Identification and Characterization of Transposable Elements of Paracoccus pantotrophus

Abstract

We studied diversity and distribution of transposable elements residing in different strains (DSM 11072, DSM 11073, DSM 65, and LMD 82.5) of a soil bacterium Paracoccus pantotrophus (␣-Proteobacteria). With application of a shuttle entrapment vector pMEC1, several novel insertion sequences (ISs) and transposons (Tns) have been identified. They were sequenced and subjected to detailed comparative analysis, which allowed their characterization (i.e., identification of transposase genes, terminal inverted repeats, as well as target sequences) and classification into the appropriate IS or Tn families. The frequency of transposition of these elements varied and ranged from 10 ؊6 to 10 ؊3 depending on the strain. The copy number, localization (plasmid or chromosome), and distribution of these elements in the Paracoccus species P. pantotrophus, P. denitrificans, P. methylutens, P. solventivorans, and P. versutus were analyzed. This allowed us to distinguish elements that are common in paracocci (ISPpa2, ISPpa3-both of the IS5 family-and ISPpa5 of IS66 family) as well as strain-specific ones (ISPpa1 of the IS256 family, ISPpa4 of the IS5 family, and Tn3434 and Tn5393 of the Tn3 family), acquired by lateral transfer events. These elements will be of a great value in the design of new genetic tools for paracocci, since only one element (IS1248 of P. denitrificans) has been described so far in this genus.

Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2

“…Transformation of E. coli TG1 was performed as described by Kushner (1978). Triparental mating experiments were conducted as previously described (Bartosik et al, 2003a). Briefly, overnight cultures (pelleted by centrifugation and washed to remove antibiotics) of the donor strain E. coli TG1 carrying the mobilizable vector, the recipient strain P. pantotrophus DSM 11072R, and E. coli DH5a carrying the helper plasmid pRK2013, were mixed at a ratio of 1 : 2 : 1.…”

“…3 and Results for details). For amplification of the internal fragment of Tn3434 (used as a probe in hybridization) the primers L3434 and R3434, previously described by Bartosik et al (2003a), were used. Amplification was performed in a Mastercycler (Eppendorf) using the above synthetic oligonucleotides, OptiTaq polymerase (Eurx) (with supplied buffer) and appropriate template DNAs.…”

“…For identification of the target site of TnPpa1 integrated within pMMB2, five nested pairs of cartridge-specific forward and reverse oligonucleotide primers (ALIS/ARIS, BLIS/BRIS, CLIS/CRIS, DLIS/DRIS, ELIS/ERIS) were used as described by Bartosik et al (2003a). The chromosomal DNA sequences adjacent to TnPpa1 within the P. pantotrophus genome were amplified with the following primers: RTN3434 (59-TCCTCGCCGCCATCATCAT-39), LTN3434 (59-GCAGATTGACACAACGACTG-39) and 147LCD2 (59-ATCGG-CAAGGCAGATTGACC-39) (see Fig.…”

“…This strain was chosen for two reasons: (i) it is rich in TEs, and carries ISPpa1, ISPpa2, ISPpa3, ISPpa5 and Tn3434, elements entrapped by pMEC1 (Bartosik et al, 2003a); and (ii) its natural plasmids are compatible with the repABC replicon of pMMB2 (data not shown).…”

“…Through the application of positiveselection entrapment vector pMEC1 (which carries a cI-tetA selective cartridge) we showed that TEs are abundant in Paracoccus spp. We identified eight novel ISs: ISPpa1, ISPso1 (IS256 family), ISPpa2, ISPpa3, ISPpa4, ISPso2, ISPso3 (IS5 family) and ISPpa5 (IS66 family), as well as two closely related Tns of the Tn3 family, cryptic Tn3434 (DSM 11072) and streptomycin-resistant Tn5393 (LMD 82.5) (Bartosik et al, 2003a, b The size of the identified TEs [400 tetracycline-resistant (Tc R ) clones analysed] did not exceed 5?3 kb (Tn5393) (Bartosik et al, 2003a). This raised the question whether the studied strains contained Tns of larger size and whether the tool used was not suitable for their capture.…”