Identification of a New Phosphatase Enzyme Potentially Involved in the Sugar Phosphate Stress Response in Pseudomonas fluorescens

The alginate-producing bacterium Pseudomonas fluorescens utilizes the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to metabolize fructose, since the upper part of its Embden-Meyerhof-Parnas pathway is defective. Our previous study indicated that perturbation of the central carbon metabolism by diminishing glucose-6-phosphate dehydrogenase activity could lead to sugar-phosphate stress when P. fluorescens was cultivated on fructose. In the present study, we demonstrate that PFLU2693, annotated as a haloacid dehalogenase-like enzyme, is a new sugar-phosphate phosphatase now designated Spp, which is able to dephosphorylate a range of phosphate substrates including glucose 6-phosphate and fructose 6-phosphate in vitro The effect of spp overexpression on growth and alginate production was investigated using both wild type and several mutant strains. The results obtained suggested that sugar-phosphate accumulation is causing diminished growth in some of the mutant strains, since this was partially relieved by spp overexpression. On the other hand, overexpression of spp in fructose-grown alginate-producing strain negatively affected both growth and alginate production. The latter implies that Spp dephosphorylates the sugar phosphates, thus depleting the pool of these important metabolites. Deletion of the spp gene did not affect growth of the wild type strain on fructose, but the gene could not be deleted in the alginate-producing strain. This may indicate that Spp is essential for relieving the cells of sugar-phosphate stress in P. fluorescens actively producing alginate. IMPORTANCE: In enteric bacteria, the sugar-phosphate phosphatase YigL is known to play an important role to combat stress caused by sugar-phosphate accumulation. In this study, we identified a sugar-phosphate phosphatase designated Spp in Pseudomonas fluorescens. Spp utilizes glucose 6-phosphate, fructose 6-phosphate and ribose 5-phosphate as substrates, and overexpression of the gene had a positive effect on growth in P. fluorescens mutants experiencing sugar-phosphate stress. The gene was localized downstream of gnd and zwf-2, which encode enzymes involved in the pentose phosphate and Entner-Doudoroff pathways. Genes encoding Spp homologues were identified in similar genetic contexts in some bacteria belonging to several phylogenetically diverse families, suggesting similar function