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Many human colonic Bacteroides spp. harbor a conjugative transposon, CTnDOT, which carries two antibiotic resistance genes, tetQ and ermF. A distinctive feature of CTnDOT is that its excision and transfer are stimulated by tetracycline. Regulation of the genes responsible for excision has been described previously. We provide here the first characterization of the regulation of CTnDOT transfer (tra) genes. Reverse transcription-PCR analysis of the region containing the tra genes showed that these genes are regulated at the transcriptional level. Surprisingly, increased production of tra gene mRNA in tetracycline-stimulated cells was mediated by the proteins encoded by the excision genes. Previous studies have shown that expression of the excision gene operon is controlled by the regulatory protein RteC. Accordingly, it was possible that RteC was also regulating tra gene expression and that the excision proteins were only accessory proteins. However, placing the excision gene operon under the control of a heterologous promoter showed that the excision proteins alone could activate tra gene expression and that RteC was not directly involved. We also found a second level of tra gene control. The transfer of CTnDOT was inhibited by a DNA segment that included only a portion of the 3' end of one of the excision genes (exc). This segment contained a small open reading frame, rteR. By replacing the codons encoding the first two amino acids of the putative protein product of this open reading frame with stop codons, we showed that the rteR gene might encode a small regulatory RNA. RteR acted in trans to reduce the number of tra transcripts in a way that was independent of the excision proteins. The repressive effect of RteR was not the result of decreased stability of the tra mRNA. Instead, RteR appears to be modulating the level of tra gene expression in some more direct fashion. The complex regulatory system that controls and links the expression of CTnDOT excision and transfer genes may be designed to ensure stable maintenance of CTnDOT in nature by reducing the fitness toll it takes on the cell that harbors it. doi: 10.1128/JB.00739-09

Early studies of the Bacteroides mobilizable transposon NBU1 established that excision, the first step in NBU1 transfer, requires exposure of the cells to tetracycline. More recently, we found that excision is also associated with growth phase; even after exposure to tetracycline, excision is detectable only after the cells enter late exponential phase. The tetracycline effect is mediated by a two-component regulatory system, RteA and RteB, which is provided in trans by an integrated self-transmissible element, CTnDOT. The rteA and rteB genes are part of a three-gene operon that also contains the tetracycline resistance gene tetQ. We report here that neither transcription nor translation of the tetQ-rteA-rteB operon is affected by growth phase. Moreover, RteA is not required for the growth phase effect, because a mutant form of RteB that does not require phosphorylation by RteA did not make excision independent of growth phase. Two conditions made NBU1 excision independent of growth phase. One was reducing the tetracycline concentration from an inhibitory concentration (1 [micro]g/ml) to a subinhibitory level (0.05 [micro]g/ml). Independence of growth phase also occurred when rteA and rteB were placed under the control of a heterologous maltose-inducible promoter, [P.sub.susA]. Our results suggest that at low concentrations of tetracycline, ribosomes are capable of translating enough RteA and RteB for excision to occur. At higher tetracycline concentrations, however, TetQ is needed to protect enough ribosomes to allow the translation of excision genes, and this protection takes time to develop. Thus, subinhibitory concentrations of tetracycline may increase the probability of gene transfer because, in contrast to inhibitory concentrations, excision can occur at all phases of growth.

A newly discovered Bacteroides conjugative transposon (CTn), CTnBST, integrates more site specifically than two other well-studied CTns, the Bacteroides CTn CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically. Also, in most of these integrases, all of the conserved amino acids are required for integration. In the case of IntBST, however, we found that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Changes in the other amino acids had little or no effect. Thus, although the CTnBST integrase still seems to be a member of the tyrosine recombinase family, it clearly differs to some extent from other members of the family in its catalytic site. We also determined the sequence requirements for CTnBST integration in the 18-bp region where the crossover occurs preferentially during integration. We found that CTnBST integrates in this preferred site about one-half of the time but can also use other sites. A consensus sequence was tentatively derived by comparison of a few secondary sites: AATCTGNNAAAT. We report here that within the consensus region, no single base change affected the frequency of integration. However, 3 bp at one end of the consensus sequence (CTG) proved to be essential for integration into the preferred site. This sequence appeared to be at one end of a 7-bp crossover region, CTGNNAA. The other bases could vary without affecting either integration frequency or specificity. Thus, in contrast to well-studied site-specific recombinases which require homology throughout the crossover region, integration of CTnBST requires homology at one end of the crossover region but not at the other end.

