Specificity determinants of conjugative DNA processing in the Enterococcus faecalis plasmid pCF10 and the Lactococcus lactis plasmid pRS01

Abstract

The DNA-processing region of the Enterococcus faecalis pheromone-responsive plasmid pCF10 is highly similar to that of the otherwise unrelated plasmid pRS01 from Lactococcus lactis. A transfer-proficient pRS01 derivative was unable to mobilize plasmids containing the pCF10 origin of transfer, oriT. In contrast, pRS01 oriT-containing plasmids could be mobilized by pCF10 at a low frequency. Relaxases PcfG and LtrB were both capable of binding to single-stranded oriT DNAs; LtrB was highly specific for its cognate oriT, whereas PcfG could recognize both pCF10 and pRS01 oriT. However, pcfG was unable to complement an ltrB insertion mutation. Genetic analysis showed that pcfF of pCF10 and ltrF of pRS01 are also essential for plasmid transfer. Purified PcfF and LtrF possess double-stranded DNA binding activities for the inverted repeat within either oriT sequence. PcfG and LtrB were recruited into their cognate F-oriT DNA complex through direct interactions with their cognate accessory protein. PcfG also could interact with LtrF when pCF10 oriT was present. In vivo cross-complementation analysis showed that ltrF partially restored the pCF10DeltapcfF mutant transfer ability when provided in trans, whereas pcfF failed to complement an ltrF mutation. Specificity of conjugative DNA processing in these plasmids involves both DNA-protein and protein-protein interactions.

Fig. 1

A. Comparison of pCF10 and pRS01 DNA-processing regions. The minimal pCF10 oriT is a 40 bp sequence located in the intergenic region between pcfE-pcfF (Staddon et al., 2006). The intergenic region between ltrD-ltrE contains the functional pRS01 oriT (Mills et al., 1998), but the minimal oriT sequence was not determined previously. Dashed lines represent homology between gene products and the percentages of amino acid sequence identity are indicated. ltrM and ltrE encode proteins with no homologues in pCF10. ltrB is interrupted by the group II intron Ll.ltrB (Mills et al., 1996). The intron insertion site in ltrB is indicated by a vertical arrow. B. Sequence alignment of the minimal pCF10 oriT with other origins of transfer (adapted from Staddon et al., 2006). Inverted repeats are shown as dashed arrows. Asterisks represent nucleotides identical to the pCF10 sequence. The nic sites in pC221, pC223 and RP4 are indicated as ⇓. The Tn5252 sequence is part of a 2 kb sequence that includes the relaxase substrate (Srinivas et al., 1997).

Fig. 2

Binding specificity of PcfG and LtrB to single-stranded oriT DNA sequences. PcfG or LtrB was incubated with 3′-end DIG-labelled oligonucleotides (16 fmol) at room temperature for 15 min. The reactions were analysed on 8% native polyacrylamide gels. The components in each lane are indicated at the top of the gel. Sequences of the oligonucleotides are listed in Table 1. PcfG and LtrB concentrations: 150 nM when using pCF10-S and pRS01-S probes; 750 nM when using pCF10-AS and pRS01-AS probes. pCF10 oriT ssDNA: S*, 3′-end DIG-labelled pCF10-S; AS*, 3′-end DIG-labelled pCF10-AS; S, 10-fold molar excess of cold pCF10-S. pRS01 oriT ssDNA: S*, 3′-end DIG-labelled pRS01-S; AS*, 3′-end DIG-labelled pRS01-AS; S, 10-fold molar excess of cold pRS01-S. A. PcfG bound to pCF10 and pRS01 sense-strand oriT ssDNA. B. LtrB bound to only pRS01 sense-strand oriT ssDNA.

Fig. 3

Site-specific ssDNA cleavage and stand-transfer catalysed by PcfG. A. Schematic representation of the cleavage and strand-transfer assays. The DIG molecules are indicated with stars. B. PcfG (755 nM) was incubated with 3′-end DIG-labelled ssDNA and the reactions were carried out as described in the Experimental procedures. Lanes 1–6: pCF10 ssDNA. S1*, 3′-end DIG-labelled pCF10-S1 (60-mer); AS1*, 3′-end DIG-labelled pCF10-AS1 (60-mer); L, pCF10-L (28 mer). Lanes 7 and 8: pRS01 ssDNA. S*, 3′-end DIG-labelled pRS01-S (60-mer); L, pRS01-L (28-mer).

Fig. 4

Interactions of PcfF and LtrF with double-stranded oriT DNA sequences. dsDNA probes pRS01-20 (8 fmol) and pCF10-20 (8 fmol) were incubated with purified LtrF or PcfF for 15 min at room temperature. The reactions were analysed on 8% native polyacrylamide gels. Lanes 1 and 11, DNA probes only. Protein concentration in each lane is as follows. For LtrF (B): lane 2, 170 nM; lane 3, 340 nM; lane 4, 680 nM; lanes 5–10, and 12–15, 680 nM. For PcfF (C): lane 2, 78 nM; lane 3, 156 nM; lane 4, 312 nM; lanes 5–10, and 12–15, 312 nM. In the competition assays (lanes 5–10, and 12–15), cold specific or non-specific dsDNA was added to the preformed F protein and oriT complexes. Unlabelled cold competitor in each lane is indicated at the top of the gel. The concentrations of cold pCF10-20 and pRS01-20 competitors (molar excess) are as following: lanes 5 and 8, 10-fold; lanes 6 and 9, 100-fold; lanes 7 and 10, 1000-fold. The concentration of cold Tn5252 oriT (putative) competitor (molar excess) is as following: lane 13, 100-fold; lane 14, 1000-fold; lane 15, 10 000-fold. A. Schematic representations of the pcfE-pcfF and ltrD-ltrE intergenic regions. Inverted repeats are indicated as pairs of arrows. The linear segments below the map represent probes used in the EMSA assays. B. LtrF specifically bound to pRS01 and pCF10 oriT dsDNA. C. PcfF specifically bound to pCF10 and pRS01 oriT dsDNA.

Fig. 5

Interactions between PcfG and PcfF/LtrF—oriT DNA complexes. dsDNA probes pRS01-180 (16 fmol) and pCF10-186 (16 fmol) were incubated with purified PcfG (150 nM) and LtrF (314 nM) or PcfF (158 nM) for 15 min at room temperature. The reactions were analysed on 8% native polyacrylamide gels. Protein components in each lane are shown on the top of the gel. PcfG concentration in lane 1 was 300 nM. A. Interactions between PcfG and PcfF—oriT DNA. B. Interactions between PcfG and LtrF—oriT DNA.

Fig. 6

Interactions between LtrB and PcfF/LtrF—oriT DNA complexes. Double-stranded probe pRS01-180 (16 fmol) and pCF10-186 (16 fmol) were incubated with purified LtrB (152 nM) and LtrF (314 nM) or PcfF (158 nM) for 15 min at room temperature. The reactions were analysed on 8% native polyacrylamide gels. Protein components in each lane are shown on the top of the gel. LtrB concentrations in A lane 4, and B lane 3 were 304 nM. A. pRS01-180 DNA probe. B. pCF10-186 DNA probe.