The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa

Williams, Steven G.; Greenwood, Jacqueline A.; Jones, Colin W.

doi:10.1099/13500872-140-11-2961

Cited by 29 publications

(28 citation statements)

References 30 publications

(14 reference statements)

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“…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 facilitator and cytoplasmic glycerol kinase, respectively (28). As previously demonstrated in E. coli (36), glycerol kinase has a propensity to associate with the cytoplasmic membrane (41). Intracellular G3P is further metabolized via a branch of the Entner-Doudoroff pathway after conversion of G3P into dihydroxyacetone phosphate by a cytoplasmic-membrane-associated, glpD-encoded G3P dehydrogenase, whose structural gene has recently been cloned and sequenced (27).…”

mentioning

confidence: 83%

“…Workers in several laboratories, including that of Williams et al (41) and ours (23,27,29), have argued the probable importance of glycerol as a source of carbon for growth and alginate synthesis within the lung. This argument was further supported by our recent discovery of glpM, which is located immediately downstream of glpD and whose product is required for efficient alginate biosynthesis from various carbon sources (29).…”

Section: Discussionmentioning

confidence: 99%

“…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 facilitator and cytoplasmic glycerol kinase, respectively (28).…”

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confidence: 99%

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

Schweizer

1996

J Bacteriol

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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...

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confidence: 83%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

Schweizer

1996

J Bacteriol

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“…Thus, glycerol uptake involves facilitated diffusion down a concentration gradient (53,54), whereas the uptake of proline is mediated by the PutP protein, which is an energy-dependent Na ϩ symporter (8,51). For the transport of glucose there are two separate inducible systems in P. aeruginosa (36); one is a low-affinity transport system which involves the extracellular oxidation of glucose to gluconate or to 2-ketogluconate prior to transport into the cytoplasm, and the second is a high-affinity system which transports glucose directly into the cytoplasm via a periplasmic binding protein-dependent ABC transport system that requires ATP as an energy source (2).…”

Section: Discussionmentioning

confidence: 99%

Role of Pseudomonas putida tol-oprL Gene Products in Uptake of Solutes through the Cytoplasmic Membrane

et al. 2003

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Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.

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“…kidney, brain and lung, represents the first mammalian water-channel homolog that selectively transports glycerol [14]. Additionally, in some organisms such as Pseudomonas aeruginosa [17] and Saccharomyces cerevisiae [28] non-saturable membrane proteins involved in glycerol uptake have been characterized and found to be integral pore proteins. We therefore tested in insect forms of T. brucei a variety of inhibitors, such as DIDS and ClHgBzOH, known to interfere with such membrane proteins; however, a reduction in glycerol uptake was not detected (Fig.…”

Section: Discussionmentioning

confidence: 99%

Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei

Wille

Schade

Duszenko

1998

European Journal of Biochemistry

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Uptake of glycerol was studied in bloodstream and insect forms of the African parasite Trypanosoma brucei using [14 C]glycerol in combination with the oil centrifugation technique. Our kinetic measurements revealed that in bloodstream forms glycerol appeared to be transported by two different mechanisms : firstly by a facilitated-diffusion carrier showing a K m of 0.17 mM and a Vmax of 44 nmol 10 Ϫ8 cells min Ϫ1 that predominates at low glycerol concentrations, and secondly by simple diffusion. The effects induced by various inhibitors suggest that uptake is neither sodium dependent nor proton-motive-force driven. The saturable component of transport was phloretin and cytochalasin B sensitive and could also be inhibited by the substrate analogue glyceraldehyde, which led to a 74% decrease in glycerol uptake. In insect forms, however, glycerol is taken up by simple diffusion only. Uptake was insensitive to mercury ions and was not influenced by a variety of different channel inhibitors. Our data show that in T. brucei glycerol transport across the plasma membrane occurs by simple diffusion. In addition, bloodstream forms express a carrier protein which promotes a rapid transport at low glycerol concentrations. Expression of this transport protein may account for a selective secretion of intracellular glycerol which otherwise could become toxic for the parasite due to its specific compartmentation of glycolysis.Keywords : glycerol uptake; facilitated diffusion; inhibitor studies; Trypanosoma brucei.Trypanosoma brucei, a protozoan parasite which causes sleeping sickness and economically important cattle diseases in Africa, is transmitted by the tsetse-fly. Insect and bloodstream forms show marked biochemical differences, especially with respect to energy metabolism. While the insect form contains a well-developed mitochondrion with both a functional citric acid cycle and respiratory chain, bloodstream forms rely exclusively on glycolysis for their ATP production [1,2]. Interestingly, in all members of the order Kinetoplastida, glycolysis occurs in a characteristic organelle: the glycosome [3]. Under anaerobic conditions, e.g. induced by salicyl hydroxamate which inhibits glycerol-3-phosphate oxidase, bloodstream-form trypanosomes produce equimolar amounts of pyruvate and glycerol [4]. At high concentrations both metabolites are toxic for the parasite and have to be removed in order to avoid cell death. Since a specific pyruvate transporter was recently demonstrated in T. brucei [5,6], we were interested in how glycerol transport across the plasma membrane occurs. The existence of a specific glycerol carrier in bloodstream-form trypanosomes would offer the possibility to interfere effectively with the parasite's metabolism Correspondence to M. Duszenko, Physiologisch-chemisches Institut,

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The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa

Cited by 29 publications

References 30 publications

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

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

Role of Pseudomonas putida tol-oprL Gene Products in Uptake of Solutes through the Cytoplasmic Membrane

Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei

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