Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon

Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.     

Integration and excision of a Bacteroides conjugative transposon, CTnDOT

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.     

Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT

Four genes have been found to be essential for excision of the Bacteroides conjugative transposon CTnDOT in vivo: intDOT, orf2c, orf2d, and exc. The intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we developed an in vitro system for the integration reaction. We have now developed an in vitro system for excision. In this system, the left and right junctions of CTnDOT, attL, and attR, are provided on separate plasmids. The excision reaction produced a cointegrate which contained the attDOT (the joined ends of CTnDOT) and attB (the chromosomal target site). Cointegrate formation was observed after electroporation of Escherichia coli with the assay mixture and was also detected directly in the assay mixture by Southern hybridization. The highest reaction frequencies (10(-3)) were obtained with a mixture that contained purified IntDOT and a cell extract from Bacteroides thetaiotaomicron 4001, which contained the excision region of CTnDOT carried on a plasmid. An unexpected finding was that the addition of purified Exc, which is essential for excision in vivo, was not required for excision in vitro, nor did it increase the frequency of cointegrate formation.     

Characterization of genes involved in modulation of conjugal transfer of the Bacteroides conjugative transposon CTnDOT

In previous studies we identified an 18-kb region of the Bacteroides conjugative transposon CTnDOT that was sufficient for mobilization of coresident plasmids and unlinked integrated elements, as well as self-transfer from Bacteroides to Escherichia coli. When this 18-kb region was cloned on a plasmid (pLYL72), the plasmid transferred itself constitutively in the absence of a coresident conjugative transposon. However, when this plasmid was present in a Bacteroides strain containing a coresident conjugative transposon, conjugal transfer was repressed in the absence of tetracycline and enhanced in the presence of tetracycline. These results suggested that a negative and a positive regulator of conjugal transfer were encoded outside the transfer region of the CTnDOT element. In this work, a minimal and inducible transfer system was constructed and used in transfer and Western blot analyses to identify the differentially regulated genes from CTnDOT responsible for the enhancement and repression of pLYL72 conjugal transfer. Both of these regulatory functions have been localized to a region of the CTnDOT element that is essential for CTn excision. In the presence of tetracycline, the regulatory protein RteC activates the expression of a putative topoisomerase gene, exc, which in turn results in an increase in transfer protein expression and a concomitant 100- to 1,000-fold increase in the frequency of pLYL72 transfer. Our results also suggest that since exc alone cannot result in enhancement of transfer, other factors encoded upstream of exc are also required. Conversely, in the absence of tetracycline, a gene located near the 3' end of exc is responsible for the repression of transfer protein expression and also results in a 100- to 1,000-fold decrease in the frequency of pLYL72 transfer.     

Translational control of tetracycline resistance and conjugation in the Bacteroides conjugative transposon CTnDOT

The tetQ-rteA-rteB operon of the Bacteroides conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of tetQ and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for tetQ. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of tetQ. Finally, comparison of the CTnDOT tetQ leader regions with upstream regions of five tetQ genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well.     

Location and characteristics of the transfer region of a Bacteroides conjugative transposon and regulation of transfer genes.

Many Bacteroides clinical isolates contain large conjugative transposons, which excise from the genome of a donor and transfer themselves to a recipient by a process that requires cell-to-cell contact. It has been suggested that the transfer intermediate of the conjugative transposons is a covalently closed circle, which is transferred by the same type of rolling circle mechanism used by conjugative plasmids, but the transfer origin of a conjugative transposon has not previously been localized and characterized. We have now identified the transfer origin (oriT) region of one of the Bacteroides conjugative transposons, TcrEmr DOT, and have shown that it is located near the middle of the conjugative transposon. We have also identified a 16-kbp region of the conjugal transposon which is necessary and sufficient for conjugal transfer of the element and which is located near the oriT. This same region proved to be sufficient for mobilization of coresident plasmids and unlinked integrated elements as well as for self-transfer, indicating that all of these activities are mediated by the same transfer system. Previously, we had reported that disruption of a gene, rteC, abolished self-transfer of the element. rteC is one of a set of rte genes that appears to mediate tetracycline induction of transfer activities of the conjugative transposons. On the basis of these and other data, we had proposed that RteC activated expression of transfer genes. We have now found, however, that when the transfer region of TcrEmr DOT was cloned as a plasmid that did not contain rteC and the plasmid (pLYL72) was tested for transfer out of a Bacteroides strain that did not have a copy of rteC in the chromosome, the plasmid was self-transmissible without tetracycline induction. This and other findings suggest that RteC is not an activator transfer genes but is stimulating transfer in some other way.     

Unexpected effect of a Bacteroides conjugative transposon, CTnDOT, on chromosomal gene expression in its bacterial host

Foreign DNA elements such as plasmids and conjugative transposons are constantly entering new bacterial hosts. A possible outcome of such events that has not been considered previously is that regulatory genes carried on some of them might affect the expression of chromosomal genes of the new host. To assess this possibility, we investigated the effect of the Bacteroides conjugative transposon CTnDOT on expression of chromosomal genes in Bacteroides thetaiotaomicron 5482 (BT4001). Most of the upregulated genes were genes of unknown function, but a number of them were associated with a region of the chromosome that contained a putative conjugative transposon, which had been tentatively designated as CTn4-bt. Upregulation of CTn4-bt genes and other chromosomal genes affected by CTnDOT was controlled by two regulatory genes on CTnDOT, rteA and rteB, which encode a two-component regulatory system. Transfer of CTn4-bt was also mediated by rteA and rteB. Three other putative CTns, CTn1-bt, CTn2-bt and CTn3-bt, were mobilized by CTnERL, a CTn closely related to CTnDOT, but genes from CTnERL other than rteA and rteB were also required. Unexpectedly, homologous recombination was required for CTn1-bt, CTn2-bt, CTn3-bt and CTn4-bt to integrate in the recipient. Our results show that regulatory genes on an incoming mobile element can have multiple effects on its new host, including the activation of previously non-transmissible elements.