Article Figures & Data
Figures
- FIG. 1.
Proteolytic profiles of AcrAhis in vitro and in vivo. (A) Schematic representation of the secondary structure of AcrA. The unique N-terminal Cys25, which is lipid modified after processing in the periplasm, is shown with an arrow. Positions of amino acid residues that form the α-β-barrel, lipoyl-binding, and α-helical hairpin domains are indicated. AcrA residues cleaved by trypsin are indicated by arrowheads. The 28.9-kDa (K46-R315) core and the 26.5-kDa fragment (K46-R294) are also indicated. (B) Purified AcrAhis (final concentration, 1.95 μM) was digested with trypsin (final concentration, 0.10 μM) at 37°C. Aliquots (10 μl) were taken at different time points, and reactions were terminated by boiling in the SDS sample buffer for 5 min. Tryptic fragments were resolved by SDS-PAGE and analyzed by silver nitrate staining. Minor fragments in the untreated control (0 min) are contaminants that copurify with AcrAhis. Lane M, molecular marker. (C) Proteolytic profiles of AcrAhis in E. coli AG100AX cells carrying pAhis and pAhisB plasmids. After treatment with increasing concentrations of trypsin for 60 min at 37°C, the whole-cell proteins were resolved by SDS-PAGE and analyzed by immunoblotting with a polyclonal anti-AcrA antibody. Masses of tryptic fragments of the C-domain of AcrAhis identified by mass spectrometry and by mobility in SDS-PAGE are indicated. O.D., optical density as determined by absorbance at 600 nm.
- FIG. 2.
Proteolytic profile of AcrA assembled into AcrAB-TolC complex differs from that of the purified or partially assembled AcrA. (A) E. coli ZK4 (wild type [WT]), ZK796 (ΔTolC), and AG102MB (ΔAcrB) were grown to the mid-exponential phase (A 600, ∼1.0). Cells were collected and digested with increasing concentrations of trypsin for 5 min (left panel) and 60 min (right panel) at 37°C. Total proteins were resolved by 12% SDS-PAGE, and AcrA fragments were visualized by immunoblotting with anti-AcrA antibody. (B) ZK796 (ΔTolC) and AG102MB (ΔAcrB) cells were transformed with pTolChis and pBhis plasmids producing TolC and AcrB, respectively, or with pUC18 vector alone. Cells were processed and analyzed as in panel A. Tryptic fragments of AcrA identified by comparison to the proteolytic profiles of the purified AcrA are indicated. Lanes M, molecular marker.
- FIG. 3.
Expression of AcrAhis and AcrB is not affected by mutations in the C-domain of AcrAhis. (A) Total E. coli W4680AE cells harboring plasmids producing AcrB and either wild-type or mutant AcrAhis were boiled in the SDS sample buffer for 5 min, separated by 10% SDS-PAGE, and analyzed by immunoblotting with the polyclonal anti-AcrA antibody. (B) Membrane fractions isolated from E. coli W4680AE harboring pAhis, pAhisB, pAhisG352CB, pAhisG356CB, and pAhisG363CB plasmids were separated by 10% SDS-PAGE and analyzed by immunoblotting with the polyclonal anti-AcrB antibody.
- FIG. 4.
The AcrAhis G363C mutant interacts with AcrB and TolC but fails to assemble into the functional AcrAB-TolC complex. (A) AcrB (top panel) and TolC (bottom panel) are copurified with the AcrAhis G363C mutant. E. coli W4680AE cells carrying pAhis, pAhisB, pAhisG352CB, and pAhisG363CB plasmids were collected at the mid-exponential phase and treated with the amino-reactive cross-linker DSP. Equal amounts of purified AcrAhis were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-AcrB and anti-TolC antibodies. (B) Trypsin digestion of E. coli W4680AE cells carrying pAhisB and its pAhisG363CB derivative. Trypsin digestion and analysis were carried out as described for Fig. 1C. O.D., optical density as determined by absorbance at 600 nm.
- FIG. 5.
Schematic representation of the mechanism of assembly of AcrAB-TolC complex. AcrA forms bipartite complexes with AcrB and TolC. The N terminus of AcrA (N) is lipid modified and inserted into the inner membrane. The lipoyl and α-β-barrel domains of AcrA interact with AcrB, whereas the α-helical hairpin domain (α-hairpin) docks with TolC. In free AcrA and bipartite complexes, the C-domain (C) is unstructured and readily cleaved by trypsin. However, upon assembly of the functional tripartite AcrAB-TolC complex, this domain is protected from trypsin attack. The G363C mutation possibly induces a structural misfit between the C-terminal domain of AcrA and AcrB during assembly of the tripartite complex. OM, outer membrane; PG, peptidoglycan; IM, inner membrane.
Tables
- TABLE 1.
Peptide masses of whole-length AcrAhis and its major tryptic fragments
Peptide Mass of AcrAhis and its peptides (Da) based on: MALDI-TOF aa sequence Whole length 41,696 41,622 N-K396 40,411 40,471 K28-K396 39,362 39,303 K46-K396 37,308 37,495 K28-K374 NDa 36,920 K46-K374 34,909 35,113 K46-K346 32,056 32,097 K46-R315 28,886 28,935 ↵ a ND, not detected.
- TABLE 2.
Antibiotic susceptibility of E. coli W4680AE cells carrying plasmids that produce the wild-type and mutant derivatives of AcrAhis
Plasmid MIC (μg/ml)a EM NFLX NB EtBr SDS pUC18 2 0.016 2 3.125 32 pAhisB 128 0.250 256 800 >65,540 pAhisG352CB 64 0.125 64 100 >65,540 pAhisG356CB 64 0.125 128 400 >65,540 pAhisG363CB 4 0.016 16 50 16,380 ↵ a All MIC measurements were done in triplicate. EM, erythromycin; NFLX, norfloxacin; NB, novobiocin; EtBr, ethidium bromide. Plasmids pAhisP309CB, pAhisV313CB, pAhisV332CB, pAhisR335CB, pAhisL353CB, pAhisD357CB, pAhisV359CB, pAhisV360CB, pAhisS362CB, pAhisK366CB, and pAhisV373CB fully complemented the drug-susceptible phenotype of W4680AE cells.
