Conjugative transfer of the integrative conjugative elements ICESt1 and ICESt3 from Streptococcus thermophilus

Abstract

Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus, ICESt1 and ICESt3. Mitomycin C, a DNA-damaging agent, derepresses ICESt3 conjugative transfer almost 25-fold. The ICESt3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICESt3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis, are site-specifically integrated into the 3' end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICESt3 was retransferred from E. faecalis to S. thermophilus. Recombinant plasmids carrying different parts of the ICESt1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.

FIG. 1.

Schematic representation of the locations and orientations of the primers used in this work to analyze the transconjugants. The locations of the recombination, conjugation, or regulation modules of the S. thermophilus ICEs are indicated by gray and white rectangles. Recombination sites are drawn as vertical rectangles: black, sequence identical in attL, attR, attI, and attB sites; checkerboards, arm of attR sites and related arm of attI sites; hatched boxes, arm of attL sites and related arm of attI sites. The recombination sites are magnified. The locations and orientations of the primers hybridizing into the ICEs (ICESt1 or ICESt3) and into the flanking sequences are indicated by black arrowheads. Each letter (A to H) indicates a class of primers hybridizing on the same locus in different species (see Table S1 in the supplemental material). The primer pairs A-B, E-F, A-F, and E-B allow the amplification of fragments carrying the attL, the attR, the attB, and the attI sites, respectively. The primer pair C-D allows the amplification of an internal fragment of the regulation module of ICESt1 or ICESt3. The amplification of an internal fragment of the fda gene using the species-specific primers G and H allows the identification of the species. The spectinomycin (spc) or the chloramphenicol (cat) resistance genes introduced in order to tag the ICEs are indicated by a large black rectangle. The sequences between the regulation module and the attL site are specific to each S. thermophilus ICE. The accession number of the untagged ICESt1 and ICESt3 nucleotide sequences are AJ278471 and AJ586568, respectively.

FIG. 2.

Map of the thermosensitive replication plasmids carrying the P6 constitutive promoter and the various fragments of the ICESt1 recombination module. The location of the thermosensitive replication origin [Ori(pWV01^TS)] is indicated by a white rectangle. The location and the orientation of the ermB gene, encoding resistance to erythromycin, is indicated by a gray arrow. The PstI and KpnI restriction sites used for molecular cloning are indicated. The location and the orientation of the P6 constitutive promoter from Lactobacillus acidophilus ATCC 4356 are indicated by a right-angle arrow. Black arrows indicate the locations and orientations of the int and xis genes, encoding the integrase and the excisionase, respectively. The location and the orientation of a fragment corresponding to the last 60 nucleotides of the fda gene is indicated by a black triangle. Recombination sites are drawn as rectangles: black, sequence identical in attL, attR, and attI sites; checkerboards, arm of attL sites and related arm of attI sites; hatched boxes, arm of attR sites and related arm of attI sites. The recombination sites are magnified. The putative rho-independent terminator downstream from the int gene is indicated by a lollipop. The locations and orientations of the primers AttI2 and AttI3 are indicated by white arrowheads (see Table S1 in the supplemental material).

FIG. 3.

Characterization by PCR of an intraspecies ICESt3 transconjugant. CNRZ385 ICESt3cat and LMG18311/pMG36e were used as the donor and the recipient, respectively. Don, Rec, and Tc indicate donor, recipient, and transconjugant, respectively. (A) ICESt3, amplification of a fragment of the regulation module. cse, amplification of the central part of the cse gene. (B and C) attR, attL, attB, and attI, amplifications of fragments carrying these attachment sites. The sizes of the PCR fragments are deduced from sequence analysis and confirmed by comigration with a DNA ladder. The classes of primer pairs used for these amplifications are indicated in parentheses, and their localizations are indicated in Fig. 1.

FIG. 4.

Characterization of an ICESt3 transconjugant of E. faecalis by PCR. S. thermophilus CNRZ385 ICESt3spc and E. faecalis JH2-2 Rif^r Fus^r were used as the donor and the recipient, respectively. Don, Rec, and Tc indicated donor, recipient, and transconjugant, respectively. (A) E. faecalis and ICESt3, amplification of an internal fda fragment specific to E. faecalis and amplification of a fragment of the ICESt3 regulation module, respectively. (B and C) attR, attL, attB, and attI, amplifications of fragments carrying these attachment sites. The sizes of the PCR fragments are deduced from sequence analysis and confirmed by comigration with a DNA ladder. The classes of primer pairs used for these amplifications are indicated in parentheses, and their localizations are indicated in he Fig. 1.

FIG. 5.

Sequences of the fda 3′ ends of the strains used as recipients and integration frequencies of pNST260+ in some of these strains. (A) Cladogram of the species used as recipients in this work. (B) The stop codon of the fda ORFs is enclosed by a rectangle. The bold and underlined nucleotides indicate the differences from the S. thermophilus sequence. The sequences in this figure correspond to those found in the sequenced strains. Only one of the two Staphylococcus aureus alleles, the most common in databases, is indicated. The second Staphylococcus aureus allele is more different from the S. thermophilus sequence: the adenine at the position 8 is replaced by a thymine. The fda 3′ end sequences available in the databases are identical for all the strains of the other species. The sequence of the E. casseliflavus fda 3′ end is not available in databases. The fda 3′ end sequences from Lactobacillus delbrueckii subsp. bulgaricus and E. coli do not display a significant identity with that of S. thermophilus. (C) pNST260+ integration frequencies in five strains used as recipients in intra- and interspecies filter mating. S. thermophilus LMG18311 and B. subtilis 168 were used in this experiment. ND, not determined.

FIG. 6.

Schematic representation of the site-specific integration of plasmid pNST260+ into the fda 3′ end and characterization of an E. faecalis transformant carrying this plasmid. (A) The locations and orientations of primers hybridizing to the chromosome and to the plasmid are indicated by black and white arrowheads, respectively. The recombination sites are magnified. The primer pairs A-AttI3, AttI2-F, A-F, and AttI3-AttI2 allow the amplification of fragments carrying the attL, the attR, the attB, and the attI sites, respectively (see Table S1 in the supplemental material). The classes of primer pairs used for these amplifications are indicated. The symbols used to represent plasmid pNST260+ are identical to those used in Fig. 3. (B) Amplifications by PCR using the DNA of E. faecalis JH2-2 and a clone of E. faecalis JH2-2 carrying plasmid pNST260+. attRç attL, and attB, amplifications of fragments carrying these attachment sites.