Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles

Abstract

Summary Cellulose, whose production is controlled by c‐di‐GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant–pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c‐di‐GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air–liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c‐di‐GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.

Biological role of EPS from Pseudomonas syringae pv. syringae UMAF0158 extracellular matrix, focusing on a Psl-like polysaccharide

“…Levan does not play a role in biofilm architecture, and it has been speculated to consist of a storage molecule that may protects cells against starvation 16 . Cellulose is a polymer composed of β-D-glucose units that constitutes one of the main components of the biofilm matrix produced by many bacteria 23 – 26 , and its biosynthesis has proven to be important for biofilm formation by Pss 9 , 27 .…”

Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides

“…Alginate is not considered essential to Pseudomonas biofilm formation (Laue et al ., 2006; Wozniak et al ., 2003), but has been implicated in epiphytic fitness and virulence in some P. syringae strains (Helmann et al ., 2019; McAtee et al ., 2018; Yu et al ., 1999). Cellulose is a homopolymer made of β-D-glucose monomers and is a primary component of the biofilm matrix of Pst and many other bacteria (Farias et al ., 2019; Prada-Ramirez et al ., 2016; Serra et al ., 2013). Cellulose has been suggested to play an important role in the transition between epiphytic and pathogenic phases of leaf association (Arrebola et al ., 2015).…”

“…However, to obtain a comprehensive understanding of plant infection, we also need to unravel the relationship between bacterial survival, stress tolerance and pathogenesis. In this study, we build on prior research on cellulose and alginate production in Pst (Farias et al ., 2019; Keith et al ., 2003; Perez-Mendoza et al ., 2019) to examine the intersecting roles of multiple Pst polysaccharide molecules in mediating bacterial infection and enabling epiphytic survival. Pst mutants in the alginate ( alg ), psl , cellulose ( wss ), α-glucan ( glg/tre ) and wapQ pathways were constructed, and their relative contributions to phenotypes including colony morphology, abiotic stress tolerance, epiphytic survival, and plant infection were defined.…”

Three biofilm types produced by a model pseudomonad are differentiated by structural characteristics and fitness advantage

“…A–L biofilm formation is commonplace amongst environmental pseudomonads [21–25] and other bacteria, suggesting that many aerobic bacteria can gain a competitive advantage by growing in the high-O 2 region of static microcosms if growth is not limited by some other resource, and in several model systems, including Bacillus subtilis 3610 [26], Pseudomonas aeruginosa PA01 and PA14 [27, 28], Pseudomonas putida KT2440 [29] and SBW25, wild-type strains are known to produce multiple mutants with different biofilm characteristics. In addition to the WS, SBW25 produces a range of other physically cohesive-class biofilm-forming lineages with similar fitness advantage.…”