Conjugative transposons (CTns) are major contributors to the spread of antibiotic resistance genes among Bacteroides species. CTnBST, a newly discovered Bacteroides conjugative transposon, carries an erythromyein resistance gene, ermB, and previously has been estimated to be about 100 kbp in size. We report here the locations and sequencing of both of its ends. We have also located and sequenced the gene that catalyzes the integration of CTnBST, intBST. The integrase gene encodes a 377-amino-acid protein that has the C-terminal R-K-H-R-H-Y motif that is characteristic of members of the tyrosine recombinase family of integrases. DNA sequence comparisons of the ends of CTnBST, the joined ends of the circular intermediate, and the preferred site into which the circular form of CTnBST had integrated revealed that the preferred integration site (attB1) contained an 18-bp sequence of identity to the crossover region, attBST, on CTnBST. Although this site was used in about one-half of the integration events, sequence analysis of these integration events revealed that both CTnBST and a miniature form of CTnBST (miniBST) integrated into a variety of other sites in the chromosome. All of the sites had two conserved regions, AATCTG and AAAT. These two regions flanked a 2-bp sequence, bp 10 and bp 11 of the 18-bp sequence, that varied in some of the different sites and sometimes in the attBST sequences. Our results suggest that CTnBST integrates site selectively and that the crossover appears to occur within a 12-bp region that contains the two regions of conserved sequences.

The conjugative transposons of Bacterroides fragilis namely, CTn86 and CTn9343 have shown similarity in their basic structures, except in the presence of an extra 7-kb region in CTn86. The DNA hybridization studies show flanking elements of pathogenicity island (PAI) similar to CTn86 in 36% strains and that is similar to CTn9343 in 36% where as the remaining strains have dissimilar structures.

CTnERL, a Bacteroides conjugative transposon, transferred DNA by an Hfr-type mechanism during conjugation when it was excision deficient due to an insertion in the integrase gene. Rescue of the conjugative transposon sequences required the recipient to be RecA proficient and to contain an integrated CTnERL. The transfer efficiency was only 10- to 30-fold lower than the normal element transfer efficiency, and the direction of transfer from the oriT gene showed that the integrase end was transferred first and that the transfer genes were transferred last.

Isolation of ermG genes in gram-positive bacteria from porcine feces and from under-barn manure pits used to store porcine wastes. It was found that porcine ermG genes were in three different gram-positive genera, which indicate that the gene is spread by horizontal gene transfer.

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.sub.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.

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.

CTnDOT is a conjugative transposon (CTn) that is found in many Bacteroides strains. Transfer of CTnDOT is stimulated 100- to 1,000-fold if the cells are first exposed to tetracycline (TET). Both excision and transfer of CTnDOT are stimulated by TET. An operon that contains a TET resistance gene, tetQ, and two regulatory genes, rteA and rteB, is essential for control of excision and transfer functions. At first, it appeared that RteA and RteB, which are members of a two-component regulatory system, might be directly responsible for the TET effect. We show here, however, that neither RteA nor RteB affected expression of the operon. TetQ, a ribosome protection type of TET resistance protein, actually reduced operon expression, possibly by interacting with ribosomes that are translating the tetQ message. Fusions of tetQ with a reporter gene, uidA, were only expressed at a high level when the fusion was cloned in frame with the first six codons of tetQ. However, out of frame fusions or fusions ending at the other five codons of tetQ showed much lower expression of the uidA gene. Moreover, reverse transcription-PCR amplification of tetQ mRNA revealed that despite the fact that the uidA gene product, [beta]-glucuronidase (GUS), was produced only when the cells were exposed to TET, tetQ mRNA was produced in both the presence and absence of TET. Computer analysis of the region upstream of the tetQ start codon predicted that the mRNA in this region could form a complex RNA hairpin structure that would prevent access of ribosomes to the ribosome binding site. Mutations that abolished base pairing in the stem that formed the base of this putative hairpin structure made GUS production as high in the absence of TET as in TET-stimulated cells. Compensatory mutations that restored the hairpin structure led to a return of regulated production of GUS. Thus, the tetQ-rteA-rteB operon appears to be regulated by a translational attenuation mechanism.

