Clustering of mutations affecting central pathway enzymes of carbohydrate catabolism in Pseudomonas aeruginosa.

Mutations in carbohydrate-negative mutants of Pseudomonas aeruginosa PAO1 individually deficient in glucose 6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), or pyruvate carboxylase (pyc) were mapped on the chromosome by plasmid R68.45-mediated conjugation and by bacteriophage F116L-mediated transduction. Loci for all three genes were located in the 45- to 55-min region of the chromosome; both zwf-1 and edd-1 were linked by transduction to nalA, whereas pyc-2 was linked by conjugation to argF10. The zwf-1 mutation exhibited cotransduction frequencies of greater than 95% with both edd-1 and the hex-9001 marker, a mutation reported to prevent growth on hexoses. The latter mutation was shown to cause a specific deficiency in 2-keto-3-deoxy-6-phosphogluconate aldolase activity and was redesignated eda-9001. These results demonstrate tight clustering of the gene loci for glucose 6-phosphate dehydrogenase and for both enzymes unique to the Entner-Doudoroff pathway in P. aeruginosa. Our evidence suggests supraoperonic clustering of these and other inducible carbohydrate catabolic genes in the 45- to 55-min region of the chromosome.

Characterization and genetic mapping of fructose phosphotransferase mutations in Pseudomonas aeruginosa.

Pseudomonas aeruginosa transports and phosphorylates fructose via a phosphoenolpyruvate-dependent fructose phosphotransferase system (PTS). Mutant strains deficient in both PTS activity and glucose-6-phosphate dehydrogenase activity were isolated and were used to select mannitol-utilizing revertant strains singly deficient in PTS activity. These mutants were unable to utilize fructose as a carbon source and failed to accumulate exogenously provided [14C]fructose, and crude cell extracts lacked phosphoenolpyruvate-dependent fructose PTS activity. Thus, the PTS was essential for the uptake and utilization of exogenously provided fructose by P. aeruginosa. Mutations at a locus designated pts, which resulted in a loss of PTS activity, exhibited 57% linkage to argF at 55 min on the chromosome in plasmid R68.45-mediated conjugational crosses. The pts mutations in four independently isolated mutant strains exhibited from 11 to 20% linkage to argF, and one of these mutations exhibited 3% linkage to lys-9015 in phage F116L-mediated transductional crosses.

Genetic locus, distant from ptsM, affecting enzyme IIA/IIB function in Escherichia coli K-12.

Most strains of Escherichia coli K-12 are unable to use the enzyme IIA/IIB (enzyme IIMan) complex of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in anaerobic growth and therefore cannot utilize glucosamine anaerobically. Introduction into these strains of a ptsG mutation, which eliminates activity of the enzyme IIIGlc/IIB' complex of the PTS, resulted in inability to grow anaerobically on glucose and mannose. Derivative strains able to grow anaerobically on glucosamine had mutations at a locus close to man, the gene coding for phosphomannose isomerase, and had higher enzyme IIA/IIB activities during anaerobic growth than did the parental strain. These results establish a locus affecting function of enzyme IIA/IIB that maps distant from ptsM, the probable structural gene for enzyme IIB.

New maltose Blu mutations in Escherichia coli K-12.

Mutations in the genes pgi, pfkA, and ptsG resulted in a maltose Blu phenotype in Escherichia coli K-12, bringing the number of known Blu alleles to six. The Blu phenotype, as visualized by staining with iodine vapor, is a convenient mutant isolation technique.

Lack of glucose phosphotransferase function in phosphofructokinase mutants of Escherichia coli.

Phosphofructokinase (pfkA) mutants of Escherichia coli are impaired in growth on all carbon sources entering glycolysis at or above the level of fructose 6-phosphate (nonpermissive carbon sources), but growth is particularly slow on sugars, such as glucose, which are normally transported and phosphorylated by the phosphoenolpyruvate, (PEP)-dependent phosphotransferase system (PTS).