Replication of Enterococcus faecalis pheromone-responding plasmid pAD1: location of the minimal replicon and oriV site and RepA involvement in initiation of replication

Abstract

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of Enterococcus faecalis. The determinants repA, repB, and repC, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The repA gene encodes an initiator protein, whereas repB and repC encode proteins related to stability and copy number. The present study focuses specifically on repA and identifies a replication origin (oriV) within a central region of the repA determinant. A small segment of repA carrying oriV was able to support replication in cis of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in trans. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only repA is sufficient for stable replication in E. faecalis. We also show that RepA binds specifically to oriV DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize oriV. An in-frame deletion within repA resulting in loss of 105 nucleotides, including at least part of oriV, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in trans. The relationship of RepA to other known initiator proteins is also discussed.

FIG. 1.

(A) pAD1 genetic map (not to scale) showing the replication and maintenance region, along with the adjacent pheromone response regulation region. Putative promoters are indicated by a “P” above the map, and the adjacent arrow indicates the direction of transcription. Transcriptional terminators are represented by t₁/t₂. Iterons and repA internal direct repeats are represented by “thick” black arrows. Thick gray arrows (above and below) represent the positions and orientations of the traB, repA, and repB primers. Different DNA fragments specifically analyzed are indicated by various lines named accordingly. (B) Nucleotide sequence of the RepA coding sequence showing the internal array of repeats. The numbers above are shown to indicate the nucleotide or amino acid position inside repA. MfeI, RsaI, and DraI restriction sites are indicated. Thin arrows correspond to the RepA internal direct repeats. Thick arrows represent the RepA internal invert repeats. The name of each repeat is also indicated as described previously (3). The asterisk indicates the location of the generated frameshift mutation in repA. Vertical arrows indicate the ends of the repA in-frame deletion obtained. The small gray arrows represent the specific primers used for the construction of clones.

FIG. 1.

(A) pAD1 genetic map (not to scale) showing the replication and maintenance region, along with the adjacent pheromone response regulation region. Putative promoters are indicated by a “P” above the map, and the adjacent arrow indicates the direction of transcription. Transcriptional terminators are represented by t₁/t₂. Iterons and repA internal direct repeats are represented by “thick” black arrows. Thick gray arrows (above and below) represent the positions and orientations of the traB, repA, and repB primers. Different DNA fragments specifically analyzed are indicated by various lines named accordingly. (B) Nucleotide sequence of the RepA coding sequence showing the internal array of repeats. The numbers above are shown to indicate the nucleotide or amino acid position inside repA. MfeI, RsaI, and DraI restriction sites are indicated. Thin arrows correspond to the RepA internal direct repeats. Thick arrows represent the RepA internal invert repeats. The name of each repeat is also indicated as described previously (3). The asterisk indicates the location of the generated frameshift mutation in repA. Vertical arrows indicate the ends of the repA in-frame deletion obtained. The small gray arrows represent the specific primers used for the construction of clones.

FIG. 2.

Analysis of the replication ability of an E. faecalis suicide vector carrying appropriate fragments of pAD1 DNA. (A) Schematic representation of the process envisioned for cointegrate formation by homologous recombination between the two plasmids pAM330 and pAM88A* carrying the pAD1 repA coding sequence. The related primers used to generate PCR products demonstrating no recovery of the cointegrate plasmid from the chloramphenicol-resistant E. faecalis JH2-2/pAM330 transformants are indicated. The positions of the relevant BamHI and EcoRI sites are also shown. The sizes of the PCR fragments obtained are listed below. N.O., no PCR fragment observed in the assay. (B) Agarose gel electrophoresis representing the BamHI (lanes 2 to 6) and EcoRI (lanes 8 to 12) restriction fragments of the plasmid DNA content of the chloramphenicol-resistant E. faecalis JH2-2/pAM330 transformants containing pAM88oriV plasmid (lanes 2 to 5 and lanes 8 to 11) or the E. coli DH5α/pAM88oriV cells used as a positive control (lanes 6 and 12). Plasmid DNA was obtained in both cases from alkaline lysis preparations as described in Materials and Methods. The molecular mass ladder 1-Kb-Plus (Invitrogen) is shown in lanes 1 and 7, and selected bands or sizes are noted on the left and right. (C) Southern blot analysis of the DNA restriction profiles shown in panel B. The EcoRI restriction fragments of pAM88oriV plasmid DNA were labeled and used as a probe. Black arrowheads indicate the BamHI or EcoRI restriction fragments corresponding to pAM88oriV, and band sizes are noted on the left (BamHI) and right (EcoRI). The asterisks indicate the corresponding pAM330 fragments carrying oriV sequences. (D) The sizes of the restriction fragments that would have been obtained from cointegration events are indicated.