Research has been conducted on Bacteroides conjugative transposon CTnDOT. The integration system allowing a plasmid with CTnDOT joined ends to integrate into the second plasmid with the target sites into which CTnDOT integrates in vivo has been developed and is described.

Research has been conducted on the Bacteroides conjugative transposon CTnDOT 18-kb region. Results demonstrate that after the cloning of this region on a plasmid, the plasmid transferres itself in the coresident conjugative transposon absence but is present in the Bacteroides strain with the coresident conjugative transposon.

Research has been conducted on the mobilizable NBU1 transposon that excises from Bacteriodes chromosomes. The regulation of the NBU1 excision has been investigated and the results are discussed.

The ermF region of the Bacteroides conjugative transposon CTnDOT is a chimera composed of a class I composite transposon and a mobilizable transposon. A study of 19 Bacteroides isolates found that ermF region is in the same location on the genose, and the composition of ermF is identical. This indicates that it was probably derived from a common ancestor.

Research has been conducted on the Bacteroides conjugative transposons (CTns). The cloning and sequencing of Bacteroides CTn ends in examining the excision and integration events have been carried out.

Research has been conducted on the Bacteroides element NBU2. The results of the NBU2 full DNA sequence examination have been discussed.

Research has been conducted on the nonreplicating Bacteroides-1 unit (NBU1). Results indicate that a large NBU1 region is required for excision.

A research study was conducted regarding the construction of a RecA strain of Bacteroides thetaiotaomicron (B thetaiotaomicron) and the RecA relation to the integration and excision of the conjugative transposons and nonrepicating Bacteroides unit 1 (NBU1). The methods used were the amplification, cloning and analysis of DNA, construction of recA disruption mutant of B thetaiotaomicron and analysis of the recA disruption on the integration and excision of conjugative transposons and NBU1. Results showed that the absence of recA activity had no effects on the integration and excision of conjugative transposons, NBU1 and on Tn4351.

NBU1, a nonreplicative Bacteroides unit, can be mobilized from Bacteroides donors to Bacteriodes recipients by conjugation. The process involves insertion of NBU1 at a frequency close to that of the recipient Bacteroides species, and is facilitated by the enzyme integrase. The NBU1 integrase gene can increase the amount of integrase available in the recipient.

The region involved in the integration of a nonreplicative Bacteroides unit termed NBU1 in Bacteroides thetaiotaomicron was determined using molecular genetic techniques. Insertion vectors identified this region as the 3' end of the Bacteroides Leu-tRNA gene, which is highly conserved in many Bacteroides species.

The reconstruction of a carboxymethyl cellulase (CMCase) gene from the ruminal bacteria Prevotella ruminicola B(sub 1)4 allows the transfer of the CMCase gene from Escherichia coli to Bacteroides uniformis 0061 and then to other P. ruminicola B(sub 1)4 cells. The reconstruction involves the addition of a cellulose binding region from a Thermomonospora fusca cellulase. The gene is transferred using a chloramphenicol and tetracycline resistance shuttle vector. B. uniformis produces CMCase in the presence of a xylanase promoter and grows if two exocellulases are added to the culture medium.

Conjugative transposons take part in spreading antibiotic resistance genes in gram-positive cocci, and other gram-positive and gram-negative bacteria. Conjugative transposons are integrated DNA elements that can dissociate themselves and reintegrate into the same cell or into another cell by conjugation. The genes that confer resistance do not need to be present on the transposon to be transferred. The host range of conjugative transposons is wide and they are capable of mobilizing plasmids and other integrated elements. The method of excision and integration is different from that found in regular transposons.

Conjugative transposons in Bacteroides can cut themselves out of chromosomes and plasmids, transfer to other bacterium via conjugation, and control the transmission of other genetic material. These transposons are 65 kbp to 150 kbp in size and their actions are stimulated by the antibiotic tetracycline. They may play a crucial role in the transfer of genes that carry drug resistance. Other conjugative transposon properties and their implications are discussed.

