Regulation of Exopolysaccharide Production by ProE, a Cyclic-Di-GMP Phosphodiesterase in Pseudomonas aeruginosa PAO1.

The ubiquitous second messenger c-di-GMP is involved in regulation of multiple biological functions including the important extracellular matrix exopolysaccharides (EPS). But how c-di-GMP metabolic proteins influence EPS and their enzymatic properties are not fully understood. Here we showed that deletion ofproE, which encodes a protein with GGDEF-EAL hybrid domains, significantly increased the transcriptional expression of the genes encoding EPS production inPseudomonas aeruginosaPAO1 and changed the bacterial colony morphology. Our data showed that ProE is a very active phosphodiesterase (PDE), with a high enzyme activity in degradation of c-di-GMP. Interestingly, the optimal activity of ProE was found in the presence of Co2+, unlike other PDEs that commonly rely on Mg(2+)or Mn(2+)for best performance. Furthermore, we identified three widely conserved novel residues that are critical for the function of ProE through site-directed mutagenesis. Subsequent study showed that ProE, together with other three key PDEs, i.e., RbdA, BifA, and DipA regulate the EPS production inP. aeruginosaPAO1. Moreover, by using the GFP-fusion approach, we observed that these four EPS associated-PDEs showed a polar localization pattern in general. Taken together, our data unveil the molecular mechanisms of ProE in regulation of EPS production, and provide a new insight on its enzymatic properties in degradation of c-di-GMP.