Effects of inducing expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase

Abstract

To evaluate whether expression of cloned genes for the Fo proton channel of the Escherichia coli F1F0 ATPase is sufficient to cause membrane proton permeability, plasmids carrying different combinations of the uncB, E, and F genes, encoding the a, c, and b subunits of the Fo sector, cloned behind the inducible lac promoter in pUC9 or pUC18, were constructed. The effects of inducing Fo synthesis in an unc deletion strain were monitored by measuring cell growth rate, quantitating Fo subunits by immunoblotting, and measuring the ability of membranes to maintain a respiration-induced proton gradient and to bind F1 and carry out energy-coupling reactions. able form and then converted to its active, proton-conducting form by specific interactions with F1 subunits (18). As one test of this hypothesis, we constructed several plasmids carrying the genes for different Fo subunits cloned behind the inducible lac promoter and measured the effects of increasing amounts of Fo subunits on cell growth, membrane proton permeability, and the ability of membranes to bind purified F1 and carry out energy coupling reactions. The results support the model described above and also suggest that overexpression of Fo genes produces unassembled subunits that inhibit cell growth without necessarily affecting membrane proton permeability. MATERIALS AND METHODSStrains and plasmids. These studies were done in E. coli JM103 A(uncB-uncD), which is strain JM103 (13) in which seven of the nine unc genes (11), including all of the Fo genes, are deleted. Plasmid pEA4, which carries the three Fo genes in pACYC184, was described previously (2). Plasmid pEA5, which carries the three Fo genes cloned behind the inducible lac promoter, was constructed by cloning the HindIII-Sall fragment from pEA4 into pUC9 (25). Plasmid pRM2 was constructed by deleting the HpaI-SmaI fragment containing uncF from pEA5. Plasmid pRM2 was then digested with BamHI and religated to delete 617 bases from uncB, producing plasmid pRM3, which codes for the c subunit only. Plasmid pRM4 was constructed by cloning the HindIII-SalI fragment from pEA4 into pUC18. The resultant plasmid carries the same insert as pEA5, but the insert is in the opposite orientation to the lac promoter. When it is important for clarity, the subunits contained in each plasmid are given within parentheses after the plasmid designation.Growth and lac induction. Cells were grown in LB medium (14) containing 100 mg of ampicillin per liter, and growth was measured by monitoring cell turbidity (optical density at 550

The product of uncI gene in F 1 F o -ATP synthase operon plays a chaperone-like role to assist c -ring assembly

“…The plasmid pTR-hF1Fo (ϩi) was used for transformation of an F1Fo-deficient E. coli strain, JM103⌬(uncBuncD) (hereafter, JM103⌬unc) (39). The transformants were aerobically grown in 2ϫYT medium supplemented with 100 g/ml ampicillin for 21 h. In the case of transformants harboring pSTV28 derivative plasmid (mentioned later), 30 g/ml chloramphenicol was also supplemented to maintain the plasmid in the cells.…”

Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase

“…1. With the exception of the Fo+8 plasmids, these plasmids were described previously (13). pRM1 contains all the genes (uncBEF) for the Fo sector in addition to the gene for the 8 subunit of F1 (uncH).…”

“…Plasmids pEA5, pWSB30.0, and pWSB33 were described previously (13,22). pRM1, which carries the Fo genes plus uncH (S subunit) cloned behind the lac promoter, was constructed by digesting pWSB30.0 with HindIll and EcoRI and ligating the resulting fragments with the pUC9 (23) vector, which had been digested with HindIIl and EcoRI.…”

Mutations in the delta subunit influence the assembly of F1F0 ATP synthase in Escherichia coli

“…,Brusilow and coworkers (1,16) have suggested that the a and 8 subunits act not upon assembly of Fo but upon activation of proton translocation.…”

“…In Escherichia coli FIFO ATP synthase, the 8 subunit is required for the binding of F1 to Fo (1,8,9,14,16,17). A narrow stalk connecting the two sectors is thought to consist of the Fo b subunits and possibly F1 subunits, including the 8 subunit (5).…”