Session X66: Physics of Microbes I

Systematic mutagenesis of oncocin reveals enhanced activity and insights into the mechanisms of antimicrobial activity

Oncocin is a proline-rich antimicrobial peptide that inhibits protein synthesis by binding to the bacterial ribosome. The aim of this work is to improve the antimicrobial activity of oncocin by systematic peptide mutagenesis and activity evaluation. We discovered that a pair of cationic substitutions (P4K and P7K/R) enhanced the activity by 2 to 4 fold (p<0.05) against multiple Gram-negative bacteria. An in vitro transcription / translation assay indicated that the increased activity was not because of stronger ribosome binding. Instead a cellular internalization assay revealed a higher internalization rate for the optimized analogs thereby suggesting a mechanism to increase potency. In addition, we found that the optimized peptides' benefit is dependent upon nutrient-depleted media conditions.   

Quantifying the Formation and Dissolution of Multilayer Regions in the Expansion of Twitching Bacterial Colonies

Type IV pili (T4P) are very thin (5-8 nm in diameter) protein filaments that can be extended and retracted by certain classes of Gram-negative bacteria including P. aeruginosa [1]. These bacteria use T4P to move across viscous interfaces, referred to as twitching motility. Twitching can occur for isolated cells or in a collective manner [2]. In collective motion, the advancing front of an expanding colony consists of finger-like protrusions consisting of many aligned bacteria, with 5 to 30 cells across the fingers, followed by cells that twitch within a lattice-like pattern. During the outward radial motion of the fingers along an agar-glass interface, cells can vertically displace the agar to form multilayer regions. Using our custom-built, temperature- and humidity-controlled environmental chamber, we have studied the formation and dissolution of multilayer regions within fingers for a range of agar concentrations. We find that there is a minimum finger width required for the stability of multilayer regions.

[1] Burrows, L.L. (2012) Annu. Rev. Microbiol. 66: 493–520.
[2] Semmler, A.B., Whitchurch, C.B., Mattick, J.S. (1999) Microbiology 145: 2863-2873.   

Magnetotactic bacterial scattering in porous media hinders transport

Swimming cells exhibit complex surface scattering behaviors in both natural and engineered porous habitats, impairing their persistent random walks. Using microfluidics experiments complemented with Langevin simulations, we study how the scattering of magnetotactic bacteria (MTB) within a lattice of obstacles modifies cell transport, and how guidance by an external field augments their mobility. MTBs are used as a model biological system, because they share motility mechanisms with many other bacterial species, and their swimming direction is easily manipulated via an external magnetic field. We show that both the diffusive and directed mobility of the cells decreases markedly in the presence of porous microstructure compared to bulk fluid. Moreover, the spacing between the obstacles and the degree of the disorder in the lattice play a key role in the magnitude of the observed reduced mobility. These results are an important step toward understanding the physical ecology of swimming cells in quiescent porous media as well as for controlling micro-robots in complex environments.
  

Cargo carrying capacity of a single bacterium

In recent times, several groups have demonstrated bio-hybrid micro-robots where bacteria are attached to “cargo” such as polystyrene beads, which are transported using the power generated by bacterial motility. A system consisting of a single bacterium pulling a load is important for a clear understanding of such bacteria powered devices. Previous reports of cargo delivery by single bacteria have relied on attaching the cells to very small particles (~ 1 micron dia.) so that statistically only one cell can bind due to steric restriction. Such an approach is not effective, as dynamics of a single cell carrying a large load, say ~ 10 micron dia., cannot be studied. Systems describing single bacterium carrying such large size cargo have not been described so far to the best of our knowledge. In this talk, we ask a very general question, namely, what is the maximum load of a spherical cargo which can be transported by a flagellated swimmer at a specified speed. We present a technique to attach single loads as large as 12 micron diameter to micron sized single bacteria. Further, results from our experiments and theoretical analysis suggests the crucial role of tuning the flagellar geometry and the torque-speed characteristics of the flagellar motor to maximize cargo carrying capacity.   

Three-dimensional imaging and force mode analysis of microflows induced by swimming Chlamydomonas reinhardtii

Understanding the fluid flow induced by microswimmers is paramount to revealing how they interact with each other and their environment. Here, we present a three-dimensional (3D) measurement and characterization of the flow field induced by motile planktonic algal cells, Chlamydomonas reinhardtii. A single alga is captured and held stationary by a micropipette, which beats its flagella in a characteristic breastroke pattern. We track the 3D flow field around the alga by employing fast holographic imaging on 1 um tracer particles, which leads to a nominal spatial resolution of ~ 54 nm along the optical axis and ~ 44 nm in the imaging plane. The method allows us to image the flow around a single alga continuously over thousands of flagellar beat cycles and show time-averaged and phase-binned 3D flow fields. We analyze these 3D flow fields and determine the dominant force modes of the flagellar motion of C. reinhardtii. Our study demonstrates the power of holography in imaging detailed microscopic flows and provides crucial information for understanding the detailed locomotion of swimming microorganisms.   

