The plasmid RK2 replication initiator protein (TrfA) binds to the sliding clamp beta subunit of DNA polymerase III: implication for the toxicity of a peptide derived from the amino-terminal portion of 33-kilodalton TrfA

Abstract

The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli beta sliding clamp (beta(2)). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with beta(2) and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and beta(2) and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1. The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the beta-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent over-initiation in E. coli through its interaction with the initiator protein, DnaA, and beta(2). Our results support a model in which T1 toxicity is caused by T1 binding to beta(2), especially when T1 is overexpressed, preventing beta(2) from interacting with host replication proteins such as Hda during the early events of chromosome replication.

FIG. 1.

Alignment of partial amino acid sequences of TrfA/RepA orthologues from plasmids related to RK2 and pMLb. Amino acids matching the beta-binding site consensus sequence are underlined. The vertical arrow indicates the start of the TrfA-33 protein. Sequences are labeled with the plasmid/mobile genetic element of origin or GenBank gi number where the plasmid/mobile genetic element has not been named.

FIG. 2.

(A) SDS-PAGE analysis of purified wild-type β₂ (lane 1), His-tagged TrfA-33 (lane 2), and His-tagged TrfA-33 (ΔLF) (lane 3) proteins. Three to five micrograms of each purified protein was electrophoresed through a 4 to 12% SDS-PAGE gel prior to being stained with Coomassie brilliant blue R-250 (Bio-Rad). Positions of size standards are indicated. (B) Interactions of β₂ with TrfA-33 proteins. Increasing concentrations of β₂ protein were added to TrfA-33 proteins immobilized onto microtiter plates (5 μg/ml, 50 μl/well). Bound β₂ proteins were detected with anti-β₂ antibody. ▪, ΤrfA-33 on plate; ▴, TrfA-33 (ΔLF) on plate; •, binding buffer, no protein on plate. (C) β₂-binding consensus peptide, peptide 14 with the sequence IG QLSLF GV, inhibited the interaction of TrfA-33 and β₂. Microtiter plate assay results showing the inhibition of TrfA-33 and β₂ interaction by addition of various amounts of peptide 14. No inhibition was observed with the control peptide, peptide 4 with the sequence IG QADMA GV. ▪, peptide 14; •, peptide 4.

FIG. 3.

Inhibition of cell growth upon overproduction of T1. E. coli TOP10 cells were transformed by various pBAD plasmids expressing TrfA-33 peptides. Cells were grown at 37°C, and protein expression was induced by adding arabinose (final concentration, 2%) to the culture. Aliquots were then withdrawn at the indicated times for measurement of the optical density (OD₆₀₀) which was used as an indicator of cell growth. □, pBAD-T1 with 2% arabinose added; ▪, pBAD-T1 with no arabinose; ▴, pBAD-T1-2 with 2% arabinose added; •, pBAD-T2 with 2% arabinose added.

FIG. 4.

Growth, DNA replication, and cell division were suppressed by overproduction of the TrfA T1 peptide but not by the TrfA T1 (ΔLF) mutant. Cells were transformed by various pBAD plasmids and grown at 37°C in LB supplemented with 100 μg/ml ampicillin and 2% arabinose. Cultures were withdrawn at the times indicated. (A) Cell growth was monitored by measuring the OD₆₀₀ of the cultures. (B) DNA replication was assessed by measuring the incorporation of [H³]dTTP into acid-insoluble materials. □, pBAD-T1; ▪, pBAD-T1 (ΔLF); •, pBAD-T2.

FIG. 5.

Defects in cell morphology in the cells with overexpressed T1 peptide and suppression of this phenotype in the cells expressing the mutant peptide. Cells transformed with pBAD-T1 and pBAD-T1 (ΔLF) were grown at 37°C in LB supplemented with 100 μg/ml ampicillin and 2% arabinose until the OD₆₀₀ reached 0.5. They were then fixed with methanol and stained with neutral red (0.5%) to obtain microphotographs. Bars, 5 μm.

FIG. 6.

T1 interacts with β₂, and mutation in β₂-binding motif disrupts the interaction. Purified H₁₀-TrfA-33, H₆-T1, H₆-T2, and H₆-T1 (ΔLF) were coupled to 50 μl of Ni-NTA resin (2 h at 4°C with rotation) and incubated with 2 μg of wild-type β₂ in 200 μl of binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) at room temperature for 30 min with constant agitation. After excessive washing with the same buffer (four times with 500 μl), the resin was resuspended in 25 μl of SDS sample buffer, and the bound proteins were separated in SDS-PAGE and probed with anti-β₂ polyclonal antibody. Arrows indicate histidine-tagged proteins, and the arrow indicates the Ni-NTA pull-down product, β_2.

FIG. 7.

T1 (ΔLF) can be expressed at a high level in E. coli cells, but T1 cannot. Coomassie-stained SDS-PAGE of total cell lysates from TOP10 cells expressing T1 (ΔLF), T2, and T1. Arrows point to His-tagged T1 (ΔLF) and T2. Protein size standards are shown on right.