The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids

Abstract

The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

Figure 1

Phylogenetic tree of RepA homologs. The RepA homologs were retrieved from the NCBI database by a standard BLAST search using pAD1 RepA as query. The tree was drawn using the CLC Combined Workbench sequence analysis software at default settings; bootstrap values are indicated at nodes. Clusters described in the text are bracketed and labeled with the associated genera. The following species abbreviations are used: Bs, Bacillus subtilis; Efm, Enterococcus faecium; Efs, Enterococcus faecalis; Lbb, Lactobacillus brevis; Lbc, Lactobacillus casei; Lbh, Lactobacillus helveticus; Lbp, Lactobacillus paracasei; Lbsak, Lactobacillus sakei; Lbsal, Lactobacillus salivarius; Lcl, Lactococcus lactis; Lm chrom, Leuconostoc mesenteroides chromosomal locus; Sa, Staphylococcus aureus; Se, Staphylococcus epidermidis; Sag chrom, Streptococcus agalactiae chromosomal locus; Sh, Staphylococcus haemolyticus; Spn, Streptococcus pneumoniae; Spy, Streptococcus pyogenes; Ss, Staphylococcus saprophyticus; S sp, undefined Staphylococcus species strain 693-2; Sth, Streptococcus thermophilus; Sw, Staphylococcus warneri. The references and accession numbers for these sequences are provided in Table 1.

Figure 2

Alignment of select RepA homologs from deeply branching groups. Representative plasmids were selected from each deeply branching group and aligned using CLC Combined Workbench at default settings. Abbreviations are as in Fig. 1 and plasmids are shown in the same order in which they appear in Fig. 1. The RepA_N consensus sequence was obtained from the NCBI website. Amino acids are colored according to a polarity coloring scheme (red = acidic, blue = basic, green = polar, black = non-polar). The final ~30 amino acids of the alignment were deleted because only very few of the proteins had C-terminal amino acids extending beyond those shown here.

Figure 3

Organization of direct and inverted repeats in repA genes of select plasmids. Large primary repeats are shown as solid arrows above the sequence. Secondary inverted repeats internal to the primary repeats are shown as open arrows over the sequence. Note that in the case of pAD1, pCF10, pSK41, pRUM, EJ-1 and pSK1 the inverted repeats converge on a poly-A tract. Although no obvious inverted repeats are present in the pSAK1 sequence, a poly-A tract is present near the center of the primary repeats.

Figure 4

Phylogenetic tree of RepB homologs. The RepB homologs were retrieved from the NCBI database by a standard BLAST search using pAD1 RepB as the query. The tree was drawn using the CLC Combined Workbench sequence analysis software at default settings; bootstrap values are indicated at nodes. The RepB homologs from Enterococcal RepA_N replicons are marked with thick arrows, from RepA_N replicons from other genera with thin arrows, and from inc18 replicons with stars. The pMP118 Lactobacillus salivarius plasmid actually contains both a Rep_3-like initiator protein and a divergent RepA_N protein that does not cluster with any of the homologs shown in Fig. 1. The following species abbreviations are used: Ba, Bacillus anthracis; Bc, Bacillus cereus; Bt, Bacillus thuringiensis; Cp, Clostridium perfringens; Efm, E. faecium; Efs, E. faecalis; Lbb, L. brevis; Lbc, L. casei; Lbpl, Lactobacillus plantarum; Lbr, Lactobacillus reuteri; Lbsal, L. salivarius; Lcl, L. lactis; Lin, Listeria innocua; Lm, L. mesenteroides; Pea, Pediococcus acidilactici; Spy, S. pyogenes; Ss, S. saprophyticus; Sw, S. warneri.

Figure 5

Organization of RepC binding sites in pAD1 and putative binding sites in other RepBC-containing plasmids. Vertically lined boxes depict repeats in the orientation shown on the right side of the figure. Horizontally lined boxes depict repeats present in the inverted orientation. Numbers in the boxes depict total number of repeats in the cluster. Boxes without numbers contain only a single repeat. Numbers between the boxes depict the size in nucleotides of non-repetitive spacer regions. pAD1 and pAM373 show two different repeat organizations in related pheromone-responsive E. faecalis plasmids. pX02 encodes a RepBC (type Ic) partition module associated with an inc18-like initiator protein (Tinsley et al. 2004). pTE15 (Lin and Chung 1999) contains the most closely related homolog of pAD1 RepB but does not have the type Ic organization of upstream and downstream repeats. The ParAB locus (par2) of E. coli plasmid pB171 is only distantly related to the pAD1 ParBC partition locus, but also possesses upstream and downstream repeats (Ebersbach and Gerdes 2001).

Figure 6

Phylogenetic tree of chromosomally-encoded RepA_N homologs. RepA_N homologs were identified using the consensus RepA_N sequence as query in a BLAST search at the NCBI database. The tree was drawn using the CLC Combined Workbench sequence analysis software at default settings; bootstrap values are indicated at nodes. Select representatives from the plasmid RepA_N replication initiators were included for reference. Two RepA_N proteins associated with known conjugative transposons are boxed. Chromosomal RepA_N proteins segregate neatly into size groups which are indicated with brackets and size range on the right.