Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Pathogenesis

Background:

• Cellulose production in bacterial biofilms is regulated by cyclic-di-GMP (c-di-GMP).
• Pseudomonas syringae pv. tomato (Pto) DC3000 synthesizes acetylated cellulose via the wssABCDEFGHI operon.
• Elevated c-di-GMP levels stimulate cellulose production and pellicle formation.

Purpose of the Study:

• To investigate the mechanisms underlying pellicle formation in Pto DC3000.
• To determine the roles of flagella, biosurfactant, and extracellular matrix components in pellicle development.
• To characterize the effects of high c-di-GMP levels on pellicle structure and cell behavior.

Main Methods:

• Comparative analysis of pellicle formation in wild-type and mutant Pto DC3000 strains (lacking flagella, biosurfactant, or specific extracellular matrix components).
• Overexpression of the heterologous diguanylate cyclase PleD to induce high c-di-GMP levels.
• Development of novel techniques for collecting and processing labile pellicles for electron microscopy (scanning and transmission).

Main Results:

• Cellulose and flagella are important but not essential for pellicle formation.
• Alginate and the biosurfactant syringafactin are not required for pellicle development.
• High c-di-GMP levels enhance pellicle cohesion, induce intracellular inclusion bodies, and promote extracellular fiber and vesicle formation.

Conclusions:

• Pellicle formation in Pto DC3000 is primarily driven by cellulose and influenced by flagella, with high c-di-GMP levels playing a crucial role in structural integrity and cellular organization.
• Novel microscopy preparation techniques facilitate the study of fragile bacterial biofilms, offering new avenues for research in other bacterial systems.