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. 2018 Jul 31;9:1779.
doi: 10.3389/fmicb.2018.01779. eCollection 2018.

Excision and Circularization of Integrative Conjugative Element Tn 5253 of Streptococcus pneumoniae

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Free PMC article

Excision and Circularization of Integrative Conjugative Element Tn 5253 of Streptococcus pneumoniae

Francesco Santoro et al. Front Microbiol. .
Free PMC article

Abstract

The integrative conjugative element (ICE) Tn5253 of Streptococcus pneumoniae, conferring resistance to tetracycline and chloramphenicol, was found integrated at a 83-bp specific target site (attB) located in the rbgA gene of the pneumococcal chromosome. PCR analysis of Tn5253-carrying strains showed evidence of precise excision of Tn5253 from the pneumococcal chromosome with production of (i) circular forms of the ICE in which the ends were joined by a 84-bp sequence (attTn), and (ii) reconstituted chromosomal attB. When integrated into the chromosome, Tn5253 was flanked by attL, identical to attB, and attR, identical to attTn. Circular forms of Tn5253 were present at a concentration of 3.8 × 10-4 copies per chromosome, whereas reconstituted attB sites were at 3.0 × 10-4 copies per chromosome. Deletion of int-xis of Tn5253 abolished production of circular forms (<7.1 × 10-6 copies per chromosome) and was associated to the lack of Tn5253 conjugal transfer suggesting, as expected, that Tn5253 circular form acts as a conjugation intermediate.

Keywords: Tn5253; attachment site; circular form; conjugation; conjugative transposon; integrative conjugative element (ICE); mobile genetic elements.

Figures

FIGURE 1
FIGURE 1
Tn5253 excises from pneumococcal chromosome producing a circular form and a reconstitution of attB insertion site. In the circular form of Tn5253 the left and right ends are joined by attTn which is identical to attR whereas the reconstituted attB site is identical to attL. att sites are represented as filled rectangles, chromosomal genes as open arrows, Tn5253 as a yellow bar. Arrowheads represent PCR primers used for circular form (dark gray) and reconstituted attB site (light gray) detection.
FIGURE 2
FIGURE 2
Sequence alignment of Tn5253 attachment sites in Streptococcus pneumoniae D39 and derivatives. Upon integration into pneumococcal chromosome, Tn5253 is flanked by attL and attR. The element excises from chromosome producing a circular form where the left and right ends are joined by attTn and restoring the attB insertion site. attB is 83-bp long and is identical to attL while attTn is 84-bp long and is identical to attR. attR-attTn contain 4 nucleotide changes and 1 insertion compared to attL-attB. attL-attB contains the first 20 nucleotides of rbgA CDS, whose deduced amino acid sequence is reported, together with the 63 nucleotides upstream of the start codon. Within the sequences, identical nucleotides are indicated by colon, changes are shaded. Amino acids are indicated using one-letter code abbreviations. Lengths of attachment sites are reported on the right.
FIGURE 3
FIGURE 3
The composite integrative conjugative element (ICE) Tn5253 contains two distinct genetic elements integrated: (i) Tn5251 which carries tet(M) and is able to transfer by conjugation autonomously; (ii) Ωcat(pC194) which contains cat, is not conjugative, but is capable of intracellular transposition. Two sets of xis/int recombinase genes are carried by Tn5253, one set (orf78/orf79) is at the right end of the element, while the other (orf21/orf22) belongs to Tn5251. ORFs and their transcription direction are indicated as arrows, sequences corresponding to Tn5251 and Ωcat(pC194) are indicated by solid bars.

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