Figure 1. Sco2149_TMD-reporter fusions to follow membrane insertion.

(A) Cartoon representations of the S. coelicolor Rieske protein, Sco2149, and the Sco2149_TMD-AmiA and Sco2149_TMD-Bla fusions. A signal peptidase I cleavage site (indicated by scissors) was introduced between the end of TMD3 and the AmiA sequence to allow release of AmiA from the membrane (Keller et al., 2012). The position of the twin-arginine motif is indicated by RR. (B) Sequence of the Sco2149 cytoplasmic loop region between TMDs 2 and 3. Amino acids predicted to be part of TMDs 2 and 3 are shown in red. The twin arginines of the Tat recognition motif are given in purple underline. Predicted α-helical secondary structure is shown with a dotted line, and alanine residues within this region that were mutated to proline are shown in pink. Negatively charged amino acids in the loop region are shown in blue, positively charged ones in grey. (C) E. coli strain MCDSSAC (which carries chromosomal deletions in the signal peptide coding regions of amiA and amiC) or an isogenic tatABC mutant containing either pSU-PROM (empty vector), or pSU-PROM producing native AmiA, Sco2149_TMD-AmiA or a variant where the twin-arginines were substituted to AD (Sco2149_TMDRRAD-AmiA), were spotted, after serial dilution, on LB medium in the absence or presence of 1% SDS. The plates were incubated for 20 hr at 37°C. (D) Representative images of M.I.C.Evaluator strip tests of strains MC4100 (tat⁺) and DADE (tat^-) harbouring pSU-PROM (empty vector), pSU-PROM Sco2149_TMD-Bla or pSU-PROM Sco2149_TMDRRAD-Bla are shown. The mean M.I.C ± s.d. for strains harbouring these constructs is given at the bottom of each test strip (where n = 4 biological replicates for each strain harbouring the empty vector, n = 5 biological replicates for each strain harbouring pSU-PROM Sco2149_TMD-Bla and n = 3 biological replicates for each strain harbouring pSU-PROM Sco2149_TMDRRAD-Bla).

DOI: http://dx.doi.org/10.7554/eLife.26577.002

Figure 1—figure supplement 1. Sequence alignment of selected actinobacterial Rieske proteins.

(A) Sequences of the Rieske (QcrA) proteins from Streptomyces coelicolor (ScoQcrA), Mycobacterium tuberculosis (MtuQcrA) and Corynebacterium glutamicum (CglQcrA) were aligned using ClustalW (http://www.ch.embnet.org/software/ClustalW.html) and Boxshade (http://www.ch.embnet.org/software/BOX_form.html). Predicted positions of the TMDs, using the SCAMPI2/TOPCONS servers (Tsirigos et al., 2015, 2016), are shown in blue. Positively charged amino acids immediately downstream of TMD3 are shown in orange. The consensus twin arginine (Tat) motif is boxed in red and conserved boxes I and II that are predicted coordinate the 2Fe-2S cluster are boxed in yellow. The positions after which reporter proteins were fused to the S. coelicolor or M. tuberculosis proteins are indicated. (B) Partial sequence alignment of the same three actinobacterial Rieske proteins with the single TMD Rieske proteins from Paracoccus denitrificans and Rhodobacter sphaeroides.

DOI: http://dx.doi.org/10.7554/eLife.26577.003

Figure 1—figure supplement 2. A twin lysine substitution of the twin arginine motif of Sco2149 still retains some interaction with the Tat pathway.

E. coli strains MCDSSAC or MCDSSAC △tatABC, as indicated containing either pSU-PROM (empty vector), or pSU-PROM producing native AmiA, Sco2149_TMD-AmiA or variants where the twin-arginines were substituted to KK (Sco2149_TMDRRKK-AmiA), AD (Sco2149_TMDRRAD-AmiA) or AA (Sco2149_TMDRRAA-AmiA) were spotted, after serial dilution, on LB medium in the absence or presence of 1% SDS. The plates were incubated for 20 hr at 37°C.

DOI: http://dx.doi.org/10.7554/eLife.26577.004

Figure 1—figure supplement 3. Sequence alignment TMD2/TMD3 loop region for a selection of actinobacterial Rieske proteins.

Sequences were aligned using ClustalW (http://www.ch.embnet.org/software/ClustalW.html) and Jalview (Cole et al., 2008). The approximate end position of TMD2 and start of TMD3 is indicated and the dotted black line indicates the position of predicted α-helical secondary structure determined using Jpred3 (http://www.compbio.dundee.ac.uk/www-jpred/index.html).

DOI: http://dx.doi.org/10.7554/eLife.26577.005

Figure 1—figure supplement 4. Effect of >35 residue truncations in the Sco2149 cytoplasmic loop region on the ability of Sco2149_TMD-AmiA to support growth on SDS.

E. coli  strains MCDSSAC or MCDSSAC △tatABC, as indicated containing pSU-PROM producing Sco2149_TMD-AmiA or variants with 35 or more residues removed (Sco2149_TMDΔ118–152-AmiA, Sco2149_TMDΔ123–157-AmiA, Sco2149_TMDΔ118–153-AmiA, Sco2149_TMDΔ118–154-AmiA, Sco2149_TMDΔ118–155-AmiA, Sco2149_TMDΔ118–156-AmiA and Sco2149_TMDΔ118–157-AmiA) were spotted after serial dilution on LB medium in absence or presence of 1% SDS. The plates were incubated for 20 hr at 37°C.

DOI: http://dx.doi.org/10.7554/eLife.26577.006