Regulation of Excision Genes of theBacteroidesConjugative Transposon CTnDOT

Abstract

The first step in the transfer of the Bacteroides conjugative transposon CTnDOT is excision of the integrated element from the chromosome to form a circular transfer intermediate. Excision occurs only after the bacteria are exposed to tetracycline. Previously, four excision genes were identified. One was the integrase gene intDOT, which appeared to be expressed constitutively. Three other genes essential for excision (orf2c, orf2d, and exc) were found located in a cluster 13 kbp downstream of intDOT. By using uidA fusions and real-time reverse transcriptase PCR, we demonstrate here that the excision genes orf2c, orf2d, and exc are part of an operon that also contains open reading frame orf3, previously shown not to be essential for excision. We also show that operon expression is regulated at the transcriptional level in response to tetracycline. The transcript start site for the operon has been localized. Three CTnDOT regulatory genes are thought to be involved in tetracycline regulation of excision, rteA, rteB, and rteC. By placing rteC under the control of a heterologous promoter, we found that RteC alone was sufficient for induction of the orf2c operon. If, however, the rteC gene was under the control of its own promoter, it was not able to induce orf2c operon expression unless rteA and rteB were present. Thus, RteA and RteB participate in excision by stimulating transcription of rteC. Using electrophoretic mobility shift analysis, we found that a purified His 6 -tagged form of RteC bound DNA upstream of the ؊33 region of the promoter. Changing the sequence in the region between bp ؊50 and ؊70 reduced the expression of the orf2c operon in vivo. Taken together, our results support the hypothesis that RteC acts as a DNA-binding protein that binds upstream of the orf2c promoter and is responsible for tetracycline-regulated transcriptional regulation of the orf2c operon.Conjugative transposons (CTns) related to the Bacteroides CTn CTnDOT have been found in a number of Bacteorides species (22,26). Members of CTnDOT family appear to be contributing significantly to transfer of antibiotic resistance genes among Bacteroides species (22,26,40). Transfer of CTnDOT occurs in three steps: excision from the chromosome to form a double-stranded circular intermediate, conjugative transfer of the circular intermediate to the recipient, and integration of the transferred circular form into the recipient's chromosome (40).Excision is stimulated by tetracycline (7,8,30). In fact, no excision is detected unless the cells carrying CTnDOT are first exposed to tetracycline (8,30). A previous study identified four genes that were essential for excision (8,30). One was the integrase gene intDOT, which is located at one end of the CTn. The other genes were located in a cluster 13 kbp downstream of intDOT (Fig. 1). Single-crossover disruptions and deletions in three of these genes, orf2c, orf2d, and exc, abolished excision. A fourth gene located in this cluster, orf3, could be deleted without affecting excision. We report here that ge...

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“…Because the B. plebeius PUL is harbored at a similar location on an otherwise very similar ICE that is present in B. thetaiotaomicron, this finding suggests that HGT via conjugative elements is the mechanism of transfer and that similar ICE vehicles are capable of acquiring new cargo (20). The observation that both the B. plebeius and B. thetaiotaomicron elements were observed to excise and circularize from the genome via site-specific recombination events supports our conclusion that we have identified the extent of these ICEs involved in PUL transfer.…”

“…Notably, the final three genes (BACPLE_01667-01669) in the PUL encode products that are homologous to RteA and RteB, which are two-component sensor histidine kinase and response regulator proteins, respectively (note that the BACPLE_01667/01668 ORFs each encode part of an RteB homolog that is split by a frameshift mutation that was validated by resequencing). RteA and RteB are important to initiate the excision and mobilization of Bacteroides conjugative transposons that carry antibiotic-resistance genes (20). Examination of the genomic region surrounding these porphyran-inducible genes further suggested that the PUL is contained within an ICE, which includes genes that encode mobilization proteins and an integrase tyrosine recombinase that lies immediately adjacent to a tRNA lys (Fig.…”

“…The apparently low excision rate, which was only detected with 36-70 additional cycles of PCR, suggests that these two example ICEs do not undergo efficient excision in the growth conditions tested, which included the cognate substrates that trigger expression of the RteA/B homologs. The frameshift mutation in the B. plebeius RteB homolog indicates that this ICE has lost the ability to excise and mobilize by the same mechanism as that of Bacteroides conjugative transposons with similar structure (20).…”

Bacteroides: the Good, the Bad, and the Nitty-Gritty

“…Tetracycline has a stimulatory effect on expression of the RteA-RteB system and, therefore, on both expression and transfer of tetracycline resistance. However, the rteA gene is not directly sensing tetracycline, and exactly what it is sensing is not yet clear (164).…”

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

“…CTnERL and related CTns have a complex regulatory network that coordinates the expression of the excision and transfer genes (5,10,17,25). The coordination of excision and transfer should be important for the survival of the CTn in natural hosts, because if the entire CTn is not transferred, the transferred CTn could not be circularized in the recipient and would thus be incapable of normal integration, which requires the joined ends of the circular form.…”