Integration and Excision of a Bacteroides Conjugative Transposon, CTnDOT

Abstract

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.

Challenging a Paradigm: the Role of DNA Homology in Tyrosine Recombinase Reactions

“…In vitro cleavage assays using phosphorothioate substrates revealed that the cleavage site on the top strand was located two bases to the left (between the T and G) within the 10-bp region of homology. Thus, the cleavage sites for IntDOT are 7 bp apart, and the attDOT-and attB-site overlap regions share 2 bp of identity on the left side (19,49) (Fig. 4).…”

“…4). Originally, the coupling sequences were thought to be 5 bp in length, since only five bases varied between sites (19). In vitro cleavage assays using phosphorothioate substrates revealed that the cleavage site on the top strand was located two bases to the left (between the T and G) within the 10-bp region of homology.…”

“…Subsequently, other mobile elements encoding tyrosine recombinases like Bacteroides CTnDOT and NBU1 were discovered. CTnDOT has a coupling sequence mechanism similar to that of Tn916 and Tn1545, and NBU1 integrase has interesting homology requirements that were not apparent from initial analyses of recombination sites (19,49,68,73). The integration of CTX phage and the capture of gene cassettes by integrons use a single strand exchange mechanism to recombine nonhomologous core sequences (11,81).…”

Fitness Costs of Plasmids: a Limit to Plasmid Transmission

“…Some ICE families target a single specific attachment site in the bacterial chromosome, usually in a tRNA gene (45), suggesting that these plasmids have evolved a strategy to avoid arbitrary disruption of the host genome during integration. However, other ICE families target multiple attachment sites (46), and some families, like Tn916, integrate almost randomly in the host chromosome (47). This nonspecific integration can disrupt protein coding or regulatory regions or can interfere in the expression of genes flanking the integration site, entailing fitness costs in the recipient bacterium.…”

A Bacteroides Conjugative Transposon, CTnERL, Can Transfer a Portion of Itself by Conjugation without Excising from the Chromosome

“…Using an unrelated suicide vector, pGW161.1, which carried ermG, a single-crossover disruption was introduced into the integrase gene (attR side) of the oriT gene of the CTnERL element in BT4104⍀traP, generating the doubleinsertion strain BT4104⍀traP⍀int. All gene disruptions were tested by PCR (⍀int) and/or Southern blot analysis (⍀traP) to confirm that the single-crossover insertions resulted in the expected gene disruption or phenotype as described previously (2,4,5). No excision was detected by PCR amplification of the joined ends of the mutant element, demonstrating that the insertion in intERL had indeed eliminated normal excision.…”