Multiple Gene Products and Sequences Required for Excision of the Mobilizable Integrated Bacteroides Element NBU1

Abstract

NBU1 is an integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision-required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision. The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRS HIII ) inhibited excision when provided in trans on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized.NBUs (nonreplicating Bacteroides units) are 10-to 12-kbp integrated elements that can be excised and mobilized in trans by tetracycline-inducible Bacteroides conjugative transposons. Two regions of NBU1, the best studied of the NBUs, have been characterized. The NBU1 integrase gene, intN1, is located near one end of the element and is transcribed away from the end. This gene and the upstream NBU1 integration region, attN1, are necessary and sufficient for integration. IntN1 is a member of the phage lambda family of site-specific integrases, although it is only distantly related to the phage lambda integrase (26). In Bacteroides species, NBU1 integration is site specific and the prima...

Isolation and Characterization of cLV25, a Bacteroides fragilis Chromosomal Transfer Factor Resembling Multiple Bacteroides sp. Mobilizable Transposons

“…transfer factors. These proteins include PrmN1 from NBU1 (62% identity), PrmN2 from NBU2 (57% identity), OrfP from the CTnDOT transfer region (41% identity), and PrmF (30% identity) from the CTnDOT ermF region (3,34,48,49). In addition, the N terminus of Orf22 (126 amino acids) has 68% identity with Tn4399 Orf1, while the C terminus (98 amino acids) has 53% identity with Tn4399 Orf3 (18).…”

“…NBU1 (10.3 kb) and NBU2 (11.1 kb) integrate at the 3Ј end of tRNA genes in Bacteroides spp. and also integrate in E. coli (32,34,48). In addition, NBU2 carries a lincosamide resistance gene, linA2 N2 , which confers a lincomycin resistance phenotype on B. thetaiotaomicron (48).…”

The Integrase of the Conjugative Transposon Tn916Directs Strand- and Sequence-Specific Cleavage of the Origin of Conjugal Transfer,oriT, by the Endonuclease Orf20

“…No other instances of a tyrosine recombinase acting as an accessory protein for a separate DNA relaxation reaction are known. However, it has previously been demonstrated that excision of the Bacteroides mobilizable element NBU1 requires a segment of DNA that includes, in addition to the end of the element and the integrase gene, the origin of conjugal transfer (32). Thus, an association between recombination and conjugation functions is not unique to Tn916.…”

Regulation of Excision Genes of theBacteroidesConjugative Transposon CTnDOT

“…For pMJF2-based clones, transconjugants were selected on TYG plates containing erythromycin (10 g/ml) and gentamicin (200 g/ml). In some experiments, plasmid pC-COW (37) or pLYL05 (27,34) was used to introduce rteC into the recipients. For pC-COW-based clones, the transconjugants were selected on TYG agar plates containing chloramphenicol (20 g/ml) and gentamicin (200 g/ml).…”

“…For pC-COW-based clones, the transconjugants were selected on TYG agar plates containing chloramphenicol (20 g/ml) and gentamicin (200 g/ml). For pLYL05-based clones, the transconjugants were selected on plates containing cefoxitin (20 g/ml) and gentamicin (200 g/ml) (27).…”