CAREER: Composition, mechanical properties, and synthesis of the Caulobacter crescentus stalk

Why cells develop and maintain a particular shape represents a fundamental question in cell biology. Understanding the maintenance of bacterial cell shape have primarily focused on rod-shaped cells. Yet we know of myriad cell shapes present in nature that are maintained by strong selective pressures. Determining the roles and underlying mechanisms of these unusual morphologies can provide great insight into a wide range of bacterial physiological properties. In particular, some bacteria synthesize unique 'appendage' structures, or localized cellular extensions. For example, the stalked bacterium Verrucomicrobium spinosum produces a large number of spikes across its entire surface and Mycoplasma pneumoniae adherence requires a membrane-bound cell extension termed the tip-structure. In order to study the regulation and synthesis of such bacterial appendages, this project focuses on the model stalked bacterium Caulobacter crescentus. Preliminary data suggest that the stalk cell wall is chemically distinct from that of the cell body; this finding is striking because it would be the first demonstration of a bacterium with differentially regulated domains containing unique chemical structures. A detailed understanding of the chemical and mechanical properties of the stalk cell wall and characterization of proteins involved in stalk elongation will provide important insight into the fundamental question of the evolution and function of bacterial shape and compartmentalization. In addition to the research goals of this project, there are several educational objectives targeting high school, undergraduate, and graduate students. New courses will be developed for students at each level to train the next generation of scientists in quantitative methods in biology including bioinformatics, quantitative image analysis, and biophysics. In particular, the high school component will involve students at the LEAP Academy in Camden, whose enrollment is 90% underrepresented minorities. Each of these programs is designed to interface with and contribute to the two research projects.

The objective of this project is to determine the mechanisms underlying localized bacterial envelope synthesis and appendage formation using the polar-stalk of Caulobacter crescentus as a model system. The central hypothesis is that Caulobacter produces a polar stalk whose peptidoglycan (PG) has distinct chemical and mechanical properties from the cell body PG. Previous studies of stalk synthesis have either focused on known PG regulating enzymes or on the localization of stalk associated proteins. In contrast, this approach is innovative because the starting point is the observation that the stalk envelope is compositionally different than that of the cell body. From this perspective, the central

hypothesis will be tested by the following specific aims: 1) Define the composition and mechanical properties of stalk peptidoglycan; 2) Targeted analysis of the stalk biosynthesis regulatory proteins; and 3) Identify novel stalk-elongation genes by genotyping stalk-phenotype mutants. Under the first aim, experimental approaches will be used to determine the chemical makeup and quantify the elastic modulus of stalk PG. The link between PG composition and mechanical properties will be further explored via computational modeling. Under the second aim, the role of MreB will be investigated using a novel MreB fluorescent fusion construct that is defective for stalk biogenesis while retaining wild type function for cell growth. Studies of the putative transpeptidase CC2105 will focus on a C-terminal domain which is uniquely conserved among the stalked bacteria of the Caulobacteraceae family. In the third aim, high school students at the LEAP Academy will use a combination of traditional genetics methods and next generation sequencing to characterize a set of recently isolated stalk-elongation mutants. The proposed research is anticipated to contribute in-depth knowledge of how stalk-specific envelope synthesis results in compartmentalized regulation of cell shape. This vertical advance in the field of bacterial cell biology suggests that the dynamic regulation of localized morphology may be a general strategy employed by bacteria to maintain physiological control over cell shape or create subcellular compartments/appendages.