Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer

Abstract

Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101-encoded proteins, the pilus-tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface-displayed TraC and Pep are required for an efficient conjugative transfer, 'extracellular complementation' potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the β-barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS-encoded, pilus-independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.

Fig. 1.

TraC and Pep stimulate intercellular contacts and mediate phage infection. Top: Strains used for the assays presented in panels A, B, and C. E. coli DH5α harbored the nontransmissible plasmids denoted in columns 1 – 8 that encode the Tra_pKM101 T4SS operon with (+) or deleted (Δ) of traC or pep;: 1. pKM101, 2. pCGR125, 3. pCGR135, 4. pCGR107, 5. pJG127, 6. pJG127 + pDA105; 7. pJG127 + pMB006; 8. pJJG127 + pMB007. Δ(c-traC, c-pep, or c-orf70): the complemented strain expresses the gene listed from the P_nahG promoter. A) Conjugation assays. Donor strains additionally harbored the transmissible plasmid poriT (pJG142), which carries pKM101’s oriT sequence and traI (relaxase), traK (accessory factor), and traJ (T4CP) mobilization genes. Donors were mated with the plasmid-free recipient (MC4100-Rif) for 2-h in liquid or on a nitrocellulose disc on LB plates (Solid-surface). Transfer frequencies depicted in the bar graph are presented as transconjugants (Tc’s)/donor. B) PRD1 and IKe phage sensitivity of E. coli strains carrying the above-listed plasmids; (S) sensitive or (R) to infection, as monitored with a plaque assay. C) Survival of E. coli strains with the above-listed plasmids when cultivated in the absence or presence of P. aeruginosa PAO1. Statistical significance is shown based on a Student’s t test corresponding to the values of plasmid-free DH5α or growth in the absence of P. aeruginosa (*P<0.05). Data presented are mean ±SE, n=3 independent replicates.

Fig. 2.

TraC and Pep localize on the E. coli cell surface. A) Upper: TraC_ST is surface-displayed in the absence of the Tra_pKM101 T4SS as determined with a dot blot assay. Strains: MC4100 producing the indicated proteins from plasmids in parantheses: TraC_ST (pCGR83), _STTraC (pCGR66), MBP_ST (pCGR84), TraF_ST (pCGR48), untagged (WT) TraC (pCGR35). Whole cells were spotted onto nitrocellulose and probed for detection of Strep-tagged proteins (with α-Strep), surface-exposed RcsF lipoprotein (α-RcsF), or the periplasmic POTRA domains of BamA (α-BamA POTRA). Lower: Lysates from cells deposited onto the nitrocellulose were analyzed for steady-state accumulation of Strep-tagged TraC, MBP, and TraF by SDS-PAGE and immunostaining with α-Strep antibodies. Left: Molecular size markers (kDa). Blots were also developed with antibodies to the RNAP β subunit as a loading control. B) TraC.V144C_ST reacts with BSA-Mal in the absence of the Tra _pKM101 T4SS. Strains: MC4100 producing TraC_ST (pDA107), TraC.Q46C_ST (pDA110), or TraC.V144C_ST (pDA112). Intact cells were treated (+) or not treated (−) with BSA-Mal, the reactions were quenched, and cell lysates were analyzed for formation of TraC_ST/BSA-Mal complexes by SDS-PAGE and immunostaining with α-FLAG antibodies. Numbers at left: molecular size markers (kDa). Positions of higher-order TraC_ST/BSA-Mal complexes and monomeric TraC_ST are indicated. C) Identification of Pep_FL in the extracellular fraction obtained by shearing of cells. Strains: MC4100 cells producing Pep_FL (from pDA103), native Pep (pDA108), or MBP_FL (pDA106). Whole cell extracts (left) or extracellular material recovered by shearing of cells (right) was analyzed for the presence of Pep_FL, and periplasmic MBP_FL as a control, by immunostaining with α-FLAG antibodies. A nonspecific crossreactive species detected in extracellular fractions from all strains served as a loading control. Immunoblots were also stained with antibodies reactive to the periplasmic POTRA domains of BamA and the cytosolic β subunit of RNA polymerase (RNAP). D) Pep is displayed on the cell surface in the dot blot assay. Strains: MC4100 producing Pep_FL, Pep, or MBP_FL from the above-listed plasmids, or Pep bearing FLAG tags immediately following the residue indicated from plasmids pDA113, 117, 115, 119, 116, 120, or 121. Whole cells were spotted onto nitrocellulose and probed with α-FLAG for detection of FLAG-tagged proteins, α-RcsF for surface-exposed RcsF lipoprotein, or α-BamA POTRA for periplasmic POTRA domains of BamA. Lysates from cells deposited onto the nitrocellulose were analyzed for Pep_FL or MBP_FL protein abundance by SDS-PAGE and immunostaining. Left: Molecular size markers (kDa). Blots were probed with α-RNAP for detection of cytosolic β-subunit of RNA polymerase as a loading control. E) PepC105_FL crosslinks with membrane-impermeable BSA-Mal. Strains: MC4100 producing Pep_FL (pDA103) or Pep.C105_FL (pMB005). Intact cells were treated (+) or not treated (−) with BSA-Mal, the reactions were quenched, and cell lysates were analyzed for formation of Pep_FL/BSA-Mal complexes by SDS-PAGE and immunostaining with α-FLAG antibodies. Right: Positions of higher-order Pep_FL/BSA-Mal complexes and monomeric Pep_FL are indicated. Left: Molecular size markers (kDa).

Fig. 3.

