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

Abstract

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 of proE, which encodes a protein with GGDEF-EAL hybrid domains, significantly increased the transcriptional expression of the genes encoding EPS production in Pseudomonas aeruginosa PAO1 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 Co 2+ , 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 in P. aeruginosa PAO1. Moreover, by using the GFPfusion 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.

Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes

“…Deletion of PA0575, PA1433 (lapD), PA1727 (mucR; Wang et al, 2015;Bellini et al, 2017;Zemke et al, 2020), PA4959 (fimX), PA5017 (dipA) and PA5442 had a far less significant impact but led to formation of smaller structures compared to PAO1. The absence of PA2567, PA3311, PA4601 (morA) and PA5295 (proE; Feng et al, 2020) did not cause any major changes in biofilm profiles. Here again, one notices that some genes that are significantly involved in initial attachment, e.g., PA2567, are of no importance for maturation of the biofilm after 72 h. Also, genes that are important for initial attachment, e.g., PA0285 and PA2567, may subsequently have quite different impacts on maturation by, respectively, failing to form mushrooms or not.…”

A library of promoter-gfpfusion reporters for studying systemic expression pattern of cyclic-di-GMP metabolism-related genes inPseudomonas aeruginosa

“…Overall, the transcription levels of most genes were relatively low at 25°C or 30°C compared to that of 37°C, while there are 10 genes, namely PA0338, PA3177, PA3343 ( hsbD ), PA4843 ( gcbA ), PA2818 ( arr ), PA1433 ( lapD ), PA4367 ( bifA ), PA4601 ( morA ), PA4959 ( fimX ), and PA5295 ( proE ), have similar transcriptional levels at three tested temperatures (Figure 3, marked with red number). Among these 10 genes, PA3177 and PA3343 ( hsbD ) encode DGCs (21, 22), PA2818 ( arr ), PA4367 ( bifA ), PA4959 ( fimX ) and PA5295 ( proE ) encode PDEs (36, 37, 39, 40), while PA4601 ( morA ) functions as both DGC and PDE. Furthermore, MorA is conserved among diverse Pseudomonas species (47, 48).…”

“…The expression of two genes, PA5295 ( proE ) and PA5442 are also induced under sub-inhibitory concentrations of tobramycin (Figure 5A). ProE is a very active PDE with high enzymatic activity in the degradation of c-di-GMP and plays an important role in regulating EPS production in P. aeruginosa PAO1 (39), and the function of PA5442 is not determined. The transcription of 7 genes (PA1107, PA4332, PA4396, PA4781, PA4601, PA4959, PA5017) were not changed by tobramycin.…”

Interplay between the cyclic di‐GMP network and the cell–cell signalling components coordinates virulence‐associated functions in Xanthomonas oryzae pv. oryzae

“…As an example in S. enterica, the DGC AdrA promotes cellulose biosynthesis by providing c-di-GMP while in Pseudomonas aeruginosa strains, Pel exopolysaccharide biosynthesis is dependent on c-di-GMP production by DGCs WspR (PA3702) and YfiN (PA1120) (Ueda and Wood, 2009). On the other hand, ProE, an active PDE in PA01 negatively regulates the transcriptional expression of pel and psl (Feng et al, 2020). In the present study, a GGDEF overexpressing construct in wild type Xoo with resultant rise in the level of c-di-GMP suppressed the EPS production to a certain magnitude (Fig.…”