10 mL starter culture, grown overnight, into a 2 L Erlenmeyer flask containing 500 mL

LBG, supplemented with the appropriate antibiotic, ampicillin (Ap) and/or

chloramphenicol (Cm). Similarly, 1 mL of a starter culture was inoculated into a nephalo

flask containing 50 mL LBG (Ap and/or Cm). The bacteria were grown at 37 oC in a

New Brunswick Scientific incubator shaker (220 rpm) and the turbidity was monitored

using a Klett-Summerson photoelectric colorimeter. IPTG (40 CIM) was added when the

turbidity reached 75 Klett units and the cells were collected when the turbidity reached

150 Klett units. The bacteria were harvested by centrifugation for 10 minutes at 8,000Xg

in a Sorvall GSA rotor. The pellets were rinsed once with TM buffer (50 mM tris-HC1,

10 mM MgSO4, pH 7.5) and then resuspended in a final volume of 10 mL TM buffer.

DNasel (10 mg/mL) was added to a final concentration of 10 Clg/mL and the bacteria

were broken by passing through a French Pressure Cell one time at 14,000 psi. Cellular

debris and unbroken cells were removed by centrifuging twice at 10,000Xg for 10

minutes. Membranes were then collected by ultracentrifugation at 150,000Xg in a

Beckman 70.1 Ti rotor for 1.5 hours. The membrane pellets were rinsed once with TM

buffer and then resuspended in TM buffer to a final volume of 2 mL using a 2 mL

Wheaton tissue grinder. For the purposes of Western blot analysis or Ni-resin

purification, one ultracentrifugation step sufficed. However, activity analysis required an

additional ultracentrifugation step in order to remove nonspecifically bound ATPases. In

this case, the membrane pellets were resuspended in a final volume of 10 mL using a 10

mL Wheaton tissue grinder and the ultracentrifugation step was duplicated.