TraC and Pep form punctate foci or patches on the cell surface. A) Left: Strain MC4100(pCGR110) producing TraC_ST in the presence of the Tra_pKM101 T4SS. TraC_ST puncta were detected by reactivity with α-Strep rabbit primary and AlexaFluor 594-conjugated goat α-rabbit secondary antibody. Cells were visualized by staining with Hoechst 3322 (blue, right panel), or the Hoechst emission channel was omitted (unstained cells, right panel). Right: Strain MC4100(pCGR127) producing Pep_FL from the kikA locus in the presence of the Tra_pKM101 T4SS. Pep_FL puncta and patches were visualized by reactivity with α-FLAG primary and AlexaFluor 488-conjugated secondary antibody, and cells by Hoechst staining as described above. B) Left and Right: MC4100 strains producing TraC_ST (from pCGR83) or Pep_FL (pDA103) in the absence of the Tra_pKM101 T4SS. TraC_ST, Pep_FL and cells were stained as described above. Scale bar = 5 μm. 3D-SIM localization patterns of TraC_ST and Pep_FL were obtained using a Nikon n-SIM Structured Illumination Super Resolution Microscope System housed at the Nikon Center of Excellence at UTHealth in the McGovern Medical School.

Fig. 4.

Extracellular TraC and Pep interact and form higher-order complexes. A and B) Whole cells of strain MC4100(pMB006), which coproduces TraC_ST and Pep_FL, were untreated (−) or treated (+) with BS³ crosslinker, and extracellular material was subjected to affinity pulldowns. Immunoblots were analyzed as indicated with α-Strep for detection of TraC_ST complexes or α-FLAG for detection of Pep_FL complexes. Monomeric forms of proteins are identified as TraC_ST or Pep_FL, crosslinked higher-order species are identified as TraC_ST* or Pep_FL*. Putative TraC_ST-Pep_FL heterodimers are denoted with black arrow heads, putative Pep_FL homodimers are denoted with a white arrow head. C and D) Cys-substituted variants of TraC and Pep form disulfide crosslinked homodimers. Strains: MC4100(pDA112) coproduces TraC.V144C_ST (V144C) and Pep_FL (WT); MC4100(pDA111) coproduces TraC.V144C_ST and Pep.C105_FL. Material from the pull-downs was analyzed by nonreducing SDS-PAGE and immunoblot development with α-Strep or α-FLAG antibodies for detection of Pep_FL or TraC_ST complexes, respectively. Monomeric TraC_ST and Pep_FL and homo- or heterodimeric complexes are indicated. Left: Molecular size markers (kDa).

Fig. 5.

TraC_ST and Pep_FL interact with components of the BAM pathway. Strain MC4100(pCGR129) produces the Tra_pKM101 T4SS plus TraC_ST and Pep_FL from the tra locus. A) TraC_ST- and Pep_FL-interacting proteins were identified by affinity pull-downs and high-resolution nano-flow LC/MS. Venn Diagram shows proteins recovered in TraC_ST or Pep_FL pulldowns, or with both sets of pulldowns. Quantitative data for this strain and the control strain MC4100(pCGR125) producing untagged forms of TraC and Pep are presented in Table S1. B and C) Detection of BS³-crosslinked proteins in material recovered from TraC_ST (B) or Pep_FL (C) pull-downs. Strains: MC4100(pCGR129) coproduces TraC_ST and Pep_FL; MC4100(pCGR125) (denoted Ut) produces untagged forms of TraC and Pep. Material recovered from both sets of pull-downs was analyzed by immunoblotting for TraC_ST with α-Strep, Pep_FL with α-FLAG, or BamA, BamC, RcsF, or OmpA with respective antibodies. Molecular size markers (in kDa) at left. Lower panels: Total cell extracts from MC4100 carrying pCGR125 or pCGR129 were analyzed for the presence of each protein indicated. Higher-order crosslinked species of TraC_ST, Pep_FL BamA, and BamC are identified with an asterisk.

Fig. 6.

Evidence for involvement of the BAM pathway for export of TraC and Pep. A) Photocrosslinking of pBPA-substituted TraC_ST variants with BamA. MC4100 strains produced TraC_ST (from pCGR83), or TraC_ST variants with pBPA incorporated at positions Q46 (pCGR86), D73 (pCGR87), L86 (pCGR88), Q191 (pCGR93), or A218 (pCGR94). Cells were treated with UV and isolated membrane fractions were analyzed for the presence of TraC_ST-BamA complexes by immunostaining with α-BamA antibodies (top panel) or total pBPA-crosslinked TraC_ST species (lower panel) by immunostaining with α-Strep antibodies. B) pBPA substitutions at the positions indicated in a space-filled model of the TraC X-ray structure (Yeo et al., 2003) formed detectable photocrosslinks with BamA. C and D) Effects of BAM pathway mutations on accumulation of extracellular complexes of TraC_ST and Pep_FL. Strains: MC4100 or indicated mutant strains producing TraC_ST from pDA107 (panel C) or Pep_FL from pDA103 (panel D). Cells were treated with BS³ (+BS³) and extracellular fractions were subjected to α-Strep or pull-downs. Material recovered from the pull-downs was analyzed for the presence of higher-order TraC_ST or Pep_FL by immunostaining. Monomeric and higher-order complexes (denoted with *) of TraC_ST and Pep_FL are indicated. Monomeric Pep_FL was detected upon overexposure (o.e.) of immunoblot (D, lower panel). Numbers below top panels correspond to relative abundance of crosslinked species compared with levels detected in MC4100(pDA107 or pDA103) cells (lane 1, set to 1), as determined by densitometry tracing of the regions of each lane demarked with] at the right of the panel using Image J software. Bottom panel: Total cellular levels (-BS³) of TraC_ST and Pep_FL in whole cell extracts of the strains listed at the top. A, C, & D) Molecular size markers (in kDa) at left.