Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene

Abstract

The operons of the glp regulon encoding the glycerol metabolic enzymes of Pseudomonas aeruginosa were hitherto believed to be positively regulated by the product of the glpR regulatory gene. During nucleotide sequence analysis of the region located upstream of the previously characterized glpD gene, encoding snglycerol-3-phosphate dehydrogenase, an open reading frame (glpR) was identified which encodes a protein of 251 amino acids that is 59% identical to the Glp repressor from Escherichia coli and could be expressed as a 28-kDa protein in a T7 expression system. Inactivation of chromosomal glpR by gene replacement resulted in constitutive expression of glycerol transport activity and glpD activity. These activities were strongly repressed after introduction of a multicopy plasmid containing the glpR gene; the same plasmid also efficiently repressed expression of a glpT-lacZ ؉ transcriptional fusion in an E. coli glpR mutant. Analysis of the glpD and glpF upstream region identified conserved palindromic sequences which were 70% identical to the E. coli glp operator consensus sequence. The results suggest that the operons of the glp regulon in P. aeruginosa are negatively regulated by the action of a glp repressor.In Escherichia coli, the catabolism of glycerol and sn-glycerol-3-phosphate (G3P) is mediated by the components of the glp regulon (5). The genes of this regulon are organized in at least five operons which map in three separate regions on the chromosome (13). The glpFK operon encodes a membrane diffusion facilitator for glycerol and a cytoplasmic glycerol kinase (37), the glpTQ operon encodes a membrane-associated G3P permease and a periplasmic glycerophosphodiester phosphodiesterase (8, 34), the glpACB operon (4) encodes anaerobic G3P dehydrogenase, the glpD gene encodes aerobic G3P dehydrogenase (1), and glpEG encodes proteins with unknown functions (22). By genetic (5) and physical means (21,22,37), it has conclusively been demonstrated that each operon is negatively regulated by a single repressor encoded by glpR, which maps adjacent to and is divergently transcribed from glpD (21, 22). The inducer for the E. coli glp regulon is G3P (5).The glp operons exhibit differential sensitivity to the glp repressor (13, 37). They are also subject to regulation by the cyclic-AMP-catabolite gene activator protein system (13, 37), and expression of the dehydrogenase genes (glpACB and glpD) is controlled by anaerobiosis (11).In Pseudomonas aeruginosa, the nature of glycerol metabolism has yet to be fully elucidated. Although biochemical and genetic data had originally demonstrated the presence of a high-affinity, energy-dependent transport system (30) associated with a periplasmic binding protein (35), subsequent experiments suggested that glycerol is transported by a high-affinity, binding protein-independent facilitated-diffusion system (41). This finding was corroborated by the recent cloning and sequencing of the glpFK operon, whose gene products exhibited ϳ80% identity to the E. coli glycerol diffusion faci...

Involvement of an Inducible Fructose Phosphotransferase Operon in Streptococcus gordonii Biofilm Formation

“…The 741-bp fruR ORF encodes a 247-amino-acid sequence with a predicted molecular mass of 27.5 kDa. The deduced amino acid sequence showed a high degree of homology to the sequences of members of the deoxyribonucleoside repressor (DeoR) family of bacterial regulatory proteins (35).…”

Structure and gene-polypeptide relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpk) of Pseudomonas aeruginosa

“…l a ) (Schweizer & Po, 1996) suggest that glpFK are the sole genes of the operon and that glpX (Truniger et al, 1992), if present in P. aeruginosa, is not a member of the glpFK operon. Previous studies showed that glpFK expression is negatively regulated by the action of a glp repressor and that the putative glpF promoter region contained a palindromic sequence that showed a 70% match with the E. coli glp operator consensus sequence (Schweizer & Po, 1996). Thus, the glpFK operon is a member of a glp regulon.…”

“…BamHI 1600 Schweizer & Po, 1994); F and K, glpF and glpK (encoding glycerol diffusion facilitator and glycerol kinase, respectively; this study); M, glpM (membrane protein affecting alginate synthesis; Schweizer et a/., 1995); R, glpR (glp regulatory gene; Schweizer & Po, 1996); X and Y, glpX and glpY, encoding a transcriptional regulator and carbohydrate kinase, respectively, of unknown function; this study); Af, Aflll; Ba, BamHI; CI, Clal; Ec, EcoRl; Ps, Pstl; Sp, Sphl. (b) Nucleotide sequence of an Aflll-Sphl fragment containing glpf and glpK is shown.…”

“…All of the enzymes involved in transport and metabolism of glycerol and G3P are inducible by both of these substrates, suggesting that their structural genes form a glp regulon (McCowen et al, 1981). More recently it was shown that glp gene expression in P. aeruginosa is negatively regulated by the action of the glpR-encoded Glp repressor (Schweizer & Po, 1996), in an analogous manner to glp expression in E. coli. Although the discovery of a number of GlpF and GlpK homologues in Gram-negative (Fleischmann et al, 1995 ;Sweet et al, 1990;Weissenborn & Larson, 1992) and Gram-positive (Claiborne et al, 1995;Holmberg et al, 1990) bacteria suggested the existence of a common mechanism for bacterial glycerol uptake, the nature of transport of this important nutrient in P. aeruginosa hitherto remained unclear.…”

AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933

“…However, generation of an agmR : : Tc r mutant of P. aeruginosa PAO1 shows no detectable glp phenotype (Schweizer, 1992;Schweizer & Po, 1996). In contrast, a Pseudomonas putida agmR : : Km r mutant (AVP2) was described to be unable to grow on decanol, ethanol and glycerol (Vrionis et al, 2002).…”