Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE 6013 Elements in Staphylococcus aureus

Abstract

ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci.IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013 A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

Keywords: Staphylococcus; Staphylococcus aureus; conjugation; gene transfer; mobile genetic elements.

FIG 1

Gene map of ICE6013. (A) Arrows show 15 ORFs of ICE6013, with eight homologs found in ICEBs1 and/or Tn916 colored as in reference . (B) Arrowheads show PCR primer sites for detecting ICE6013 circular forms (purple) and Tn insertion sites for various mutants used in this study (red). (C) Blocks show sites that encode conserved domains. The putative product or function of ORFs is noted in parentheses; annotation is based on strain DAR6247.

FIG 2

Detection of excised ICE6013 in wild-type strain JE2 under various growth conditions. (A) PCR amplification of 291-bp regions around the coupling sequence of ICE6013 circular forms (top gel) and 142-bp gyrB control amplicons (bottom gel) using 25 cycles. Ladder, 100-bp ladder (Promega). Other lanes show amplicons for growth conditions that differ by growth phase, temperature, medium pH, or UV irradiation dose or from a recA mutant. (B) qPCR was used to measure the empty integration site of ICE6013 relative to a 100% excision control (strain RN4220) grown under the same conditions and relative to gyrB as described in Materials and Methods. At least three biological replicates were performed for each condition, and the means and standard deviations are shown on a log scale. Statistical significance was assessed by comparison to the wild type under standard growth conditions (growth at 37°C in BHI to an OD₆₀₀ of ∼0.6) using a two-tailed t test. None of the comparisons were statistically significant.

FIG 3

Detection of excised ICE6013 in various strains under standard growth conditions (growth at 37°C in BHI to an OD₆₀₀ of ∼0.6). (A) PCR amplification of 291-bp regions around the coupling sequence of ICE6013 circular forms (top gel) and 142-bp gyrB control amplicons (bottom gel) using 25 cycles. Ladder, 100-bp ladder (Promega); WT, wild-type strain JE2. Other lanes show various mutants of JE2 with Tn insertions in ICE6013. (B) qPCR was used to measure the empty integration site of ICE6013 relative to a 100% excision control (strain RN4220) grown under the same conditions and relative to gyrB as described in Materials and Methods. At least three biological replicates were performed for each strain, and the means and standard deviations are shown on a log scale. Statistical significance was assessed by comparison to the wild type under standard growth conditions using a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

FIG 4

Identification of sequence bias in 58 unique, unambiguous annotated integration sites of ICE6013 from serially passaged transconjugants. The logogram shows nucleotides that are over- and underrepresented as above and below the y axis 0 line, respectively. Nucleotides with significant bias (P < 0.05) are shown in color. The 3-bp target site that is duplicated upon integration of ICE6013 is shown at the center.

FIG 5

Identification of ICE6013 subfamilies. The tree depicts gross differences between full-length ICE6013 sequences and was constructed from a matrix of pairwise average nucleotide identities (ANI) transformed into a distance, (100 − mean reciprocal ANI)/100, and clustered with the neighbor-joining algorithm. The sequences are labeled to indicate the staphylococcal species (Sau, S. aureus; Sag, S. agnetis; Sar, S. argenteus; Sha, S. haemolyticus; Sin, S. intermedius; Smi, S. microti; Sps, S. pseudintermedius; Ssa, S. saprophyticus; and Ssc, S. schleiferi), strain, other element name or chromosomal position, and eukaryotic host species. The adjacent gene map shows ORFs of ICE6013 as arrows, with eight homologs found in ICEBs1 and/or Tn916 colored as in Fig. 1. The vertical red arrowheads indicate the positions of a Tn552 insertion and a mariner Tn insertion in strain HDG2 and the mock wild type (DAR6247), respectively, which were removed for comparison purposes. The percentages of nucleotide identity along the sequences are indicated with gray shading.