Sima Setayeshgar

Physiochemical Properties of Caulobacter crescentus Holdfast: A Localized Bacterial Adhesive

To colonize surfaces, the bacterium Caulobacter crescentus employs a polar polysaccharide, the holdfast, located at the end of a thin, long stalk protruding from the cell body. Unlike many other bacteria which adhere through an extended extracellular polymeric network, the holdfast footprint area is tens of thousands times smaller than that of the total bacterium cross-sectional surface, making for some very demanding adhesion requirements. At present, the mechanism of holdfast adhesion remains poorly understood. We explore it here along three lines of investigation: (a) the impact of environmental conditions on holdfast binding affinity, (b) adhesion kinetics by dynamic force spectroscopy, and (c) kinetic modeling of the attachment process to interpret the observed time-dependence of the adhesion force at short and long time scales. A picture emerged in which discrete molecular units called adhesins are responsible for initial holdfast adhesion, by acting in a cooperative manner.

Novel Pseudotaxis Mechanisms Improve Migration of Straight-Swimming Bacterial Mutants Through a Porous Environment

ABSTRACT Bacterial locomotion driven by flagella is given directionality by the chemotaxis signal transduction network. In the classic plate assays of migration in porous motility agar, efficient motility is compromised in chemotaxis mutants of diverse bacteria. Nonchemotactic mutants become trapped within the agar matrix. Suppressor mutations that prevent this entanglement but do not restore chemotaxis, a phenomenon designated pseudotaxis, were first reported to arise for Escherichia coli. In this study, novel mechanisms of pseudotaxis have been identified for the plant-pathogenic alphaproteobacterium Agrobacterium tumefaciens. Mutants with chemotaxis mutation suppressor (cms) mutations that impart enhanced migration in motility agar compared to that of their straight-swimming, nonchemotactic parent were isolated. We find that pseudotaxis in A. tumefaciens occurs most commonly via mutations in the D1 domain of the flagellar hook protein, FlgE, but it can also be found less frequently to be due to mutations in the hook length regulator, FliK, or in the motor protein, MotA. Single-cell-tracking studies of cms mutants in bulk medium clearly reveal frequent changes in the direction of swimming, similar to the swimming of strains that are proficient for chemotaxis, but independent of a sensory mechanism. Our results suggest that the tumbling process can be tuned through mutation and evolution to optimize migration through complex, porous environments. IMPORTANCE Chemotaxis sensory networks control direct bacterial motility by modulating flagellar rotary motion, alternating cellular movement between runs and tumbles. The straight-swimming phenotype of chemotaxis-deficient cells yields nonexpanding colonies in motility agar. Enhanced, chemotaxis-independent spreading, dubbed pseudotaxis, has been observed in Escherichia coli mutants. We have identified novel pseudotaxis mutations in Agrobacterium tumefaciens that alter the flagellar hook structure or motor, leading to randomly occurring reorientations observed in single-cell tracking studies in bulk medium. These directional changes allow the cells to migrate more efficiently than the parent strain through the agar matrix, independently of the chemotaxis process. These findings reveal that tumbling can be tuned for effective navigation in complex porous environments, analogous to the natural habitats for many bacteria, and provide evidence for the strong selective pressure exerted by the external environment on the basal pattern of motility, even in the absence of chemotaxis. Chemotaxis sensory networks control direct bacterial motility by modulating flagellar rotary motion, alternating cellular movement between runs and tumbles. The straight-swimming phenotype of chemotaxis-deficient cells yields nonexpanding colonies in motility agar. Enhanced, chemotaxis-independent spreading, dubbed pseudotaxis, has been observed in Escherichia coli mutants. We have identified novel pseudotaxis mutations in Agrobacterium tumefaciens that alter the flagellar hook structure or motor, leading to randomly occurring reorientations observed in single-cell tracking studies in bulk medium. These directional changes allow the cells to migrate more efficiently than the parent strain through the agar matrix, independently of the chemotaxis process. These findings reveal that tumbling can be tuned for effective navigation in complex porous environments, analogous to the natural habitats for many bacteria, and provide evidence for the strong selective pressure exerted by the external environment on the basal pattern of motility, even in the absence of chemotaxis.