Length regulation of multiple flagella that self-assemble from a shared pool of components

The single cell biflagellate Chlamydomonas reinhardtii has proven to be a very useful model organism for studies of size control. We consider a model of flagellar length control whose key assumption is that proteins responsible for the intraflagellar transport (IFT) of tubulin are present in limiting amounts. We show that this limiting-pool assumption and simple reasoning based on the law of mass action leads to an inverse relationship between the rate at which a flagellum grows and its length, which has been observed experimentally, and has been shown theoretically to provide a mechanism for length control. We extend our length-control model to two flagella by considering different mechanisms of their coupling. Within our theoretical framework we conclude that, if tubulin and IFT proteins are freely exchanged between flagella, simultaneous length control is not possible if the disassembly rate is constant. However, if disassembly depends on the concentration of IFT proteins at the tip of the flagellum, simultaneous length control can be achieved.   

Encounter rates between motile bacteria and sinking particles

Many marine microbes rely on sinking particles of organic matter as their food source. Once attached to a particle, bacteria can solubilize its organic matter and convert it into biomass. This process hinders carbon from sinking to the deep ocean (a process known as the ‘biological pump’) and thus affects the magnitude of carbon flux in the ocean. Carbon consumption by bacteria is preceded by their encounter with sinking particles, whose quantification is needed to understand the biological pump. Here, we theoretically predict these encounter rates by combining the Stokes flow around the particle with Jeffrey's equation for a rod in flow. We show that elongated bacteria - unlike spherical particles often used in encounter rate estimates – break the fore-aft symmetry of the flow streamlines, with major consequences on encounter rates. Specifically, for small to intermediate sinking speeds, this symmetry-breaking implies that elongated swimmers are up to hundred times more likely to encounter the sinking particle than spherical bacteria with the same volume. We find that the mechanism for this encounter rate enhancement is the hydrodynamic focusing of elongated swimmers downstream of the sinking particle, which leads to their preferential attachment to the back of the particle.   

Collective bacterial vision

Bacteria are well studied primitive organisms and an ideal system for physical scientists to study how simple systems perform complex tasks. Swarming is one such complex collective phenomenon of producing surfactant to ease their motility on a semi solid surface (nutrient supplemented 0.6% agar). We perturbed the swarming pattern of Pseudomonas aeruginosa (PA) by incorporating inert obstacles (PDMS) in the agar plate thus creating local depletion of nutrient and water rich agar surface. To our surprise, we noticed that the bacteria as they colonize the surfaces with such obstacles effectively avoid them by changing the course of their path at a distance as far as 5 mm thus ‘seeing’ (sensing) them at ∼ thousand body lengths. We refer to this phenomenon as “collective bacterial vision”. We demonstrate a fluid dynamic model that can guide them around these obstacles. Our model leads to further questions such as, is it possible for a single bacterium to possess “bacterial vision”? What is the grouping cost and ideal group size? Is there a need for information transfer in the group? Is this phenomenon a completely active or completely passive or a delicate balance between them? We will describe our attempts to address some of the above questions though most of them are still open.   

Bacterial growth curves are predictable from cellular Raman spectra

Raman microscopy is an imaging technique that has been applied to cells to obtain Raman spectra that reflect the abundances of various biomolecules. It allows distinguishing cellular states in a label-free and non-destructive manner. Previously, employing spontaneous Raman scattering, we showed that cellular Raman spectra and transcriptomes could be linked in both S. pombe and E. coli, and that the transcriptomes could be reconstructed from Raman spectra (Kobayashi-Kirschvink et al., Cell Systems, 2018). Considering that omics information, which characterizes cellular states with molecular resolution, is linked to Raman spectra, we next asked whether Raman spectra could be linked to macroscopic quantities of cellular states. Our recent experimental and computational study indicates that different cellular states of single-gene knockout E. coli strains were distinguished by Raman spectra, and the entire population growth curves of different strains could be predicted by Raman spectra from cells in exponential phase. These results suggest that cellular Raman spectra have the potential to integrate macroscopic and microscopic characterizations of cellular states.   

