Mechanisms of polar growth in the alphaproteobacterial order rhizobiales
All bacteria elongate and divide to faithfully reproduce their cell shape. Understanding the mechanisms that drive bacterial morphology requires an intimate knowledge of how the cell wall is synthesized. During cell division, most bacteria synthesize new cell wall at mid-cell and the mechanism underlying this process is highly conserved. In contrast, there is a high degree of diversity in bacterial growth patterning during elongation. Bacteria in the Rhizobiales exhibit an atypical form of unipolar elongation, and the molecular mechanisms of how new cell wall is synthesized during growth and division currently remains unexplored. Using microfluidics and fluorescent cell wall probes we first investigated whether polar growth is conserved in a morphologically complex bacterium, Prosthecomicrobium hirschii. We showed that P. hirschii has a dimorphic lifestyle and can switch between a long-stalked, non-motile form and a short-stalked, motile form. Furthermore, we found that all morphotypes of P. hirschii elongate using polar growth, suggesting the polar elongation is a widespread feature of bacteria in this order. Next, we used the rod-shaped bacterium Agrobacterium tumefaciens as a model to investigate the precise mechanisms that drive polar elongation. We characterized a comprehensive set of cell wall synthesis enzymes in A. tumefaciens and identified penicillin-binding protein 3a (PBP3a) and PBP3b as a synthetic lethal pair that function during cell division, and PBP1a as an essential enzyme required for polar growth and maintenance of rod shape. Compositional analysis of the PBP1a depletion, suggested that LD-transpeptidase (LDT) enzymes may play an important role in polar growth. We identified three LDTs that likely function in polar growth. We also observed subpolar localization of LDTs, suggesting bacteria in the Rhizobiales may insert or remodel cell wall material in a subpolar zone during growth. Finally, we used RNA-seq to explore changes in gene expression during PBP1a depletion, revealing that that loss of PBP1a induces a lifestyle switch which mimics the switch from a free-living bacterium into a plant-associated state. The change in lifestyle is characterized by increased exopolysaccharide production and Type VI Secretion System activity and a decrease in flagella-mediated motility. This finding indicates that bacteria have a mechanism to sense changes in cell wall composition or integrity due to the loss of PBP1a and respond through changes in gene expression that impact physiology and behavior. This finding opens the door to future studies on the link between changes in cell wall composition and complex bacterial behaviors and lifestyles. Overall, this research provides mechanistic insights about the roles of cell wall synthesis during cell growth and division in the A. tumefaciens, which are conserved in other Rhizobiales, including agriculturally and medically species such as Sinorhizobium and Brucella.
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