Loss of PopZ(At) activity in Agrobacterium tumefaciens by Deletion or Depletion Leads to Multiple Growth Poles, Minicells, and Growth Defects

Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ(At)), we created deletions of the five PopZ(At) domains and assayed their localization. In addition, we created a popZ(At) deletion strain (ΔpopZ(At)) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ(At) strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZ(At) cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ(At), to stabilize growth pole accumulation of PopZ(At).