Integrative and sequence characteristics of a novel genetic element, ICE6013, in Staphylococcus aureus - PubMed (original) (raw)

Integrative and sequence characteristics of a novel genetic element, ICE6013, in Staphylococcus aureus

Davida S Smyth et al. J Bacteriol. 2009 Oct.

Abstract

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant Staphylococcus aureus (MRSA) revealed a novel genetic element, ICE6013. The element is 13,354 bp in length, excluding a 6,551-bp Tn552 insertion. ICE6013 is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced S. aureus strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE6013 were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE6013 is predicted to encode 15 open reading frames, including an IS30-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE6013 is more closely related to ICEBs1 from Bacillus subtilis than to the only other potential integrative conjugative element known from S. aureus, Tn5801. Evidence of recombination between ICE6013 elements is also presented. In summary, ICE6013 is the first member of a new family of active, integrative genetic elements that are widely dispersed within S. aureus strains.

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Figures

FIG. 1.

FIG. 1.

Chromosomal insertion in strain HDG2. The intergenic region between loci homologous to SAR2664 and SAR2665 contains a 19,905-bp insertion in ST239 strain HDG2 that is absent from ST36 strain EMRSA16. Dotted lines point to the junctions of this insertion. Three-base-pair direct repeats are underlined, and 8-bp imperfect inverted repeats are italicized. The insertion represents a novel genetic element, ICE_6013_. The direction of transcription for each predicted ORF is shown with large arrows. The boxed region indicates a Tn_552_ insertion.

FIG. 2.

FIG. 2.

Southern blotting of EcoRV-digested genomic DNA using an ICE_6013_ probe. Lanes 1 and 11, DIG-labeled HindIII-digested lambda ladder (Roche); lanes 2 and 10, blank; lanes 3 to 9, seven strains. The arrow indicates the expected 9.6-kb fragment from strain HDG2.

FIG. 3.

FIG. 3.

Circularization and precise excision of ICE_6013_. (A) Schematic of outward-directed PCR to detect ICE_6013_ circular forms. Black triangles indicate flanking direct repeats. White triangles indicate imperfect inverted repeats. Primer pair O135/O136 is in the correct configuration to produce a PCR amplicon when ICE_6013_ is circularized or tandemly repeated. (B) Agarose gels (1%) above and below show PCR amplicons from genomic and chromosome-free DNA templates, respectively. Lanes 1 and 16, 100-bp ladder (Promega); lanes 2 to 15, alternate loading of circular-form and aroE amplicons for seven strains. Arrows indicate the expected 536-bp aroE products and 291-bp circular-form products. (C) Sequence from outward-directed PCR amplicons. The sequence for strain EMRSA16 is from an amplicon from a genomic DNA template; all other sequences are from amplicons from chromosome-free DNA templates. Three-base-pair coupling sequences are underlined, and 8-bp imperfect inverted repeats are italicized.

FIG. 4.

FIG. 4.

Select sequences of ICE_6013_ in comparison to other genetic elements. (A) Alignment of a predicted DDE motif from ICE_6013_, the Tra8 conserved domain, and the IS_30_ Tpase. Conserved residues are in boldface type (34). Spacings between residues are in parentheses. (B) Alignment of a predicted oriT sequence from ICE_6013_ with sequences from ICE_Bs1_, Tn_916_, and Tn_5801_. A predicted nick site occurs between the 2 bp in boldface type (47). Seven-base-pair imperfect inverted repeats are italicized. Locations of these sequences are in parentheses.

FIG. 5.

FIG. 5.

Comparisons of ICE_6013_ with other genetic elements. (A) The direction of transcription for each predicted ORF is shown with large arrows. The boxed region in ICE_6013_ indicates a Tn_552_ insertion. Colored arrows indicate that the ICE_Bs1_, Tn_916_, or Tn_5801_ ORF matches the corresponding ICE_6013_ ORF in pairwise BLASTP comparisons. The two outlined arrows, representing ICE_Bs1_ YddC and Tn_916_ Orf15, indicate a global alignment length that spans <50% of the ICE_6013_ ORF. (B) Phylogenetic tree of concatenated amino acid sequences of ICE_6013_ Orf6, Orf7, Orf8, Orf12, and Orf14 and their homologs from the other genetic elements.

FIG. 6.

FIG. 6.

Recombination in ICE_6013_ sequences. (A) Alignment of 76 parsimony-informative nucleotide sites from third-codon positions and intergenic regions of seven ICE_6013_ elements. Dots indicate nucleotide identity with the top sequence. Numbering above the alignment indicates nucleotide positions in a 13,284-bp gap-free alignment. Numbering below the alignment indicates ORFs. “i” indicates an intergenic site. (B) Phylogenetic compatibility analysis of the 76 sites. White boxes indicate pairwise compatible sites. Black boxes indicate pairwise incompatible sites. (C) Matrix showing percentages of pairwise compatible sites among ICE_6013 orf5_ to orf8.

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