The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916

Tribble, Gena D.; Parker, A C; Smith, C. Jeffrey

doi:10.1128/jb.179.8.2731-2739.1997

Cited by 33 publications

(45 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since the DNA sequence of NBU1 is 86% identical to Tn4555 in this region, the NBU1 nic site is probably in the same place. Although NBU1 and Tn4555 have high DNA sequence identity in the oriT-mob region (78%), they appear to be quite different outside this region (16,26,39).We report here the complete sequence of NBU1 and define the region of NBU1 that is necessary for its excision and for formation of the circular transfer intermediate. This region proved to be unexpectedly large and contains six open reading frames.…”

mentioning

confidence: 88%

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

2000

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 88%

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

2000

View full text Add to dashboard Cite

show abstract

“…Parentheses indicate that the site was the same as the site above it. (19,27,30,31). Accordingly, we provided in trans a plasmid, pAMS9 (29), that carried tetQ, rteA, rteB, and rteC in the strain that had pDJE2.6 integrated into its chromosome.…”

Section: Vol 182 2000mentioning

confidence: 99%

“…NBU1 excision more closely resembles excision of phage lambda in that there is an att site formed by the joined ends that integrates into an identical site in the chromosome. Excision of Tn4555 is more similar to that of Tn916 (31). Although the mobilizable transposons rely absolutely on the CTns for transfer, there is so far no evidence for any significant sequence similarity between them and the CTns.…”

mentioning

confidence: 97%

Integration and Excision of a Bacteroides Conjugative Transposon, CTnDOT

et al. 2000

View full text Add to dashboard Cite

Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most Bacteroides CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF), intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of intDOT designated orf2. Thus, excision involves other genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that intDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.

show abstract

“…The ends of Tn4555 consist of a 12-bp imperfect inverted repeat, and a 6-bp variable coupling sequence is utilized as a crossover region for recombination. In contrast to cLV25, transposition of Tn4555 does not result in a target site repeat (41). Data to support probable transposition …”

Section: Discussionmentioning

confidence: 90%

“…cLV25 insertions in pGAT400⌬BglII were site specific and resulted in an 8-bp target site repeat. Additionally, there was an extra 8 bp associated with the left end of cLV25, which may represent a coupling sequence similar to that required for transposition of Tn4555 (41). The ends of Tn4555 consist of a 12-bp imperfect inverted repeat, and a 6-bp variable coupling sequence is utilized as a crossover region for recombination.…”

Section: Discussionmentioning

confidence: 99%

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

Bass

Hecht

2002

J Bacteriol

View full text Add to dashboard Cite

Horizontal DNA transfer contributes significantly to the dissemination of antibiotic resistance genes in Bacteroides fragilis. To further our understanding of DNA transfer in B. fragilis, we isolated and characterized a new transfer factor, cLV25. cLV25 was isolated from B. fragilis LV25 by its capture on the nonmobilizable Escherichia coli-Bacteroides shuttle vector pGAT400⌬BglII. Similar to other Bacteroides sp. transfer factors, cLV25 was mobilized in E. coli by the conjugative plasmid R751. Using Tn1000 mutagenesis and deletion analysis of cLV25, two mobilization genes, bmgA and bmgB, were identified, whose predicted proteins have similarity to DNA relaxases and mobilization proteins, respectively. In particular, BmgA and BmgB were homologous to MocA and MocB, respectively, the two mobilization proteins of the B. fragilis mobilizable transposon Tn4399. A cis-acting origin of transfer (oriT) was localized to a 353-bp region that included nearly all of the intergenic region between bmgB and orf22 and overlapped with the 3 end of orf22. This oriT contained a putative nic site sequence but showed no significant similarity to the oriT regions of other transfer factors, including Tn4399. Despite the lack of sequence similarity between the oriTs of cLV25 and Tn4399, a mutation in the cLV25 putative DNA relaxase, bmgA, was partially complemented by Tn4399. In addition to the functional cross-reaction with Tn4399, a second distinguishing feature of cLV25 is that predicted proteins have similarity to proteins encoded not only by Tn4399 but by several Bacteroides sp. transfer factors, including NBU1, NBU2, CTnDOT, Tn4555, and Tn5520.Bacteroides fragilis is an obligate anaerobe of the colon and a significant opportunistic pathogen. Antibiotic resistance among Bacteroides spp. is rapidly increasing, largely due to the dissemination of DNA transfer factors (plasmids and transposons) harbored by members of this genus. Transfer factors can be divided into two classes, conjugative and mobilizable. Conjugative plasmids and transposons are self-transmissible, encoding both functions of DNA transfer, that is, DNA processing events for transfer initiation and mating apparatus formation, which brings donor and recipient bacteria into stable cell-cell contact. In contrast, mobilizable plasmids and transposons carry mobilization genes only for transfer initiation, utilizing a mating apparatus provided by a coresident conjugative transfer factor.All transfer factors also contain a cis-acting origin of transfer (oriT), where transfer is initiated. DNA processing events at the oriT consist of specific protein-DNA and protein-protein interactions that result in the formation of a relaxed DNAprotein complex called the relaxosome (see references 7, 9, 14, and 25 for detailed descriptions). In brief, mobilization proteins assemble at the oriT, with one protein, the DNA relaxase, nicking a single DNA strand at the nic site. The nicked DNA strand is then transferred with 5Ј-to-3Ј polarity to a recipient bacterium via a mating bridge that is...

show abstract

The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916

Cited by 33 publications

References 46 publications

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

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

Integration and Excision of a Bacteroides Conjugative Transposon, CTnDOT

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

Contact Info

Product

Resources

About