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. 2007 Dec 7;374(4-5):877–882. doi: 10.1016/j.jmb.2007.09.080

Fig. 1.

Fig. 1

Membrane accumulation of early and late subunits in the pathway attenuated by enzymatically impaired FliI ATPase. (a) FliC export, assayed by immunoblotting, filtered supernatants from midexponential Luria broth (LB) cultures (A600 = 1.0) of ΔfliIflgKflgM cells (made by P22 transduction combined with the method of Datsenko and Wanner27) expressing in trans either wild-type FliI (FliIWT) or variant FliIE211A (FliIEA) from pBAD33 (0.1% arabinose). A ΔfliIflgKflgM strain containing empty pBAD33 was shown to be nonmotile and attenuated in the export of early FliK subunit and late FliC subunit (data not shown). (b) Salmonella fliIflgKflgM cultures expressing wild-type FliIWT or variant FliIEA separated into membrane (m) and cytoplasmic (c) fractions.11,16 Immunoblotted for FliI ATPase, FlgN chaperone and subunits. (c) Separation of the membrane fractions into outer membrane (OMP; Coomassie stained) and inner membrane (NADH oxidase marker) by sucrose gradient ultracentrifugation (0.8–2.0 M11,16 top and bottom of the gradient indicated). Proteins immunoblotted using antisera described above.