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Pseudomonas syringae  pv. tomato DC3000 carries the  wssABCDEFGHI  operon for the synthesis of acetylated cellulose, whose production is stimulated by increasing the intracellular levels of the second messenger c-di-GMP. This enhances air-liquid biofilm formation and generates a wrinkly colony morphotype in solid media. In the present study we show that cellulose production is a complex process regulated at multiple levels and involving different players in this bacterium. Using different  in vitro  approaches, including  E lectrophoretic  M obility  S hift  A ssay (EMSA) and footprint analysis, we demonstrated the interrelated role of two transcriptional regulators, AmrZ and FleQ, over cellulose production in Pto DC3000 and the influence of c-di-GMP in this process. Under physiological c-di-GMP levels, both regulators bind directly to adjacent regions at the  wss  promoter inhibiting its expression. However, just FleQ responds to c-di-GMP releasing from its  wss  operator site and converting from a repressor to an activator of cellulose production. The additive effect of the double  amrZ/fleQ  mutation on the expression of  wss , together with the fact that they are not cross-regulated at the transcriptional level, suggest that FleQ and AmrZ behave as independent regulators, unlike what has been described in other  Pseudomonas  species. Furthermore, this dual co-regulation exerted by AmrZ and FleQ is not limited to cellulose production, but also affects other important phenotypes in Pto DC3000, such as motility and virulence.

AmrZ and FleQ Co-regulate Cellulose Production in Pseudomonas syringae pv. Tomato DC3000