Morphogenesis of bacterial colonies in polymeric environments.

Many bacteria live in polymeric fluids, such as mucus, environmental polysaccharides, and extracellular polymers in biofilms. However, lab studies typically focus on cells in polymer-free fluids. Here, we show that interactions with polymers shape a fundamental feature of bacterial life-how they proliferate in space in multicellular colonies. Using experiments, we find that when polymer is sufficiently concentrated, cells generically and reversibly form large serpentine "cables" as they proliferate. By combining experiments with biophysical theory and simulations, we demonstrate that this distinctive form of colony morphogenesis arises from an interplay between polymer-induced entropic attraction between neighboring cells and their hindered ability to diffusely separate from each other in a viscous polymer solution. Our work thus reveals a pivotal role of polymers in sculpting proliferating bacterial colonies, with implications for how they interact with hosts and with the natural environment, and uncovers quantitative principles governing colony morphogenesis in such complex environments.

A minimalistic cyclic ice-binding peptide from phage display.

Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Rather than relying on the (limited) existing structure-property relationships that have been established for IBPs, here we report the use of phage display for the identification of short peptide mimics of IBPs. To this end, an ice-affinity selection protocol is developed, which enables the selection of a cyclic ice-binding peptide containing just 14 amino acids. Mutational analysis identifies three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics simulations reveal that the side chain of Thr10 hydrophobically binds to ice revealing a potential mechanism. To demonstrate the biotechnological potential of this peptide, it is expressed as a fusion ('Ice-Tag') with mCherry and used to purify proteins directly from cell lysate.

Self-assembly of protein-polymer conjugates for drug delivery.

Protein-polymer conjugates are a class of molecules that combine the stability of polymers with the diversity, specificity, and functionality of biomolecules. These bioconjugates can result in hybrid materials that display properties not found in their individual components and can be particularly relevant for drug delivery applications. Engineering amphiphilicity into these bioconjugate materials can lead to phase separation and the assembly of high-order structures. The assembly, termed self-assembly, of these hierarchical structures entails multiple levels of organization: at each level, new properties emerge, which are, in turn, influenced by lower levels. Here, we provide a critical review of protein-polymer conjugate self-assembly and how these materials can be used for therapeutic applications and drug delivery. In addition, we discuss central bioconjugate design questions and propose future perspectives for the field of protein-polymer conjugate self-assembly.

Facile actuation of aqueous droplets on a superhydrophobic surface using magnetotactic bacteria for digital microfluidic applications.

Magnetic actuation provides a low-cost, simple method for droplet manipulation on a digital microfluidic platform. The impetus to move the droplets on a low friction surface can come from internal superparamagnetic particles or paramagnetic salts. Recently, the use of microbes for bio-actuation has been established, where the thrust produced by the microbes can be exploited to exert the force required for droplet movement. This study presents biologically-driven magnetic actuation of droplets on a superhydrophobic surface using magnetotactic bacteria (MTB). MTB-droplets were impelled along various trajectories such as rectangular and figure-of-eight-shaped paths. Droplets were reproducibly actuated with speeds up of to 30 mm s-1. We demonstrated the ability to sequentially merge and mix multiple droplets by merging a 10 µL MTB droplet with two 4 µL colored droplets. The reorientation of MTB in the droplet enhanced mixing rate of the merged fluids by ∼40% compared with the control experiment where no actuation was used. Biologically-driven magnetic actuation was compared with actuation by superparamagnetic particles and paramagnetic salts, in terms of controllability and speed. MTB droplet was moved with the same average speed as other two methods and showed higher response time as the magnet acceleration increased. Lastly, MTB were used to perform a phosphatase assay using endogenous enzyme. The relative absorbance at 405 nm, indicating the production of the yellow product, increased over time and levels off after 75 min.

RTX Adhesins are Key Bacterial Surface Megaproteins in the Formation of Biofilms.

Gram-negative bacteria produce repeats-in-toxin adhesion proteins (RTX adhesins) to facilitate microbial adhesion. These large, multidomain proteins share a common architecture comprised of four regions. First to emerge from the bacterium, C terminal end leading, is the RTX export sequence that directs the protein through the type 1 secretion system (T1SS). This is followed by the ligand-binding region responsible for host adhesion and cohesion, which contains diverse ligand-binding domains. These serve a zip code function to direct bacteria to a particular environmental niche. Thereafter is a large extension region consisting of tens to hundreds of tandem bacterial immunoglobulin-like (BIg) domains, whose function is to extend the reach of the ligand-binding domains away from the bacterial surface. Lastly, there is a conserved N terminal cell-membrane-anchor region that retains the adhesin within the secretion system. This is also a site of in situ proteolysis, when nutrients are scarce, that enables the bacterium to leave the biofilm. In this review, the four regions of RTX adhesins are presented in the order in which they emerge from the cell during synthesis and retention.

Erratum: Publisher's Note: "Migration of magnetotactic bacteria in porous media" [Biomicrofluidics 12, 011101 (2018)].

[This corrects the article DOI: 10.1063/1.5024508.].

Migration of magnetotactic bacteria in porous media.

Magnetotactic bacteria (MTB) migrate in complex porous sediments where fluid flow is ubiquitous. Here, we demonstrate that magnetotaxis enables MTB to migrate effectively through porous micromodels. Directed MTB can circumvent curved obstacles by traveling along the boundaries and pass flat obstacles by repeatedly switching between forward and backward runs. Magnetotaxis enables directed motion of MTB through heterogeneous porous media, overcoming tortuous flow fields with local velocities as high as 250 µm s-1. Our findings bring new insights into the migration behaviour of MTB in their natural habitats and their potential in vivo applications as microbiorobots.

Magnetotaxis Enables Magnetotactic Bacteria to Navigate in Flow.

Magnetotactic bacteria (MTB) play an important role in Earth's biogeochemical cycles by transporting minerals in aquatic ecosystems, and have shown promise for controlled transport of microscale objects in flow conditions. However, how MTB traverse complex flow environments is not clear. Here, using microfluidics and high-speed imaging, it is revealed that magnetotaxis enables directed motion of Magnetospirillum magneticum over long distances in flow velocities ranging from 2 to 1260 µm s-1 , corresponding to shear rates ranging from 0.2 to 142 s-1 -a range relevant to both aquatic environments and biomedical applications. The ability of MTB to overcome a current is influenced by the flow, the magnetic field, and their relative orientation. MTB can overcome 2.3-fold higher flow velocities when directed to swim perpendicular to the flow as compared to upstream, as the latter orientation induces higher drag. The results indicate a threshold drag of 9.5 pN, corresponding to a flow velocity of 550 µm s-1 , where magnetotaxis enables MTB to overcome counterdirectional flow. These findings bring new insights into the interactions of MTB with complex flow environments relevant to aquatic ecosystems, while suggesting opportunities for in vivo applications of MTB in microbiorobotics and targeted drug delivery.

Structure of a 1.5-MDa adhesin that binds its Antarctic bacterium to diatoms and ice.

Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-µm-long adhesin using a "dissect and build" structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like ß-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several "repeats-in-toxin" motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.

Peptide backbone circularization enhances antifreeze protein thermostability.

Antifreeze proteins (AFPs) are a class of ice-binding proteins that promote survival of a variety of cold-adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large-scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein-mediated N- and C-terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20-Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice-binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice-affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild-type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs.

Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities.

Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.

Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.