Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1.

A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H+-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotide-sequence level of transposable elements on 30 independent derivatives of pKY159 .

F1ATPase of Escherichia coli: a mutation (uncA401) located in the middle of the alpha subunit affects the conformation essential for F1 activity.

F1ATPase from the Escherichia coli mutant of H+-ATPase, AN120 (uncA401), has less than 1% of the wild type activity and has been shown to be defective in the alpha subunit by in vitro reconstitution experiments. In the present study, the mutation site was located within a domain of the subunit by recombinant DNA technology. For this, a series of recombinant plasmids carrying various portions of the alpha subunit gene were constructed and used for genetic recombination with AN120.

Intracistronic mapping of the defective site and the biochemical properties of beta subunit mutants of Escherichia coli H+-ATPase: correlation of structural domains with functions of the beta subunit.

Sixteen mutants of Escherichia coli defective in H+-ATPase (proton-translocating ATPase) were tested for their ability to recombine with hybrid plasmids carrying various portions of the beta subunit cistron. Twelve mutations were mapped within the carboxyl half of the cistron corresponding to amino acid residues 279 to 459 (domain II), while four mutations were mapped within residues 17 to 278 (domain I). The biochemical properties of these mutants were analyzed in terms of the proton permeability of their membranes and the assembly properties of their F1F0 complex.

Escherichia coli mutants defective in the gamma subunit of proton-translocating ATPase: intracistronic mapping of the defective site and the biochemical properties of the mutants.

Various hybrid plasmids carrying a portion of the gene for the gamma subunit of the H+-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the gamma subunit. Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the gamma subunit as follows: KF10 and NR70, KF1, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the gamma subunit. The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F1.

Conformational change of the alpha subunit of Escherichia coli F1 ATPase: ATP changes the trypsin sensitivity of the subunit.

Conformational change in the alpha subunit of Escherichia coli proton-translocating ATPase was studied using trypsin. The subunit was cleaved with a small amount of trypsin (1 microgram/mg subunit) to peptides of less than 8000 daltons. On the other hand, the subunit was cleaved to two main polypeptides (30,000 and 25,000 daltons) in the presence of sufficient ATP (1 mM-0.

Reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP, with E. coli F1-ATPase and its subunits: the roles of the high affinity binding site in the alpha subunit and the low affinity binding site in the beta subunit.

We performed kinetic studies on the reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP (DNS-ATP), with E. coli F1-ATPase (EF1) and its subunits, to clarify the role of each subunit in the ATPase reaction. The following results were obtained.

Hybrid ATPase's formed from subunits of coupling factor F1's of Escherichia coli and thermophilic bacterium PS3.

ATPase complexes were reconstituted from homologous and heterologous combinations of alpha, beta, and gamma subunits of coupling factor ATPase TF1 of thermophilic bacterium PS3 and EF1 of Escherichia coli. TF1 and alpha beta gamma complex reconstituted from TF1 subunits were thermostable and activated by methanol, sodium dodecyl sulfate and anions and they were halophilic, whereas EF1 and its three-subunit complex did not show these properties. The hybrid ATPase alpha T beta T gamma E (complex of the alpha and beta subunits of TF1 and the gamma subunit of EF1) showed closely similar properties to TF1 except for thermostability, while alpha E beta E gamma T (alpha and beta from EF1 and gamma from TF1) had similar properties to EF1.

Structure and function of H+-ATPase: what we have learned from Escherichia coli H+-ATPase.

We have identified a transducing phage lambda asn5 carrying a set of structure genes coding for F1F0. New transducing phages and plasmids carrying a part of the DNA fragment in lambda asn5 were isolated and assayed by genetic complementation with mutants of F1F0. After analysis of DNA from these phages and plasmids, we mapped the genes for F1F0 within a physically defined segment of DNA of 4.

Role of inducer exclusion in preferential utilization of glucose over melibiose in diauxic growth of Escherichia coli.

The role of inducer exclusion in diauxic growth of Escherichia coli on glucose and melibiose was investigated. The amounts of glucose and melibiose in the culture medium were determined during the diauxie. Glucose was consumed during the first growth cycle of the diauxie, and melibiose was consumed during the second cycle.

Subunits of the H+-ATPase of Escherichia coli. Overproduction of an eight-subunit F1F0-ATPase following induction of a lambda-transducing phage carrying the unc operon.

The proton-translocating ATPase complex (F1F0) of Escherichia coli was purified after inductin of a lambda-transducing phage (lambda asn5) carrying the ATPase genes of th unc operon. ATPase activity of membranes prepared from the induced lambda-unc lysogen was 6-fold greater than the activity of membranes prepared from strains lacking the unc-transducing phage, confirming the report of Kanazawa et al. (1979) Proc.

Organization of unc gene cluster of Escherichia coli coding for proton-translocating ATPase of oxidative phosphorylation.

The proton-translocating ATPase (F1-F0) of oxidative phosphorylation (ATP phosphohydrolase, EC 3.6.1.

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.

Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli.

The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.