Mutations in the C-terminal region of TraM provide evidence for in vivo TraM-TraD interactions during F-plasmid conjugation

Abstract

Conjugation is a major mechanism for disseminating genetic information in bacterial populations, but the signal that triggers it is poorly understood in gram-negative bacteria. F-plasmid-mediated conjugation requires TraM, a homotetramer, which binds cooperatively to three binding sites within the origin of transfer. Using in vitro assays, TraM has previously been shown to interact with the coupling protein TraD. Here we present evidence that F conjugation also requires TraM-TraD interactions in vivo. A three-plasmid system was used to select mutations in TraM that are defective for F conjugation but competent for tetramerization and cooperative DNA binding to the traM promoter region. One mutation, K99E, was particularly defective in conjugation and was further characterized by affinity chromatography and coimmunoprecipitation assays that suggested it was defective in interacting with TraD. A C-terminal deletion (S79*, where the asterisk represents a stop codon) and a missense mutation (F121S), which affects tetramerization, also reduced the affinity of TraM for TraD. We propose that the C-terminal region of TraM interacts with TraD, whereas its N-terminal domain is involved in DNA binding. This arrangement of functional domains could in part allow TraM to receive the mating signal generated by donor-recipient contact and transfer it to the relaxosome, thereby triggering DNA transfer.

FIG. 1.

Characterization of the wild type and selected mutants of TraM. (A) Comparison of levels of TraM and its mutants by immunoblot analysis with anti-TraM antiserum when expressed from its native promoters (P_traM) or from an attenuated foreign promoter (P_traJ). N.A., not applicable. (B) Autorepression of P_traM was determined by assaying the β-galactosidase activity of cells containing pACPM24fs::lacZ and pJLM102 and its derivative pJLM105 (K99E). Complementation of pOX38-MK3 was determined by measuring the donor ability of cells containing pOX38-MK3 and pJLM102 or pJLM105. <10⁻⁷ means that there was no detectable donor ability. Negative dominance was assayed by measuring the donor ability of pOX38-Km and pJLM102 or pJLM105. pT7-4 was the vector control.

FIG. 2.

Analytical SEC fractions of purified TraM and K99E. Ten microliters from each fraction was separated by 15% SDS-polyacrylamide gels and visualized by immunoblot analysis with anti-TraM antiserum. Fraction numbers are indicated above each lane. The positions of different marker proteins are shown above the figure. BSA is bovine serum albumin monomer (66 kDa); CEA represents chicken egg albumin (45 kDa); CA represents carbonic anhydrase (29 kDa).

FIG. 3.

Binding of TraM or K99E to sbmABC as determined by EMSA. Increasing concentrations of TraM or K99E (in nM) are shown above the figures. Each reaction contained 40 nM of sbmABC, which contains all three TraM binding sites.

FIG. 4.

His₆-TraD and TraM (or its mutant proteins) interaction as determined by affinity chromatography. Equivalent amounts of purified TraM or selected mutants were incubated with BSA and His₆-TraD (or His₆-TraK)-saturated Ni-NTA agarose resin as indicated. Eluted protein was detected by immunoblotting using anti-TraD antiserum (top panel) or anti-TraM antiserum (bottom panel). Protein molecular weight markers are indicated to the right of the figure. Lane 1, His₆-TraD and BSA; lane 2, mixture in lane 1 plus S79*; lane 3, mixture in lane 1 plus TraM; lane 4, mixture in lane 1 plus K99E; lane 5, mixture in lane 1 plus F121S; lane 6, His₆-TraK, BSA, and TraM. +, present; −, not present.

FIG. 5.

TraD and TraM interactions assayed by coimmunoprecipitation. TraD and FLAG-tagged TraM (or one of its mutants) were coexpressed in E. coli cells. The complex of TraD and FLAG-tagged TraM was coprecipitated by anti-FLAG M2 agarose from the cell extract. The amounts of TraD and FLAG-tagged TraM (or one of its mutants) were determined by immunoblotting with anti-TraD antiserum (top panel) or anti-TraM antiserum (bottom panel). Protein molecular weight markers are indicated to the right of the figure. Lane 1, TraD; lane 2, FLAG-TraM; lane 3, TraD and FLAG-S79*; lane 4, TraD and FLAG-K99E; lane 5, TraD and FLAG-TraM; lane 6, TraD and FLAG-F121S. +, interaction; −, no interaction.