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Summary: The fmdA and fmdB genes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacterium Methylophilus methylotrophus were recloned with additional flanking DNA (pSW1). fmdC, encoding a weakly hydrophilic protein containing an N-terminal signal sequence, was identified upstream of fmdAB. The derived amino acid sequence of mature FmdC (Mr 39204) showed that it was rich in -sheet and aromatic amino acids, and exhibited significant similarities to several outer-membrane porins from other bacteria. Cell fractionation studies showed that the protein was located in the outer membrane. Mature FmdC was purified and shown to consist of a single type of subunit (M r 40000) with the predicted N-terminal amino acid sequence (GATISF-). SDS-PAGE and Western blotting of cells grown in continuous culture under various conditions showed that mature FmdC was induced by formamide, acetamide and urea, repressed by excess ammonia, and over-expressed during prolonged growth under formamide limitation. It is concluded that mature FmdC is a porin involved in the transport of short-chain amides and urea through the outer membrane of M. methylotrophus under conditions where these nitrogen sources are present at very low concentration.

Pseudomonas aeruginosa M60, a mucoid strain, was grown in continuous culture (D 0-05 h−1) under ammonia limitation with glucose as the carbon source. Steady-state alginate production occurred for only 1-2 d under these conditions [q alginate 0.097 g alginate h−1 (g dry wt cells)−1], after which time the percentage of mucoid cells and the alginate concentration in the culture decreased in parallel and approached zero after approximately 10 d. These changes were accompanied by similar decreases in the activities of the alginate biosynthetic enzymes (represented by phosphomannomutase and GDP-mannose dehydrogenase) and by a large increase in the activity of the first enzyme of the ‘external’ non-phosphorylative pathway of glucose metabolism, glucose dehydrogenase. In contrast, the activities of other enzymes associated with this pathway (gluconate dehydrogenase, 2-ketogluconate kinase plus 2-ketogluconate-6-phosphate reductase) or with the ‘internal’ phosphorylative pathway of glucose metabolism (glucokinase and glucose-6-phosphate dehydrogenase) remained essentially unchanged. The loss of mucoidy and alginate production was accompanied by the appearance of low concentrations of intracellular polyhydroxyalkanoate (PHA) and of extracellular gluconate and 2-ketogluconate (partly at the expense of alginate production and partly as a result of increased glucose consumption). It is suggested that ammonia-limited, glucose-excess cultures of P. aeruginosa growing at low dilution rate are unable fully to regulate the rate at which glucose and/or its ‘external’ pathway metabolites are taken up by the cell, and therefore form copious amounts of alginate in order both to overcome the potentially deleterious osmotic effects of accumulating surplus intracellular metabolites and to consume the surplus ATP generated by the further oxidation of these metabolites. The loss of mucoidy invokes the use of an alternative, but analogous, strategy via which non-mucoid cells produce an osmotically inactive intracellular product (PHA) plus increased amounts of the extracellular metabolites gluconate and 2-ketogluconate via the low-energy-yielding and, under these conditions, largely dead-end ‘external’ metabolic pathway.

Washed cells of Agrobacterium radiobacter prepared from a fructose-limited continuous culture (D 0.045 h-1) transported D(-)[U-14C]fructose in a linear manner for up to 4 min at a rate several-fold higher than the rate of fructose utilization by the growing culture. D(-)[U-14C]Fructose transport exhibited a high affinity for fructose (KT1 microM) and was inhibited to varying extents by osmotic shock, by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and by unlabelled sugars (D-fructose/D-mannoseD-riboseD-sorboseD-glucose/D-galactose/D-xylose; no inhibition by D-arabinose). Prolonged growth of A. radiobacter in fructose-limited continuous culture led to the selection of a novel strain (AR100) which overproduced a fructose-binding protein (FBP) and showed an increased rate of fructose transport. FBP was purified from osmotic-shock fluid using anion-exchange fast protein liquid chromatography (FPLC). The monomeric protein (M(r) 34,200 by SDS-PAGE and 37,700 by gel-filtration FPLC) bound D-[U-14C]-fructose stoichiometrically (1.17 nmol nmol FBP-1) and with high affinity (KD 0.49 microM) as shown by equilibrium dialysis. Binding of D-[U-14C]fructose by FBP was variably inhibited by unlabelled sugars (D-fructose/D-mannoseD-riboseD-sorbose; no inhibition by D-glucose, D-galactose or D-arabinose). The N-terminal amino acid sequence of FBP (ADTSVCLI-) was similar to that of several sugar-binding proteins from other species of bacteria. Fructose transport and FBP were variably induced in batch cultures of A. radiobacter by growth on different carbon sources (D-fructoseD-ribose/D-mannoseD-glucose; no induction by succinate). An immunologically similar protein to FBP was produced by Agrobacterium tumefaciens and various species of Rhizobium following growth on fructose. It is concluded that fructose is transported into A. radiobacter and related organisms via a periplasmic fructose/mannose-binding-protein-dependent active-transport system, in contrast to the phosphotransferase system used by many other species of bacteria.

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 (Km < or = 2 microM) 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 48,000 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.