coliandSalmonella(11, 12) making use of the nonmetabolizable glucose analog -methyl glucoside (MG), which is taken up efficiently by PtsG and represses cell growth. strains. The results obtained suggested that sugar phosphate accumulation caused diminished growth in some of the mutant strains, since this was partially relieved bysppoverexpression. On the other hand, overexpression ofsppin fructose-grown alginate-producing strains 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 thesppgene did not affect growth of the wild-type strain on fructose, but the gene could not be deleted in the alginate-producing strain. This indicates that Spp is essential for relieving the cells of sugar phosphate stress inP. fluorescens actively producing alginate. IMPORTANCEIn enteric bacteria, the sugar phosphate phosphatase YigL is known to play an important role in combating stress caused by sugar phosphate accumulation. In this study, we identified a sugar phosphate phosphatase, designated Spp, inPseudomonas 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 inP. fluorescensmutants experiencing sugar phosphate stress. The gene was localized downstream ofgndandzwf-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 functions. KEYWORDS: Pseudomonas fluorescens, alginate biosynthesis, sugar phosphate stress response, phosphatase, PFLU2693, YigL, sugar phosphate phosphatase == INTRODUCTION == Pseudomonascentral carbon metabolism has been well studied during recent decades (13). UnlikeEscherichia coli, which utilizes the phosphotransferase system type G (PtsG) as the major glucose transporter (4), most pseudomonads import glucose through an active transport system (5). In contrast, fructose transport is mediated by the phosphoenolpyruvate (PEP)-dependent phosphotransferase system in these organisms. Pseudomonasspecies lack the 6-phosphofructokinase gene required for glycolysis, and glucose is converted to glyceraldehyde-3-phosphate (GAP) and pyruvate (Pyr) via the Entner-Doudoroff Sstr5 (ED) pathway. In addition , several studies have already shown that fructose is mainly metabolized through the ED route by converting fructose-1, 6-bisphosphate (FBP) to fructose-6-phosphate (F6P) and then to glucose-6-phosphate (G6P) (1, 6) (Fig. 1). == FIG 1 . == Glucose and fructose metabolic pathways inP. fluorescens. Abbreviations: G6P, glucose-6-phosphate; 6PGA, 6-phosphogluconate; 2KGP, 2-keto-6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; F6P, fructose-6-phosphate; FBP, fructose-1, 6-bisphosphate; F1P, fructose-1-phosphate; GAP, glyceraldehyde-3-phosphate; Pyr, pyruvate; DPGA, 1, 3-diphosphoglycerate; 3PGA, 3-phosphoglycerate; 2PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; OA, oxaloacetate; Mal, malate; Gad, gluconate Betaxolol dehydrogenase; Gcd, glucose dehydrogenase; Kgk, 2-ketogluconate kinase; Gnk, gluconokinase; Glk, glucokinase; Kgr, 2-KGP reductase; Gnd, 6PGA dehydrogenase; Zwf1-2, G6P dehydrogenases; Pgi, phosphoglucose isomerase; Pgl, 6-phosphogluconolaktonase: Edd, 6PGA dehydratase; Eda, KDPG aldolase; 1Fpk, 1-phosphofructokinase; Fdp, fructose-1, 6-bisphosphatase; Fda, fructose-1, 6-bisphosphate aldolase. Figure abridged from reference10. Alginate is a polysaccharide with a wide variety of industrial and medical applications, and Betaxolol it is composed of variable ratios of -d-mannuronic acid (M) and -l-guluronic acid (G). Two genera of bacteria, PseudomonasandAzotobacter, are capable of producing alginate. Alginate biosynthesis uses F6P as a precursor to generate GDP-mannuronic acid, which then is polymerized, transported through the periplasm, and secreted through the AlgE porin in the outer membrane (7, 8). Pseudomonas fluorescensstrain Pf201 is a nonpathogenic mutant strain that was developed in our group by random mutagenesis of the wild-type strain NCIMB10525 and studied as a stable alginate producer (9). In our recent study focusing on the role of genes involved in central carbon metabolism of Pf201 on alginate production (10), it was hypothesized that the observed growth deficiencies of the glucose-6-phosphate dehydrogenase (G6PD) (encoded byzwf-1andzwf-2) mutants of the NCIMB10525 strain grown on fructose resulted from accumulation of hexose phosphates (G6P and Betaxolol F6P) causing sugar phosphate stress. It was shown that the growth defect was less severe in azwf-1mutant of an alginate-producing strain, possibly because alginate production may drain away a portion of the excessive sugar phosphates (10). This hypothesis motivated our current study to investigate any possible sugar phosphate stress response in Pf201 further. A sugar phosphate stress response has been studied inE. coliandSalmonella(11, 12) using the nonmetabolizable glucose analog -methyl glucoside (MG), which is taken up efficiently by PtsG and represses cell growth. Recently, Papenfort et al. provided strong evidence that the phosphatase YigL inSalmonella entericaserovar Typhimurium is needed for dephosphorylation of accumulated hexose phosphate prior to efflux by an unidentified efflux pump (11). Sugar phosphate stress is still not well understood, but current evidence indicates that accumulation of sugar phosphates leads to depletion of.