Table 5-1. Aerobic growth properties and membrane-associated ATP hydrolysis activity of mutants expressing uncF(b) mutations at the amino terminus Strains Description Growth' Activt KM2/pKAM14 (+) bwt, Apr +++ 1.46 & 0.03 KM2/pBR322 (-) Ab, Apr 0.17 & 0.02 KM2/pAWH243 basn24ala, thr64ala_ glnl04ala, Apr -0.83 & 0.09 KM2/pTAM43 basn24ala, Apr +++ 1.49 & 0.09 KM2/pTAM44 bthr64ala, Apr +++ 1.13 & 0.07 KM2/pTAM45 bginlonala, Apr +++ 1.15 & 0.04 E. coli strains were grown aerobically on succinate minimal medium supplemented with 40 CLM IPTG. Colony size was scored after 72-hr incubation at 37oC as: +++, 1.0 mm: ++, 0.3-0.5 mm: +, ~0.1 mm: -, no growth. SE. coli strains were grown in LBG supplemented with the appropriate antibiotics and 40 CLM IPTG. ATPase activities were measured as described under "Materials and Methods." Units were calculated from the slope of the line based on three measurements with incubations for 12 minutes. SWork accomplished by Andrew W. Hardy. mutant to be completely defective; however, membrane-associated ATP hydrolysis activity and immunoblot analysis revealed that the triple mutant was forming intact F1Fo ATP synthase complexes, suggesting that the mutant was incapable of performing coupled proton translocation (202). As a result of these observations, the alanine substitutions were studied individually. The amino acids expressed by codons 2, 6 and 10 were individually mutated to express an alanine, resulting in plasmids pTAM43 (basn24ala), pTAM44 (bthr64ala), and pTAM45 (bgintoala (Table 5-1). The effects of the mutations were studied by the ability of the plasmids to compliment E. coli strain KM2 (Ab) (218). Growth on succinate minimal media was used as an initial qualitative gauge of enzyme activity in vivo since E. coli strains lacking F1Fo ATP synthase cannot derive energy from nonfermentable sources. In each case, the strains expressing the different alanine substitutions grew comparably to the wild type strain (Table 5-1).