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

Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK

Author for correspondence: Colin W. Jones. Tel: +44 533 523458. Fax: +44 533 523369.

ABSTRACT

Pseudomonas aeruginosa NM48, a non-mucoid derivative of an alginate-producing strain isolated from a cystic fibrosis patient, was grown in batch culture with glycerol, glucose or succinate as carbon source, and in continuous culture (D 0.05 h^-1) under glycerol or glucose limitation. Glycerol uptake, glycerol kinase and glycerol-3-phosphate dehydrogenase were induced by glycerol, but not by glucose or succinate. Linear uptake of [¹⁴C]glycerol by washed cells (K_m 2 µM) was inhibited by unlabelled glycerol and glyceraldehyde, but not by cyanide or the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and was accompanied by substantial intracellular accumulation of glycerol-3-phosphate and/or dihydroxyacetone phosphate but not glycerol. Prolonged growth under glycerol limitation led to substantial increases in the activities and/or concentrations of the enzymes catalysing glycerol uptake and metabolism, together with a 48000 M_r outer-membrane protein which was also over-expressed following prolonged growth under glucose limitation. The N-terminal amino acid sequence (AEAFSPN-) and electrophoretic properties of this protein were the same as those of the previously characterized glucose porin (OprB) from P. aeruginosa, indicating that this porin is active with both glucose and glycerol. It is concluded that during growth under glycerol limitation, glycerol is transported into P. aeruginosa NM48 via OprB and a high-affinity, binding-protein-independent facilitated-diffusion system.

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