Imaging the emergence of collective swarming in light-controlled bacteria

Collective motions of active matter as illustrated by bird flocks, fish schools and bacterial swarms demonstrate the intriguing emergent behaviors of living systems. While moving independently at low density, active entities move collectively with its neighbors at high density, exhibiting orientational order at a scale larger than their individual sizes. Although such a disorder-order nonequilibrium phase transition has been previously studied, detailed kinetics of this transition has not been explored in experiments. Here, using engineered E. coli, whose locomotion can be reversibly controlled by light, we experimentally study the kinetic pathway of the swarming transition in 3D bacterial suspensions. We trigger bacterial swarming by tuning light intensity and image the emergence of collective motion. We map the phase diagram of bacterial swarming as functions of bacterial concentration, swimmer velocity and the number fraction of active swimmers. Moreover, we find that the swarming transition occurs in a nucleation manner and characterize the incubation time of the transition. Our results reveal the microscopic dynamics of the emergence of bacterial swarming and provide insights into the nonequilibrium phase transition in active matter.   

Topological defect driven thickness changes in layers of bacteria

Myxococcus xanthus is a rod shaped soil bacterium that lives in collectives of many
millions of cells. These groups of cells exhibit different collective behaviors
which benefit the bacteria depending on the external environment. When M. xanthus are in favorable
conditions, colonies will swarm and spread out to explore new territory, resulting in thin
layers only a few cells thick. Our goal is to understand how the motility
of individual bacteria along with local interactions can lead to the collective behaviors
that exist on length scales significantly larger that of the individuals. Using a
confocal laser scanning microscope we image layers of cells with single cell
resolution while simultaneously obtaining height maps of the layer thickness across the
sample. From these images we are able to extract flow and director fields of the bacteria
over time. Topological defects in director fields are connected to pressure fluctuations
in active nematic systems which can lead to local changes in layer thickness. By combining
height maps with director and flow fields, we are able to examine how the active nematic
nature of the system can result in thickness changes in bacteria layers.   

Self-organization of swimmers drives long-range fluid transport in bacterial colonies

Microbes commonly live in structured communities that affect human health and influence ecological systems. Colony mode of bacterial growth on solid substrates (e.g. food products) is closely related to biofilm development, and it is a main approach to study structured microbial communities. Heterogeneous populations, such as non-motile and motile populations, often coexist in bacteria colonies. Here we discovered that motile cells in sessile colonies of peritrichously flagellated bacteria can self-organize into motile bands that can drive long-range fluid transport at a constant speed of ~30 μm/s, providing a stable high-speed avenue for material transport at the colony scale. These findings present a unique form of large-scale self-organization and active transport in bacterial colonies.   

Bacterial Surface Motility is Modulated by An Abiotic Jamming Transition and is Independent of Chemotaxis and Individual Motility

The need to travel over surfaces is ubiquitous in the microbial world, where bacterial groups move at speeds of ~30 um/s despite their low Reynolds number environment. Bacillus subtilis is a model organism for the study of directed, collective motion over surfaces with groups exhibiting motility on length scales three orders of magnitude larger than themselves in a few doubling times. While genetic and chemical studies clearly show that surface tension gradients and water availability are required for this ‘ultrafast’ group motility, the relative importance of chemosensing, exogenous nutrient gradients, and individual motility are largely unknown from an experimental viewpoint. We demonstrate that contrary to simulations of bacterial growth on surfaces, B. subtilis does not rely on chemotaxis for direction, that the rate of dendritic expansion of the colony is faster when bacteria are motile but that the same type of group motility is possible even with non-motile cells, and that water availability is likely a control parameter modulating an abiotic jamming transition that determines whether the group remains fluidized and therefore motile.   

Emergent behaviors of motile bacteria in a dynamic oxygen gradient

Despite decades of microbiology research, we lack a practical understanding of how bacteria migrate through host tissues in disease and through soils in bioremediation to consume environmental pollutants. Models of bacterial motility neglect how the impact of cells on their microenvironment dynamically influences subsequent motion of cells. We aim to unravel how the multi-scale dynamic feedback between bacterial motility and nutrient consumption gives rise to emergent behaviors. We find that E. coli suspension droplets in two-dimensional confinement spontaneously separate into two populations according to a self-generated oxygen gradient: immotile cells cluster in a droplet center and motile cells congregate near an outer air interface. We show how cluster formation and size depend on the interplay between oxygen consumption, diffusion, and bacterial motility. Understanding the impact of gradient-consumption feedback on population scale dynamics will better inform population level microbial behaviors like biofilm formation and migration.