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Pseudomonas fluorescens ATCC 13525 and a particulate glucose oxidase (d-glucose:oxygen oxidoreductase, EC 1.1.3.4) mutant of this organism, gox-7, were examined to determine if glucose oxidation via particulate glucose oxidase is a required first step for glucose uptake. Initial [(14)C]glucose-uptake rates in parent and gox-7 cells were qualitatively similar. Initial [(14)C]glucose-uptake product analysis revealed that glucose was accumulated via active transport and was rapidly metabolized to glucose-6-phosphate and gluconate-6-phosphate in both parent and gox-7 cells. Cell extracts contained soluble adenosine 5'-triphosphate specific kinase activity for phosphorylation of glucose. Glucose uptake was induced by glucose and not gluconate, thus, establishing independent regulation of glucose transport and glucose catabolism in p. fluorescens. The results prove that glucose oxidase was not an obligatory reaction for glucose carbon permeation in P. fluorescens. A general unifying scheme for glucose utilization in the aerobic fluorescent pseudomonads is suggested for the purpose of clarifying glucose uptake in these bacteria.

Two mutations that affect expression of the Salmonella typhimurium leu operon were investigated. leu operon DNA from these mutant strains was cloned, and nucleotide sequences of the leu control regions were determined. leu-500, which eliminates expression of all four leu genes simultaneously, is a point mutation in the -10 region of the leu promoter. leu-2012 is a point mutation within the -35 region of the leu promoter. leu-2012 suppressed leucine auxotrophy caused by leu-500 only when the medium contained a carbon source that does not cause catabolite repression. A cya mutation (adenylate cyclase deficiency) introduced into the leu-500 leu-2012 strain caused leu enzymes to be made only if cAMP was supplied exogenously. A leu-500 leu-2012 strain containing a crp mutation (cAMP receptor protein deficiency), on the other hand, could not make leu enzymes even in the presence of cAMP. In vitro transcription experiments demonstrated that the leu-2012 mutation created a new transcription initiation site. RNA polymerase utilized this site in vitro in the absence of added cAMP receptor protein and cAMP.

Induction of Entner-Doudoroff pathway enzymes in Pseudomonas fluorescens was investigated to study the role of gluconate as a possible inducer. Glucose oxidase-deficient mutants were isolated and characterized. One of these mutants, gox-7, was deficient in particulate glucose oxidase; another mutant, gox-17, was deficient in particulate glucose and gluconate oxidase activities. Gluconate, but not glucose, induced synthesis of gluconokinase and 6-phosphogluconate dehydratase in both mutants. High constitutive levels of 2-keto-3-deoxy-6-phosphogluconate aldolase were found when both mutants were grown on glucose. Growth of parent and both mutant strains on glycerol also resulted in high levels of Entner-Doudoroff pathway enzymes. It was concluded that glucose cannot serve as an inducer molecule for derepression of Entner-Doudoroff pathway enzymes in P. fluorescens. Evidence presented provides good support for gluconate being the true inducer of this pathway in P. fluorescens. A relationship is presented for explaining distribution of the Entner-Doudoroff pathway in certain groups of bacteria.

A mutation, GD-1, in the leucine operon imposed unusual growth characteristics upon a leucine auxotrophic strain bearing the leucine operator mutation, leu-500. The strain with the GD-1 mutation was able to grow on a minimal salts medium when citrate was the sole carbon source, but required leucine when glucose was present. Tests with a large number of carbohydrates suggest that in the strain bearing the GD-1 mutation the leucine biosynthetic enzymes are under catabolite repressor control. Recombination studies indicate that the GD-1 mutation is a secondary alteration of the leucine operator at or very close to the site of the leu-500 mutation. Mutations at the supX locus (previously termed su leu 500 and located on the chromosome between the cysteine B and tryptophan gene clusters) result in elimination of the catabolite repression effect. The data are interpreted as an indication that the GD-1 and leu-500 mutations alter the leucine operator with respect to its specificity of response to repressors.