Bacterial transport of colloids in liquid crystalline environments.

We describe the controlled transport and delivery of non-motile eukaryotic cells and polymer microparticles by swimming bacteria suspended in nematic liquid crystals. The bacteria push reversibly attached cargo in a stable, unidirectional path (or along a complex patterned director field) over exceptionally long distances. Numerical simulations and analytical predictions for swimming speeds provide a mechanistic insight into the hydrodynamics of the system. This study lays the foundation for using cargo-carrying bacteria in engineering applications and for understanding interspecies interactions in polymicrobial communities.

Effects of confinement, surface-induced orientations and strain on dynamical behaviors of bacteria in thin liquid crystalline films.

We report on the organization and dynamics of bacteria (Proteus mirabilis) dispersed within lyotropic liquid crystal (LC) films confined by pairs of surfaces that induce homeotropic (perpendicular) or hybrid (homeotropic and parallel orientations at each surface) anchoring of the LC. By using motile vegetative bacteria (3 µm in length) and homeotropically aligned LC films with thicknesses that exceed the length of the rod-shaped cells, a key finding reported in this paper is that elastic torques generated by the LC are sufficiently large to overcome wall-induced hydrodynamic torques acting on the cells, thus leading to LC-guided bacterial motion near surfaces that orient LCs. This result extends to bacteria within LC films with hybrid anchoring, and leads to the observation that asymmetric strain within a hybrid aligned LC rectifies motions of motile cells. In contrast, when the LC film thickness is sufficiently small that confinement prevents alignment of the bacteria cells along a homeotropically aligned LC director (achieved using swarm cells of length 10-60 µm), the bacterial cells propel in directions orthogonal to the director, generating transient distortions in the LC that have striking "comet-like" optical signatures. In this limit, for hybrid LC films, we find LC elastic stresses deform the bodies of swarm cells into bent configurations that follow the LC director, thus unmasking a coupling between bacterial shape and LC strain. Overall, these results provide new insight into the influence of surface-oriented LCs on dynamical bacterial behaviors and hint at novel ways to manipulate bacteria using confined LC phases that are not possible in isotropic solutions.

Using liquid crystals to reveal how mechanical anisotropy changes interfacial behaviors of motile bacteria.

Bacteria often inhabit and exhibit distinct dynamical behaviors at interfaces, but the physical mechanisms by which interfaces cue bacteria are still poorly understood. In this work, we use interfaces formed between coexisting isotropic and liquid crystal (LC) phases to provide insight into how mechanical anisotropy and defects in LC ordering influence fundamental bacterial behaviors. Specifically, we measure the anisotropic elasticity of the LC to change fundamental behaviors of motile, rod-shaped Proteus mirabilis cells (3 µm in length) adsorbed to the LC interface, including the orientation, speed, and direction of motion of the cells (the cells follow the director of the LC at the interface), transient multicellular self-association, and dynamical escape from the interface. In this latter context, we measure motile bacteria to escape from the interfaces preferentially into the isotropic phase, consistent with the predicted effects of an elastic penalty associated with strain of the LC about the bacteria when escape occurs into the nematic phase. We also observe boojums (surface topological defects) present at the interfaces of droplets of nematic LC (tactoids) to play a central role in mediating the escape of motile bacteria from the LC interface. Whereas the bacteria escape the interface of nematic droplets via a mechanism that involved nematic director-guided motion through one of the two boojums, for isotropic droplets in a continuous nematic phase, the elasticity of the LC generally prevented single bacteria from escaping. Instead, assemblies of bacteria piled up at boojums and escape occurred through a cooperative, multicellular phenomenon. Overall, our studies show that the dynamical behaviors of motile bacteria at anisotropic LC interfaces can be understood within a conceptual framework that reflects the interplay of LC elasticity, surface-induced order, and topological defects.

Dynamic self-assembly of motile bacteria in liquid crystals.

