Effects of carbon source on expression of F0 genes and on the stoichiometry of the c subunit in the F1F0 ATPase of Escherichia coli

Abstract

Expression of the genes for the membrane-bound F0 sector of the Escherichia coli F1F0 proton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric F0 proton channel. Transcriptional and translational lacZ fusions to the promoter and to two F0 genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of uncB, encoding the a subunit of F0, are higher than during growth on glucose. In contrast, translation of the uncE gene, encoding the c subunit of F0, is higher during growth on glucose than during growth on succinate. Translation rates of both uncB and uncE change as culture density increases, but transcription rates do not. Quantitation of the c stoichiometry shows that more c subunits are assembled into the F1F0 ATPase in cells grown on glucose than in cells grown on succinate. E. coli therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances.

FIG. 1

The start of the unc operon indicating the locations of fusions to lacZ. The promoter (P) and the first three genes of the unc operon are indicated with restriction enzyme recognition sites used to make these fusions in plasmids. The translational unB′-′lacZ fusions in pDKWH107 and pKS104 and the translational uncE′-′lacZ fusion in pKS105 have been described previously (9, 19). The transcriptional fusion in pWSB56 was constructed for this study by cloning the SspI-BamHI fragment from the pKS105 fusion into the promoter detection plasmid pRZ5255, which carries a trp-lac fusion containing the entire lacZ gene (14). β-Galactosidase activity produced by this construction is dependent upon a cloned promoter, but lacZ translation is initiated from the translational initiation region of lacZ, not the uncB gene. The horizontal lines indicate the amount of unc DNA cloned either into the transcriptional fusion vector to make the WSB56 fusion or into translational fusion vectors to make the DKWH107, KS104, and KS105 fusions, and the number following each line indicates exactly how many bases of each gene are present in each fusion construction. The BamHI* site is not normally present in uncB but was constructed for the purpose of making the fusion in DKWH107 (9).

FIG. 2

The fusions described for Fig. 1 were transferred from plasmids into λRZ5, integrated into the λ att site of MC1000 Unc⁺, and assayed as described in Materials and Methods. The points on these plots are the averages of duplicate assays, which typically varied by less than 5%. The effects of glucose and succinate on promoter activity are shown in the graph of the WSB56 lysogen, effects of carbon source on translation of uncB (a subunit) are shown in the graphs of the DKWH107 and KS104 fusions, and effects of carbon source on translation of uncE (c subunit) are shown in the graph of the KS105 fusion. ▪, succinate-grown cultures; ▴, glucose-grown cultures.

FIG. 3

Immunoblot of F₁F_o complexes purified from cells grown on either minimal-glucose medium or minimal-succinate medium and immunoprecipitated with anti-F₁. This procedure has been shown to precipitate F_o subunits which are associated with F₁ subunits but not F_o subunits alone (15). Lanes: 1, purified F₁; 2, F₁F_o purified from cells grown on minimal-glucose medium; 3, control (with control serum) immunoprecipitation of glucose F₁F_o; 4, immunoprecipitation (with anti-F₁ antiserum) of glucose F₁F_o; 5, F₁F_o purified from cells grown on minimal-succinate medium; 6, control (with control serum) immunoprecipitation of succinate F₁F_o; 7, immunoprecipitation (with anti-F₁ antiserum) of succinate F₁F_o. This blot was developed with dilute preparations of antiserum in order to minimize the background. The overall reactivity of the c subunit is therefore much less than in the experiments described for Table 1.

FIG. 4

Possible effect of a variable c stoichiometry. This figure shows the model for rotational catalysis as described by Duncan et al. (3). The F_o subunits a, b, and c are shown, as is the F₁ γ subunit. The α and β subunits are depicted as a trimer of αβ dimers. The rotation of the c oligomer in response to a proton motive force produces rotation of the γ subunit within the trimer of αβ dimers. If the cogging of the c oligomer into and out of contact with the a subunit to form the actual proton channel is rate limiting, then, for a given proton motive force, the rate of ATP synthesis or hydrolysis will depend on the size of the c oligomer. Lower stoichiometries would produce higher rates.