One monomer is coloured pink the other 2 are coloured white. (B) Ribbon diagram of trimeric TolC (PDB: 1EK9) from E. gingivalis operon encodes 2 putative TolC-like proteins the most downstream is depicted in Fig 4B. Green (regulatory protein), pink (TolC-like protein), yellow (periplasmic adaptor protein), purple (cytoplasmic membrane channel component), white (hypothetical protein). The NCBI accession number is shown below the indicated bacterial strains, and the locus tags for each gene are also shown. (A) The genetic organisation of the 12 selected efflux pumps depicted in Fig 4B are shown (not to scale). S6 Fig: Identification of tripartite efflux pumps in monodermic Firmicutes. (B-C) Uncropped images are presented in S1 Raw Images original autoradiographs (including relevant replicates) are presented in S2 Raw Images. The presence of native TamA is indicated as a cartoon to the left (panel B only). The position of FimD, TamA, and its fragments A, B, and C are indicated to the right of the autoradiograms, and protein standards are indicated on the left (sizes are in kDa). Total protein was analysed by SDS-PAGE and storage phosphor imaging. (B-C) Aliquots were taken at 10 s (0 min), 2, 4, 8, 16, and 32 min (panel B) or 8 min only (panel C) and treated with (+) or without (−) proteinase K. Media were supplemented with the indicated reducing or oxidising agent, or they were not supplemented (i.e., nonreducing) as indicated. coli BL21 Star (DE3) Δ tamA harbouring pKS02 and the plasmid encoding the indicated TamA cysteine mutants. (C) FimD assembly was monitored as per panel B, except all strains were E. coli BL21 Star (DE3) harbouring pKS02 ( fimD expression vector) and either pACYCDuet-1 (base vector) or pCJS69 ( tamA complementation vector). (B) FimD assembly was monitored over time by pulse chase analysis in the indicated strains of E. ![]() On addition of extracellular protease (PK, yellow), protease-sensitive radiolabelled proteins can be detected once they are localised to the outer membrane through the accumulation of degradation products or a reduction in full-length protein. Cells are pulsed (45 s) with -methionine and -cysteine before chase media (containing -methionine and -cysteine) is added. Cells are then subjected to rifampicin treatment (1 h) to block native RNA transcription before IPTG induction (5 min) allows transcription from the T7 RNA polymerase (which is not sensitive to rifampicin) promoter upstream from the plasmid-encoded gene of interest. coli BL21 Star (DE3) cells harbouring a plasmid with the gene of interest (orange arrow) are subjected to sulphur starvation to deplete sulphur-containing amino acids. ![]() (A) Schematic of the pulse chase experiment based on. S2 Fig: FimD assembly as a readout of TamA function.
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