Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.
In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.
Daniels
G A
GA
Department of Biology, University of California, San Diego, La Jolla 92093.
Drews
G
G
Saier
M H
MH
D001692:Biological Transport; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004790:Enzyme Induction; D005632:Fructose / Q000378:metabolism*; D005796:Genes; D005798:Genes, Bacterial; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics* / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor); D012244:Rhodospirillaceae / Q000201:enzymology / Q000235:genetics*
D004251:DNA Transposable Elements; D005632:Fructose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D010732:Phosphofructokinase-1; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase