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The effects of specific inhibitors on the high-affinity transport system of cyclitols and on the respiration of Pseudomonas putida shows that the transport activity is dependent on high energy phosphate bond.

A soil isolate of a Pseudomonas sp. can utilize myo-inositol (MI) as the sole carbon source. In this strain, MI is transported through the membrane by a high-affinity transport system in which a periplasmic binding protein is involved. Mutants impaired in the transport system were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently identified by their slow growth rate at low MI concentrations. Strains with a low linear initial rate of MI uptake were analyzed. Using a broad-host-range cosmid cloning system, we have constructed a gene bank of the wild-type Pseudomonas sp. in an Escherichia coli recA-host. A rapid mating technique enabled us to screen the gene library for clones which are able to restore the active transport of MI in the mutant. An 11.5-kilobase segment containing genes involved in the MI transport has been isolated, and its restriction enzyme cleavage map has been determined.

A myo-inositol-binding protein was isolated from a Pseudomonas sp. soil isolate and was purified to homogeneity. Its molecular weight is 30,000, and it has a single binding site. The amino acid analysis showed that the protein contains three tryptophan residues and no cysteine. Tryptophan residues seem to be involved in the binding of the ligand, as shown by the modification of the fluorescence spectra and by the fact that oxidation of tryptophan residues with N-bromosuccinimide abolished the binding of myo-inositol. Sequence analysis of the N-terminal segment of 37 amino acids showed that 13 are conserved when compared with the galactose-binding protein of Escherichia coli.

The kinetic features of the myo-inositol transport system in Pseudomonas putida are reported. The system is sensitive to osmotic shock, is not operative in membrane vesicles, and does not involved substrate phosphorylation. Line-weaver-Burk plots indicate the presence of two different systems, whose Kt are 5 micrometer and 0.43 mM and whose V max are 7.9 and 27 nml/mg per min, respectively. Transport activity of glucose-grown cells is very low. myo-Inositol-grown cells lose the high-affinity system upon osmotic shock; concentrated shock fluid possesses myo-inositol-binding activity. The system is very specific for the myo-configuration of the cyclitol.