(A) Schematic representation of a polytopic predicted molybdenum cofactor (MoCo) binding protein (Sco3746, left) a polytopic polyferredoxin (PFD, centre) and a polytopic metallophosphoesterase of the YkuE family (right) identified bioinformatically as candidate dual-inserted membrane poteins. The twin arginines of the Tat recognition sequence are highlighted in yellow. Four histidines in the TMDs of Sco3746 and homologues that are predicted to ligate two b hemes are shown in red. Note that three of these histidines are conserved (Figure 5) but the one at the N-terminal end of TMD3 (H?) is not. (B) Fusions of the TMDs of Sco3746 and of Q1NSB0 (PFD) to maltose binding protein (MBP) or AmiA are shown as cartoons. As before, a signal peptidase I cleavage site (indicated by scissors) was introduced at the end of predicted TMD5 to allow release of AmiA from the membrane (Keller et al., 2012). (C) E. coli tat+ strain HS3018-A (that lacks chromosomally-encoded MBP) harboring pSU18 (empty vector), or the same vector producing native MBP, Sco3746TMD-MBP, PFDTMD-MBP or the twin-arginine substituted variants Sco3746TMDRRKK-MBP and PFDTMDRRKK-MBP, as indicated, was cultured overnight, resuspended in minimal medium containing 1% Maltose and 0.002% Bromocresol purple. Cells were diluted either 2.5 fold (left hand panel) or 10 fold (right hand panel) in the same medium and incubated without shaking at 37°C for 24 hr (left hand panel) or 48 hr (right hand panel). (D) E. coli strains MCDSSAC or an isogenic tatABC mutant harboring pSU18 (empty vector), pSU18 producing native AmiA, or pSU18 producing either Sco3746TMD-AmiA or PFDTMD-AmiA fusion proteins, or variants of these where the twin-arginine motif was substituted to twin lysine (Sco3746TMDRRKK-AmiA/PFDTMDRRKK-AmiA) were serially diluted and spotted onto LB or LB containing 2% SDS. The plates were incubated for 20 hr at 37°C.