Article Figures & Data
Figures
- FIG. 1.
Steady-state levels of TonB protein in strains used for [55Fe]ferrichrome uptake. Aliquots (0.5 ml) of cells grown to mid-exponential phase (as described in Table 3, footnote a) were harvested just prior to the addition of [55Fe]ferrichrome, precipitated in 10% trichloroacetic acid at 4°C for 15 min, centrifuged, washed in 100 mM Tris-Cl (pH 8.0), suspended in 25 μl of Laemmli sample buffer, incubated at 98°C for 5 min, resolved on a sodium dodecyl sulfate-11% polyacrylamide gel (30), transferred to a polyvinylidene fluoride membrane, and probed using the TonB-specific monoclonal antibody 4F1 (33), with subsequent visualization by enhanced chemiluminescence as previously described (48). Following immunoblot analysis, membranes were stained for total protein with Coomassie blue and visually examined to confirm equivalent sample loading of all lanes. In this experiment, the wild-type (ExbB+ ExbD+ TolQ+ TolR+) sample was two- to threefold underloaded relative to the other samples. Strains are identified by their relevant phenotypes at the top, and the positions of molecular mass standards are indicated on the right. B, ExbB; D, ExbD; Q, TolQ; R, TolR.
- FIG. 2.
In vivo chemical cross-linking. Cells were grown in T-broth supplemented with 100 μg ml−1 ampicillin to mid-exponential phase, harvested in 1.0-ml aliquots, centrifuged, and suspended in 938 μl of 100 mM phosphate buffer (pH 6.8). Then 62 μl of 16% paraformaldehyde was added, and suspensions were incubated at room temperature for 15 min, centrifuged, and suspended in 25 μl of Laemmli sample buffer. Samples were incubated at 60°C for 5 min and then resolved on a sodium dodecyl sulfate-11% polyacrylamide gel, and a subsequent immunoblot analysis was performed as described in the legend to Fig. 1. Strains are identified by their relevant phenotypes at the top; the positions of molecular mass standards are indicated on the right, and the relative positions of monomeric TonB and the specific TonB-containing complexes are indicated on the left. B, ExbB; D, ExbD; Q, TolQ; R, TolR.
- FIG. 3.
Stability of TonB under steady-state conditions. All strains were grown to mid-exponential phase in LB broth supplemented with 100 μg ml−1 ampicillin. Chloramphenicol was then added to a final concentration of 100 μg ml−1 to halt protein synthesis. Samples were taken at 0, 15, 30, 60, and 120 min and precipitated in 10% (wt/vol) trichloroacetic acid. Samples were washed in 100 mM Tris-Cl (pH 8.0) and then suspended in 25 μl of Laemmli sample buffer, incubated at 98°C for 5 min, and resolved on sodium dodecyl sulfate-11% polyacrylamide gels, and a subsequent immunoblot analysis was performed as described in the legend to Fig. 1. Strains are identified by their relevant phenotypes on the left, sample times (in minutes) are indicated at the top, and the positions of molecular mass standards are indicated on the right. B, ExbB; D, ExbD; Q, TolQ; R, TolR.
Tables
- TABLE 1.
E. coli strains and plasmids
Strain or plasmida Relevant genotype and/or phenotypeb Reference Strains BW25113 lacIq rrnB T14 ΔlacZ wj16 hsdR514 ΔaraBAD AH3 3 ΔrhaBAD LD78 7 W3110 F-IN(rrnD-rrnE)1 23 KP1456 W3110 ΔexbBD tolQ(Am)37 18 RA1003 W3110 ΔexbBD::kan 32 RA1034 W3110 ΔexbBD::kan ΔtolR This study RA1035 W3110 ΔtolQR This study RA1044 W3110 ΔexbB ΔtolR This study RA1045 W3110 ΔtolQR ΔexbD This study RA1046 W3110 ΔtolQR ΔexbB This study RA1051 W3110 ΔexbBD::kan ΔtolQR This study Plasmids pBAD24 araBAD promoter, AraC, Ampr 16 pKP392 pBAD24 araBAD-regulated exbB 18 pKP393 pBAD24 araBAD-regulated exbD This study pRA001 pBAD24 araBAD-regulated tolQ This study pRA002 pBAD24 araBAD-regulated tolR This study pRA003 pBAD24 araBAD-regulated tolQR This study ↵ a The identity of each plasmid construct generated in this study was confirmed by sequence determination.
