Article Figures & Data
Figures
- FIG. 1.
In the absence of an identifiable TolB-binding sequence, colicin K translocation is TolB dependent. (A) Sequence alignment of colicin K and three TolB-dependent colicins (A, E2, and E9). Conserved residues are indicated by red letters. The characterized TolB binding sequence is indicated by the green box (defined in references 12 and 27). (B) Colicin spot assays using serial dilutions of colicins A (TolB dependent), E1 (TolB independent), and K on a wild-type (WT) strain and its tolB derivative (from left to right, 100, 10, 1, and 0.1 ng of colicins have been spotted, respectively).
- FIG. 2.
The colicin K N-terminal domain is tethered to the membrane fraction through interaction with Tol components. Total (T) wild-type (WT) (A and B) and tolQRABPal (ΔQRABP) (C) cells producing KT were subjected to fractionation to separate the periplasm (P), cytoplasm (C), and membrane (M) fractions. Membrane proteins from the M fraction of panel A were separated into integral membrane proteins (iM) and peripherally associated membrane proteins (pM) by Na2CO3 treatment (B). Samples were loaded onto a 12.5% acrylamide SDS-PAGE gel and subjected to immunodetection with antibodies raised against the cytoplasmic EF-Tu, the periplasmic MalE, the peripherally membrane-associated TolB, the integral membrane TolR and AcrA proteins, and the FLAG epitope carried by the colicin K translocation domain (KT). Molecular mass markers (in kDa) are indicated on the left of each panel.
- FIG. 3.
Periplasmic production of KT induces tol phenotype. Comparison between wild-type cells carrying the empty vector pIN-III-ompA2 (vector) or pKT (KT) for periplasm content release (A) (C, cell pellet; S, culture supernatant); RNase I release (B, upper panels); deoxycholate (DOC) susceptibility (B, lower panels) (from top to bottom, 106, 105, and 104 cells were spotted, respectively); release of outer membrane vesicles (C) (arrows indicate vesicles); and susceptibility to colicins K, A, E1, E2, and 5 (D) (from left to right, 100, 10, and 1 ng of colicins have been spotted, respectively). Quantitative survival measurements in the presence of various concentrations of sodium dodecyl sulfate and colicins are shown in Fig. S2 in the supplemental material and reported in Table 1.
- FIG. 4.
KT interacts in vitro with TolA, TolB, TolQ, and TolR. Copurification experiments using histidine-tagged purified proteins immobilized on ion metal affinity chromatography columns and periplasmic extracts of tolQRABPal cells carrying the empty vector pIN-III-ompA2 (−) or pKT (+). The immobilized protein is indicated (A3, C-terminal domain of the TolA protein; B, full-length TolB protein; R2-3, periplasmic domain of the TolR protein; Q, full-length TolQ protein; Pal, Pal lipoprotein devoid of its acylated N-terminal cysteine residue; B2-3, periplasmic domain of the TonB protein; none, no protein immobilized). Immunodetected His-tagged proteins are indicated by asterisks. Molecular mass markers (in kDa) are indicated on the left.
- FIG. 5.
KT interacts in vivo with TolA, TolB, TolQ, and TolR. Detergent-solubilized extracts of wild-type cells producing TolQHA and carrying the empty vector pIN-III-ompA2 (vector) or pKT (KT) producing the FLAG epitope-tagged colicin K N-terminal domain were subjected to immunoprecipitation using the anti-FLAG antibody (A). The total solubilized material (input [Inp]) and the precipitated material (IP) were loaded on a 12.5% acrylamide SDS-PAGE gel and immunodetected with the anti-HA and anti-FLAG monoclonal antibodies and with the anti-Pal, -TolA, -TolB, and -TolR polyclonal antibodies. Detergent-solubilized extracts of the same strains were subjected to immunoprecipitation using anti-TolA (B), anti-TolB (C), anti-TolR (D), and anti-HA (E) (precipitation of the HA epitope-tagged TolQ protein) antibodies. The corresponding mutant strain carrying the KT-producing vector has been used as an additional control for specificity. Immunodetected proteins are indicated on the right. Asterisks indicate heavy or light chains of immunoglobulins. Molecular mass markers (in kDa) are indicated on the left.
- FIG. 6.
The TolQ periplasmic loop L164 residue is required for TolQ-KT interaction. (A) Colicin spot assays using serial dilutions of colicin K (from top to bottom, 1,000, 100, 10, and 1 ng of colicins have been spotted) on tolQ cells producing HA epitope-tagged TolQ (WT) or cysteine variants (V159C to A168C). (B) Detergent-solubilized extracts of tolQ cells coproducing TolQHA or its Cys derivatives and the FLAG epitope-tagged colicin K N-terminal domain were subjected to immunoprecipitation using the anti-FLAG antibody. The total solubilized material (input [Inp]) and the precipitated material (IP) were loaded on a 12.5% acrylamide SDS-PAGE gel and immunodetected with the anti-HA (TolQHA) and anti-FLAG (KT) monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular mass markers (in kDa) are indicated on the left.
Tables
- TABLE 1.
Phenotypic consequences of the periplasmic production of the colicin K translocation domain
Strain and IPTG inductiona Outer membrane stability Infection by: DOCb SDSc RNased Colicine fd bacteriophagef ColK ColA ColE1 ColE2 ColIa Col5 WT pIN-III-ompA2 Noninduced 94.0 2 − 0.07 0.07 0.07 0.04 0.05 0.04 2.8 × 10−1 Induced 92.0 2 − 0.07 0.07 0.07 0.04 0.05 0.04 3.1 × 10−1 WT pKT Noninduced 3.0 0.35 + >500 >500 7 500 0.05 0.04 2.1 × 10−2 Induced 1.3 0.35 + >500 >500 8 500 0.05 0.04 1.5 × 10−2 ↵ a Induction with 10 mM IPTG.
↵ b Percent survival on 1% sodium deoxycholate (DOC) plates.
↵ c Percent sodium dodecyl sulfate (SDS) concentration required to inhibit 50% of bacterial growth after 120 min (50% lethal dose) (from Fig. S2A in the supplemental material).
↵ d Presence (+) or absence (−) of the halo surrounding the bacterial spot on RNA plates.
↵ e Colicin concentration (ng/ml) required to inhibit 50% of bacterial growth after 120 min (50% lethal dose) (from Fig. S2B).
↵ f Infection frequency, i.e., number of infected cells per total cells.
- TABLE 2.
Protection by the KT domain against exogenous colicins or bacteriophage
Colicin or bacteriophage Protectiona ColK >7,000 ColA >7,000 ColE1 100 ColE2 12,500 ColIa 1 Col5 1 fd bacteriophage 20 ↵ a Protection by the KT domain against exogenous colicins or bacteriophage is reported as the ratio of 50% lethal dose of colicin or bacteriophage for WT pKT cells to 50% lethal dose of colicin or bacteriophage for WT pIN-III-ompA2 cells (Table 1).
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1, sequence aligment of colicins K, 5, and 10
PDF file, 13K. - Supplemental file 2
-
Fig. S2, survival measurements of cells producing periplasmic KT
Zipped MS PowerPoint file, 126k.
- Supplemental file 1 -
Fig. S1, sequence aligment of colicins K, 5, and 10