FIG. 3.

Analysis of the pAD1 minimal replicon. (A) Schematic diagram showing the construction of pAM434brepA and unique restriction sites are indicated (21); (B) agarose gel electrophoresis representing the BamHI/NsiI (lanes 2 and 3) restriction fragments (black arrowheads) of the DNA plasmid content (pAM434brepA) of two independently obtained erythromycin-resistant E. faecalis JH2-2 transformants. The molecular mass ladder 1-Kb-Plus (Invitrogen) is shown in lane 1. Representative sizes are indicated on the left and right. *, Per Flannagan and Clewell (21).

FIG. 4.

Gel mobility shift assays showing in vitro RepA-DNA-binding properties. PCR or end-labeled dsDNA or ssDNA fragments containing the oriV or iteron repeats were incubated with 0.5 μg of purified RepA, purified RepB, or empty vector (pASK60)-derived control protein extracts in the absence or presence of increasing concentrations of unlabeled competitor DNA fragments. (A) Mobility shift assays showing RepA-specific binding to dsDNA. The substrate DNA used was as follows: lanes 1 to 10, dsDNA corresponding to the repA MfeI/RsaI internal sequence (oriV); lanes 11 to 16, dsDNA corresponding to the iteron repeats upstream of the repB coding sequence (It3′). Purified protein added in each lane is indicated at the top of the figure. The addition of a 1-, 10-, 50-, or 100-fold excess of unlabeled dsDNA fragments (competitor DNA) is also indicated at the top of the figure. (B) Mobility shift assays showing RepA N-terminal domain specific binding to dsDNA. Lanes 1 to 3, dsDNA corresponding to oriV (MfeI/RsaI repA internal fragment); lanes 4 and 5, dsDNA corresponding to the iteron repeats upstream of the repA coding sequence (It5′ PCR product). Purified protein domains (N and C terminal) added in each lane are indicated at the top of the figure. (C) Mobility shift assays showing RepA nonspecific binding to ssDNA. Lanes 1 to 4 represent It3′ PCR product containing both dsDNA and ssDNA forms. In contrast to panels A and B, the DNA used in panel C had not been exposed to S1 nuclease. Lanes 5 to 8 represent oriV PCR product containing both dsDNA and ssDNA forms. Purified protein fractions in each lane are also indicated at the top of the figure. Free DNA forms and RepA-DNA complexes are indicated.

FIG. 5.

Gel mobility shift assays showing the importance of the IR-1 repeats in the putative RepA recognition site. (A) Nucleotide sequence of the repA MfeI/RsaI segment containing the oriV site. IR-1 repeats are indicated by the arrows above the sequence and are numbered 1 through 5 (correlating with specific mutations generated). (B) Gel mobility shift assays with end-labeled dsDNA fragments containing the IR-1 (wild type) (lanes 1 and 2), IR-1* (four point mutations) (lanes 3 and 4), or iteron (ItC) (lanes 5 and 6) repeats. Lanes 1, 3, and 5 represent control dsDNA fragments. Lanes 2, 4, and 6 represent dsDNA plus purified RepA protein. (C) Competition of RepA binding to the MfeI/RsaI oriV dsDNA fragment with increasing concentrations (same as Fig. 4) of unlabeled IR-1 (lanes 3 to 6) or IR-1* (lanes 7 to 10) dsDNA fragments. Lane 1, control oriV DNA; lane 2, RepA control binding reaction to oriV dsDNA; lanes 3 to 10, competition reactions as indicated at the top of the figure.

FIG. 6.

Map of RepA indicating strongly conserved amino acids among the related family of replication proteins. Identical and similar amino acids conserved in the N-terminal region of all proteins (38 proteins in the database compared) are noted in capital letters. (Similar residues are according to conservative substitutions [V/I/L, T/S, D/E, N/Q, Y/F, and R/K].) Positions of putative motifs reflecting conserved residues are noted by the hashed boxes below the N terminus. Amino acid residues that are strongly conserved among RepA homologues of Enterococcus, Staphylococcus, and Lactococcus spp. are indicated by lowercase letters above the C-terminal region. The “u” denotes hydrophobic amino acids in the noted position. The repeats present in the central region are noted in gray. The white box under the N terminus indicates the fragment shown to have specific DNA-binding properties.