↵ b The specific nature of each of the deletions involving exbB, exbD, tolQ, and tolR is as follows. The ΔexbBD deletion of KP1456 is a large, incompletely characterized deletion involving the exbBD operon and the adjacent metC gene, originally isolated by Guterman and Dann (15). The ΔexbBD::kan mutation involves the replacement of all but the initiation codon of exbB (codons 2 to 244) and its TAA termination codon, the six intervening nucleotides, and all of the codons of exbD (codons 1 to 141) by the kan gene from pACYC177, as previously described (32). The ΔexbB deletion removed exbB codons 1 to 244, the TAA termination codon, and the six additional nucleotides that occur prior to the exbD initiation codon. The ΔexbD deletion removed exbD codons 1 to 141 and the TAA termination codon. The ΔtolQR deletion removed tolQ codons 1 to 230, the TAA termination codon, the three nucleotides that occur prior to the tolR initiation codon, all 142 tolR codons, and the TAA termination codon. The ΔtolR deletion removed tolR codons 1 to 142 and the TAA termination codon.
- TABLE 2.
Susceptibility to group A and B colicins and phage φ80
Straina Phenotype Susceptibility tob: ColA ColB φ80 W3110(pBAD24) ExbB+ ExbD+ TolQ+ TolR+ 7, 7, 7 7, 7, 7 8, 8, 8 RA1051(pBAD24) ExbB− ExbD− TolQ− TolR− R, R, R R, R, R R, R, R RA1003(pBAD24) ExbB− ExbD− TolQ+ TolR+ 7, 7, 6 5, 5, 4 7, 6, 6 RA1034(pRA002) ExbB− ExbD− TolQ+ pRA002+ 6, 6, 6 6, 6, 7 7, 7, 6 RA1035(pBAD24) ExbB+ ExbD+ TolQ− TolR− R, R, 3 7, 8, 7 7, 8, 9 RA1045(pRA002) ExbB+ ExbD− TolQ− pRA002+ R, R, R R, R, R 4, 4, 2 RA1044(pBAD24) ExbB− ExbD+ TolQ+ TolR− R, R, R R, R, R R, R, R RA1045(pBAD24) ExbB+ ExbD− TolQ− TolR− R, R, R R, R, R R, R, R RA1046(pBAD24) ExbB− ExbD+ TolQ− TolR− R, R, R R, R, R R, R, R RA1034(pBAD24) ExbB− ExbD− TolQ+ TolR− R, R, R R, R, R R, R, R RA1051(pRA002) ExbB− ExbD− TolQ− pRA002+ R, R, R R, R, R R, R, R ↵ a Most strains carried plasmid pBAD24; the exceptions were the strains which carried plasmid pRA002, which encodes TolR. Cells were grown at 37°C with aeration in LB broth supplemented with 100 μg ml−1 ampicillin to mid-exponential phase (an A 550 of 0.4, as determined with a Spectronic 20 spectophotometer with a path length of 1.5 cm) and then plated in T-top medium supplemented with 100 μg ml−1 ampicillin and 0.01% (wt/vol) l-arabinose on similarly supplemented T-medium plates. Colicin and phage dilutions were added as 5-μl aliquots, and the plates were incubated at 37°C for 18 h and then scored for clearing.
↵ b The values are the highest numbers of dilutions (fivefold dilutions for colicins and 10-fold dilutions for φ80) of the agent that resulted in an evident zone of clearing in the cell lawn. R indicates resistance (i.e., no clearing) with the undiluted colicin or phage stock. The values for three platings are shown for each strain-agent pair.
- TABLE 3.
TonB-dependent ferrichrome transport
Straina Phenotype [55Fe]ferrichrome uptakeb W3110(pBAD24) ExbB+ ExbD+ TolQ+ TolR+ 1,982.5 ± 273 RA1035(pBAD24) ExbB+ ExbD+ TolQ− TolR− 1,752.0 ± 33 RA1003(pBAD24) ExbB− ExbD− TolQ+ TolR+ 11.3 ± 4.3 RA1045(pRA002) ExbB+ ExbD− TolQ− pRA002+ −4.5 ± 2.1 RA1044(pBAD24) ExbB− ExbD+ TolQ+ TolR− −6.7 ± 2.9 RA1051(pBAD24) ExbB− ExbD− TolQ− TolR− −6.9 ± 6 ↵ a Most strains carried plasmid pBAD24; the exception was the strain which carried plasmid pRA002, which encodes TolR. Cells were grown with aeration at 37°C to mid-exponential phase in T-broth supplemented with 100 μg ml−1 ampicillin and then centrifuged and suspended at a concentration of 2 × 108 CFU ml−1 in M9 salts containing 0.1 mM nitrilotriacetate. Transport was initiated by addition of 150 pmol [55Fe]ferrichrome as previously described (34).
↵ b Uptake is expressed in cpm 55Fe transported per 1 × 109 cells min−1. The values are means ± standard deviations of triplicate assays.
