Influence of (p)ppGpp on biofilm regulation in Pseudomonas putida KT2440

The global regulatory molecule (p)ppGpp is synthesized under limited nutrition conditions and involves in many cellular processes in bacteria. (p)ppGpp has been reported to affect biofilm formation in several bacterial species. Here, we found that deletion of (p)ppGpp synthase genes of Pseudomonas putida KT2440 led to enhanced biofilm formation in polystyrene microtitre plates. Besides, the pellicle of this mutant formed at the air -liquid interface lost the robust structure and became frail. The biofilm formation and its structure are mainly determined by exopolysaccharides (EPSs) and adhesins. Transcriptional analysis of four EPS operons designated as pea, peb, alg and bcs and two adhesin genes nominated as lapA and lapF showed that the deletion of (p)ppGpp synthase genes increased the expression of peb, bcs and lapA but repressed the expression of pea and lapF. Furthermore, expression of the regulation factor FleQ was significantly augmented in (p)ppGpp-synthase mutants while the expression of sigma factor RpoS was reduced. Since FleQ and RpoS play important roles in regulating expression of EPS and adhesin genes, (p)ppGpp may mediate the synthesis of biofilm matrix via influencing these regulators to control the biofilm formation and pellicle structure.