Diffusion of Bacterial Cells in Porous Media

The chemotaxis signal transduction network regulates the biased random walk of many bacteria in favorable directions and away from harmful ones through modulating the frequency of directional reorientations. In mutants of diverse bacteria lacking the chemotaxis response, migration in classic motility agar, which constitutes a fluid-filled porous medium, is compromised; straight-swimming cells unable to tumble become trapped within the agar matrix. Spontaneous mutations that restore spreading have been previously observed in the enteric bacterium Escherichia coli, and recent work in other bacterial species has isolated and quantified different classes of nonchemotacting mutants exhibiting the same spreading phenotype. We present a theoretical description of bacterial diffusion in a porous medium-the natural habitat for many cell types-which elucidates how diverse modifications of the motility apparatus resulting in a nonzero tumbling frequency allows for unjamming of otherwise straight-swimming cells at internal boundaries and leads to net migration. A unique result of our analysis is increasing diffusive spread with increasing tumbling frequency in the small pore limit, consistent with earlier experimental observations but not captured by previous models. Our theoretical results, combined with a simple model of bacterial diffusion and growth in agar, are compared with our experimental measurements of swim ring expansion as a function of time, demonstrating good quantitative agreement. Our results suggest that the details of the cellular tumbling process may be adapted to enable bacteria to propagate efficiently through complex environments. For engineered, self-propelled microswimmers that navigate via alternating straight runs and changes in direction, these results suggest an optimal reorientation strategy for efficient migration in a porous environment with a given microarchitecture.

Layered Structure and Complex Mechanochemistry Underlie Strength and Versatility in a Bacterial Adhesive

ABSTRACT While designing synthetic adhesives that perform in aqueous environments has proven challenging, microorganisms commonly produce bioadhesives that efficiently attach to a variety of substrates, including wet surfaces. The aquatic bacterium Caulobacter crescentus uses a discrete polysaccharide complex, the holdfast, to strongly attach to surfaces and resist flow. The holdfast is extremely versatile and has impressive adhesive strength. Here, we used atomic force microscopy in conjunction with superresolution microscopy and enzymatic assays to unravel the complex structure of the holdfast and to characterize its chemical constituents and their role in adhesion. Our data support a model whereby the holdfast is a heterogeneous material organized as two layers: a stiffer nanoscopic core layer wrapped into a sparse, far-reaching, flexible brush layer. Moreover, we found that the elastic response of the holdfast evolves after surface contact from initially heterogeneous to more homogeneous. From a composition point of view, besides N-acetyl-d-glucosamine (NAG), the only component that had been identified to date, our data show that the holdfast contains peptides and DNA. We hypothesize that, while polypeptides are the most important components for adhesive force, the presence of DNA mainly impacts the brush layer and the strength of initial adhesion, with NAG playing a primarily structural role within the core. The unanticipated complexity of both the structure and composition of the holdfast likely underlies its versatility as a wet adhesive and its distinctive strength. Continued improvements in understanding of the mechanochemistry of this bioadhesive could provide new insights into how bacteria attach to surfaces and could inform the development of new adhesives. IMPORTANCE There is an urgent need for strong, biocompatible bioadhesives that perform underwater. To strongly adhere to surfaces and resist flow underwater, the bacterium Caulobacter crescentus produces an adhesive called the holdfast, the mechanochemistry of which remains undefined. We show that the holdfast is a layered structure with a stiff core layer and a polymeric brush layer and consists of polysaccharides, polypeptides, and DNA. The DNA appears to play a role in the structure of the brush layer and initial adhesion, the peptides in adhesive strength, and the polysaccharides in the structure of the core. The complex, multilayer organization and diverse chemistry described here underlie the distinctive adhesive properties of the holdfast and will provide important insights into the mechanisms of bacterial adhesion and bioadhesive applications. There is an urgent need for strong, biocompatible bioadhesives that perform underwater. To strongly adhere to surfaces and resist flow underwater, the bacterium Caulobacter crescentus produces an adhesive called the holdfast, the mechanochemistry of which remains undefined. We show that the holdfast is a layered structure with a stiff core layer and a polymeric brush layer and consists of polysaccharides, polypeptides, and DNA. The DNA appears to play a role in the structure of the brush layer and initial adhesion, the peptides in adhesive strength, and the polysaccharides in the structure of the core. The complex, multilayer organization and diverse chemistry described here underlie the distinctive adhesive properties of the holdfast and will provide important insights into the mechanisms of bacterial adhesion and bioadhesive applications.