This paper reports an investigation of dynamical behaviors of motile rod-shaped bacteria within anisotropic viscoelastic environments defined by lyotropic liquid crystals (LCs). In contrast to passive microparticles (including non-motile bacteria) that associate irreversibly in LCs via elasticity-mediated forces, we report that motile Proteus mirabilis bacteria form dynamic and reversible multi-cellular assemblies when dispersed in a lyotropic LC. By measuring the velocity of the bacteria through the LC (8.8 ± 0.2 µm s(-1)) and by characterizing the ordering of the LC about the rod-shaped bacteria (tangential anchoring), we conclude that the reversibility of the inter-bacterial interaction emerges from the interplay of forces generated by the flagella of the bacteria and the elasticity of the LC, both of which are comparable in magnitude (tens of pN) for motile Proteus mirabilis cells. We also measured the dissociation process, which occurs in a direction determined by the LC, to bias the size distribution of multi-cellular bacterial complexes in a population of motile Proteus mirabilis relative to a population of non-motile cells. Overall, these observations and others reported in this paper provide insight into the fundamental dynamic behaviors of bacteria in complex anisotropic environments and suggest that motile bacteria in LCs are an exciting model system for exploration of principles for the design of active materials.

Divin: a small molecule inhibitor of bacterial divisome assembly.

Bacterial cell division involves the dynamic assembly of division proteins and coordinated constriction of the cell envelope. A wide range of factors regulates cell division--including growth and environmental stresses--and the targeting of the division machinery has been a widely discussed approach for antimicrobial therapies. This paper introduces divin, a small molecule inhibitor of bacterial cell division that may facilitate mechanistic studies of this process. Divin disrupts the assembly of late division proteins, reduces peptidoglycan remodeling at the division site, and blocks compartmentalization of the cytoplasm. In contrast to other division inhibitors, divin does not interact with the tubulin homologue FtsZ, affect chromosome segregation, or activate regulatory mechanisms that inhibit cell division indirectly. Our studies of bacterial cell division using divin as a probe suggest that dividing bacteria proceed through several morphological stages of the cell envelope, and FtsZ is required but not sufficient to compartmentalize the cytoplasmic membrane at the division site. Divin is only moderately toxic to mammalian cells at concentrations that inhibit the growth of clinical pathogens. These characteristics make divin a useful probe for studying bacterial cell division and a starting point for the development of new classes of therapeutic agents.

Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness.

The stiffness of bacteria prevents cells from bursting due to the large osmotic pressure across the cell wall. Many successful antibiotic chemotherapies target elements that alter mechanical properties of bacteria, and yet a global view of the biochemistry underlying the regulation of bacterial cell stiffness is still emerging. This connection is particularly interesting in opportunistic human pathogens such as Pseudomonas aeruginosa that have a large (80%) proportion of genes of unknown function and low susceptibility to different families of antibiotics, including beta-lactams, aminoglycosides, and quinolones. We used a high-throughput technique to study a library of 5,790 loss-of-function mutants covering ~80% of the nonessential genes and correlated P. aeruginosa individual genes with cell stiffness. We identified 42 genes coding for proteins with diverse functions that, when deleted individually, decreased cell stiffness by >20%. This approach enabled us to construct a "mechanical genome" for P. aeruginosa d-Alanine dehydrogenase (DadA) is an enzyme that converts d-Ala to pyruvate that was included among the hits; when DadA was deleted, cell stiffness decreased by 18% (using multiple assays to measure mechanics). An increase in the concentration of d-Ala in cells downregulated the expression of genes in peptidoglycan (PG) biosynthesis, including the peptidoglycan-cross-linking transpeptidase genes ponA and dacC Consistent with this observation, ultraperformance liquid chromatography-mass spectrometry analysis of murein from P. aeruginosa cells revealed that dadA deletion mutants contained PG with reduced cross-linking and altered composition compared to wild-type cells.IMPORTANCE The mechanical properties of bacteria are important for protecting cells against physical stress. The cell wall is the best-characterized cellular element contributing to bacterial cell mechanics; however, the biochemistry underlying its regulation and assembly is still not completely understood. Using a unique high-throughput biophysical assay, we identified genes coding proteins that modulate cell stiffness in the opportunistic human pathogen Pseudomonas aeruginosa This approach enabled us to discover proteins with roles in a diverse range of biochemical pathways that influence the stiffness of P. aeruginosa cells. We demonstrate that d-Ala-a component of the peptidoglycan-is tightly regulated in cells and that its accumulation reduces expression of machinery that cross-links this material and decreases cell stiffness. This research demonstrates that there is much to learn about mechanical regulation in bacteria, and these studies revealed new nonessential P. aeruginosa targets that may enhance antibacterial chemotherapies or lead to new approaches.