionary experiments to identify the abiotic and biotic forces that shape the evolution of these different dispersal mechanisms, with particular focus on the theoretically important role of kin competition and the structure of the abiotic environment.

Flagella, the helical propellers that extend from the bacterial cell surface, illustrate how complex nanomachines assemble outside the cell. The sequential construction of the flagellar rod, hook, and filament requires export of thousands of structural subunits across the cell membrane and this is achieved by a specialised flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. The fT3SS imposes a crude ordering of subunits, with filament subunits only exported once the rod and hook are complete. This “export specificity switch” is controlled by the FlhB component of the fT3SS export gate in response to a signal from the exported molecular ruler FliK, which monitors the length of the growing hook. This study seeks to clarify how rod and hook subunits interact with FlhB, and how FlhB switches export specificity. Rod and hook subunits possess a conserved gate recognition motif (GRM; Fxxxφ, with φ being any hydrophobic residue) that is proposed to bind a surface-exposed hydrophobic patch on the FlhB cytosolic domain. Mutation of the GRM phenylalanine and the final hydrophobic residue resulted in impaired subunit export and decreased cell motility. Isothermal titration calorimetry was performed to assess whether subunit export order is imposed at FlhB. These experiments showed that rod and hook subunits bind to FlhB with micromolar dissociation constants (5-45 μM), suggesting transient interactions. There was no clear correlation between subunit affinity for FlhB and the order of subunit assembly in the nascent flagellum. Solution-state nuclear magnetic resonance (NMR) spectroscopy supported prior data showing that rod and hook subunits interact with FlhB’s surface-exposed hydrophobic patch. NMR also indicated that residues away from the patch undergo a conformational change on subunit binding. FlhB autocleaves rapidly in its cytosolic domain, and the resulting polypeptides (FlhBCN and FlhBCC) are held together by non-covalent interactions between b-strands that encompass the autocleavage site. The autocleavage event is a prerequisite for the export specificity switch, but its function is unclear. Analysis of the cellular localization of FlhBCN and FlhBCC revealed that FlhBCC dissociated from the membrane export machinery, but only in the presence of FliK. Biochemical and biophysical studies of FlhB variants that undergo export specificity switching in the absence of FliK showed that these FlhB “autonomous switchers” were less stable than wildtype FlhB and their FlhBCC domain could dissociate from the export machinery in the absence of FliK. The results suggest that the export specificity switch involves a FliK-dependent loss of FlhBCC from the export machinery, eliminating the binding site for rod and hook subunits.

Bacterial motility, and in particular repulsion or attraction towards specific chemicals, has been a subject of investigation for over 100 years, resulting in detailed understanding of bacterial chemotaxis and the corresponding sensory network in many bacterial species including Escherichia coli. E. Coli swims by rotating a bundle of flagellar filaments, each powered by an individual rotary motor located in the cell membrane. When all motors rotate counter-clockwise (CCW), a stable bundle forms and propels the cell forward. When one or more motors switch to clock-wise (CW) rotation, their respective filaments fall out of the bundle, leading to the cell changing orientation. Upon switching back to CCW, the bundle reforms and propels the cell in a new direction. Chemotaxis is performed by the bacterium through prolonging runs by suppressing CW rotation when moving towards nutrients and facilitating reorientation by increasing CW bias when close to a source of a harmful substance. Chemicals are sensed through interaction with membrane bound chemosensors. These proteins can interact with a very specific set of chemicals and the concentrations they are able to sense are in the range between 10-⁶ and 10-² M. However, experiments have shown that the osmotic pressure exerted by large (> 10-¹ M) concentrations of solutes, which have no specificity for binding to chemosensors (e.g. sucrose), is able to send a signal down the chemotactic network. Additionally, clearing of bacterial density away from sources of high osmolarity has been previously observed in experiments with agar plates. This behaviour has been termed osmotaxis. The aim of this doctoral thesis work is to understand how different environmental cues influence the tactic response and ultimately, combine at the network output to direct bacterial swimming. As tactic responses to chemical stimuli have been extensively studied, I focus purely on the response to non-specific osmotic stimuli, using sucrose to elevate osmolarity. I monitor the chemotactic network output, the rotation of a single bacterial flagellar motor, using Back Focal Plane Interferometry over a variety of osmotic conditions. Additionally, in collaboration with Vincent Martinez, I studied the effect of elevated osmolality on swimming speed of large (104) bacterial populations, using differential dynamic microscopy (DDM). I have found that sudden increases in media osmolarity lead to changes of both motor speed and motor clockwise bias, which is the fraction of time it spends rotating clockwise. Changes in CW Bias proceed in two phases. Initially, after elevating the osmolarity, CW Bias drops to zero, indicating that the motor is exclusively in the ‘cell run’ mode. This phase lasts from 2-5 minutes depending on the magnitude of the change in solute concentration. What follows then is a distinct second phase where the CW Bias is elevated with respect to the initial levels and this phase lasts longer than 15-20 minutes. In comparison, for defined chemical stimuli, the motor output resets after several seconds, a behaviour termed perfect adaptation. For changes of 100 mOsm/kg and 200 mOsm/kg in magnitude the motors speed up, often by as much as a factor of two, before experiencing a gradual slow down. Despite the slow down, motors still rotate faster 15-20 minutes after the change in osmolarity, than they did before. For changes of 400 mOsm/Kg in magnitude the motors decrease sharply in speed, coming to a near halt, recovering after 5 minutes and eventually, on average, speeding up. DDM studies of free swimming bacteria have shown that elevated osmolality leads to higher swimming speeds, in agreement with single motor data. Using theoretical models of bacterial swimming from the literature, it is discussed how this motor output, although different to what is expected for chemotaxis, is able to drive bacteria away from regions of space with high osmolalities. Additionally, I have started extending the work done with sucrose, to another solute often used to elevate osmolality, sodium chloride. While sucrose is outer membrane impermeable, NaCl can cross the outer membrane into the periplasmic space. Another layer of complexity is that NaCl has some specificty for the chemoreceptors. The preliminary results are shown and qualitatively agree with those obtain with sucrose.