(ball, Apr) and pAUL47 (b it, Apr), respectively, and have been described previously

(193, 194, 203). The plasmids encoding the uncF(b) gene were used to compliment

Escherichia coli (E. coli) strain KM2 (Ab) carrying a chromosomal deletion of the gene.

Plasmid pJLG1 (a, c, b, Cmr), generated by Dr. James Gardner in our lab, was used as a

temporary vector in order to facilitate cloning due to its extra unique restriction enzyme

sites. The wild type unc operon expression plasmid, pAES9 (upyisabc, Cmr), was used

to create a cysteine-less F1Fo ATP synthase complex and has been described previously

(178). The plasmids encoding the unc operon (upyGsabc) were used to compliment E.

coli strain 1100ABC (BApyisabc) carrying a chromosomal deletion for the entire operon.

All strains were streaked onto plates containing Minimal A media supplemented with

succinate (0.2% w/v), to determine enzyme viability. Cells harvested for membrane

preparation were grown in Luria Broth supplemented with glucose (0.2% w/v) (LBG).

Isopropyl-1 -thio-P-D-galactopyranoside (IPTG)(40 Clg/ml), ampicillin (Ap) (100Clg/ml),

and chloramphenicol (Cm) (25 Clg/ml) were added to media as needed. All cultures were

incubated at 37oC for the appropriate duration.

Recombinant DNA Techniques

 Plasmid DNA was purified with the Qiagen Mini-Prep and Maxi-Prep kits.

Restriction endonuclease digestions, ligations, and transformations were performed

according to the recommendations of the manufacturers (New England Biolabs,

Stratagene and Life Technologies, Inc.). Site-directed mutagenesis was performed either

by means of a Stratagene Quikchange kit or by ligation-mediated mutagenesis. DNA

fragments were separated in 0.8 % agarose gel by electrophoresis and purified using a