Progress toward an In Vitro System for Tn916 Transposition .
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, Tc r Em r 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 Tc r Em r DOT was cloned on 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 of transfer genes but is stimulating transfer in some other way.Conjugative transposons are integrated elements that can excise themselves from the genome in which they are integrated, transfer themselves by conjugation into a recipient cell, and integrate into the recipient's genome (1,3,14,18). Conjugative transposons have been found in a variety of species of gram-positive and gram-negative bacteria. The best studied of these conjugative transposons are Tn916, the closely related Tn1545, and the Bacteroides conjugative transposons. The Bacteroides conjugative transposons are much larger than Tn916 (18 kbp) and Tn1545 (25 kbp). Although the Bacteroides conjugative transposons range in size from 65 to over 150 kbp, most of them are in the 70-to 80-kbp range (1 In addition to transferring themselves, Bacteroides conjugative transposons can mobilize coresident plasmids, both in trans and in cis, and they can excise and transfer in trans unlinked integrated elements called NBUs (stands for nonreplicating Bacteroides units) (9,17,19,21,27). The transfer intermediate of the NBUs is a covalently closed circle with an internal oriT (9). Although it seemed likely that coresident plasmids and NBU circle forms ...
Human colonic Bacteroides species harbor a family of large conjugative transposons, called tetracycline resistance (Tcr) elements. Activities of these elements are enhanced by pregrowth of bacteria in medium containing tetracycline, indicating that at least some Tcr element genes are regulated by tetracycline. Previously, we identified a central regulatory locus on the Tcr elements that contained two genes, rteA and rteB, which appeared to encode a two-component regulatory system (A. M. Stevens, J. M. Sanders, N. B. Shoemaker, and A. A. Salyers, J. Bacteriol. 174:2935-2942, 1992). In the present study, we describe a gene which is located downstream of rteB in a separate transcriptional unit and which requires RteB for expression. Sequence analysis of this gene showed that it encoded a 217-amino-acid protein, which had no significant sequence similarity to any proteins in the GenBank or EMBL data base. An insertional disruption in the gene abolished self-transfer of the Tcr element to Bacteroides recipients, indicating that the gene was essential for self-transfer. The disruption also affected mobilization of coresident plasmids. Mobilization frequency was reduced 100- to 1,000-fold if the recipient was Escherichia coli but was not affected to the same extent if the recipient was an isogenic Bacteroides strain. The complex phenotype of the disruption mutant suggested that the newly identified gene, like rteA and rteB, had a regulatory function. Accordingly, it has been designated rteC. Our results indicate that regulation of Tc(r) element functions is unexpectedly complex and may involve a cascade of regulators, with RteA and RteB exerting central control over secondary regulators like RteC, which in turn control subsets of Tcr element structural genes.
Many Bacteroides clinical isolates carry large conjugative transposons that, in addition to transferring themselves, excise, circularize, and transfer smaller, unlinked chromosomal DNA segments called NBUs (nonreplicating Bacteroides units). We report the localization and DNA sequence of a region of one of the NBUs, NBU1, that was necessary and sufficient for mobilization by Bacteroides conjugative transposons and by IncP plasmids. The fact that the mobilization region was internal to NBU1 indicates that the circular form of NBU1 is the form that is mobilized. The NBU1 mobilization region contained a single large (1.4-kbp) (19,24,27). The Tcr elements have an unusual activity. They mediate the excision and circularization of discrete unlinked 10-to 12-kbp segments of chromosomal DNA (32,38). Since these circle forms do not replicate, they were designated NBUs (nonreplicating Bacteroides units). Two NBUs, NBU1 and NBU2, have been characterized to date (32). They differed in restriction pattern and size but shared some cross-hybridizing DNA, which was located in the interior of each element (30, 32). Many Bacteroides strains contain DNA that cross-hybridizes with NBU1, indicating that carriage of NBU-like elements may be widespread in the Bacteroides group.Previously, we had noted that NBUs were sometimes cotransferred with a Tcr element (la). Thus, it appeared that the Tcr elements could mobilize NBUs as well as excising them from the chromosome. Further evidence that NBUs were mobilizable was obtained when Shoemaker et al. (30) isolated three hybrid plasmids, designated Y5, Y11, and Y17, that consisted of a mobilization-deficient plasmid (pEG920) inserted into different sites on NBU1 (30, 34). Y5 and Y1 1 were mobilized from Bacteroides donors by a Tcr element (Tcr ERL) at a much higher frequency than pEG920 alone. Y5 and Y11 were also mobilized from Escherichia coli donors by the IncP plasmid R751 at a 1,000-fold-higher frequency than pEG920.These results suggested that NBU1 carried a mobilization (mob)
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