The transfer origin (oriT) of the conjugative transposon of Bacteroides has been identified. The oriT is located near the middle of the TcrEmrDOT transposon. There is also a 16-kbp region located close to oriT that is involved in conjugal transfer. The rte genes do not appear to be necessary for conjugal transfer.

The oriT mobilization regions of two nonreplicating Bacteroides units (NBUs), NBU1 and NBU2, have almost identical sequences up to the stop codon of the 'mob' gene, indicating that the mobilization region may be on some type of a cassette. Bacteroides carry integrated self-transmissible elements known as conjugative transposons, which can excise and mobilize smaller unlinked DNA elements, the NBUs. The NBUs are excised from the chromosome to form a double-stranded circle during transfer.

The newly identified conjugative transposon, Tcr Emr 7853, characterizes a group of Bacteroide conjugative transposons that exhibit high sequence similarity with the TCr Emr DOT-type conjugative transposons. TCr Emr 7853 possesses the tetracycline (Tcr) and Erythromycin (Emr) resistance genes but its transfer is not modulated by tetracyclin and rteA and rteB regulatory gene do not govern its transfer genes.

Comparison of the DNA sequences from Prevotella bacterial species in the intestinal tracts and rumens of farm animals and from Bacteriodes in the human colonic microflora revealed the transfer of a horizontal tetracycline resistance gene, tet Q, between the bacterial genera in farm animals and man. The bacterial isolates with different geographic origin and taxonomy contained identical tet Q sequences.

The mobilization region of a Bacteroides insertion element NBU1 is located using a vector that is not mobilized by tetracycline resistance (Tcr) elements, and is characterized using an open reading frame. NBUs are mobilized and excised at times with a Tcr element. Bacteroides are gram-negative anaerobic bacteria present in the human colon.

A nonreplicating Bacteroides unit, NBU, is mobilized, transferred, integrated and analyzed. NBU mobilization and excision, unlike NBU integration into the recipient, require the presence of a tetracycline resistance gene. NBU1 insertion site, its DNA sequences and NBU1-chromosome junction are different from those of the cryptic tetracycline gene. NBU mobilization is similar to plasmid mobilization.

A novel tetracycline resistant (Tcr) element gene, rte C, which is controlled by two other genes, rte A and rte B, and could depict a second layer of modification of Tcr element genes, was examined. This gene encoded a 217-amino acid protein, and it was crucial for self-transfer. Like rteA and rteB, rtc C also has a monitoring mechanism. Various regulators, such as RteA and RteB, which monitor secondary regulators such as Rte C, and the latter, which controls subsets of Tcr element structural genes, are included in the complicated control of Tcr element mechanisms.

Molecular genetic studies were done on self-transmissible chromosomal tetracycline resistance (Tcr) genes found in colonic Bacteroides strains. A segment of the Tcr gene which mediates excision and circularization of nonreplicating Bacteroides units was localized and sequenced. This gene segment contained three open reading frames; one was the Tcr gene (tetQ) and the other two (rteA and rteB) shared amino acid homology with two-component regulatory systems suggesting an operon system. Introduction of tetracycline induced transcription from the tetQ promoter region.

Naturally occuring chromosomal elements in Bacteroidesfragilis can be transferred to Prevotella ruminicola cells under laboratory conditions. B. frgailis is an anaerobic human colonic organism while P. ruminicola is a bovine ruminal anaerobe. Results show that such transfer is notonly possible but also reversible. These results support the theory that such transfer occurs in nature and is responsible for the presence of identical tetracycline resistance genes on Bacteroides sp. and P. ruminicola cells.

The insertion of the self-transmissible Tc(super R) elements, which carry a tetracycline resistance gene, in the Bacteroides chromosome was studied. With one exception, all of the Tc(super R) elements were found to be about 70 to 80 kbp in size. They had three to seven sites of insertion, while the nonreplicating Bacteroides units (NBUs) they cotransferred were also relatively site selective, although inserted at different sites of that of the Tc(super R)s transfer sites. Characterization of XBU4422, a cryptic element showing homology to Tc(super R) showed that its excision occured within an eight-bp palindrome identical to a known recombinational hot spot.

The full DNA sequence of conjugative transposon (CTn) CTnBST was obtained for assessing the possible origin of the CTn. Findings reveal that CTnBST is a hybrid element that acquired a portion of its coding region from gram-positive bacteria.