The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons.

CTnDOT is a 65kbp integrative and conjugative element (ICE) that carries genes encoding both tetracycline and erythromycin resistances. The excision operon of this element encodes Xis2c, Xis2d, and Exc proteins involved in the excision of CTnDOT from host chromosomes. These proteins are also required in the complex transcriptional regulation of the divergently transcribed transfer (tra) and mobilization (mob) operons of CTnDOT. Transcription of the tra operon is positively regulated by Xis2c and Xis2d, whereas, transcription of the mob operon is positively regulated by Xis2d and Exc. Xis2d is the only protein that is involved in the excision reaction, as well as the transcriptional regulation of both the mob and tra operons. This paper helps establish how Xis2d binds the DNA in the mob and tra region. Unlike other excisionase proteins, Xis2d binds a region of dyad symmetry. The binding site is located in the intergenic region between the mob and tra promoters, and once bound Xis2d induces a bend in the DNA. Xis2d binding to this region could be the preliminary step for the activation of both operons. Then the other proteins, like Exc, can interact with Xis2d and form higher order complexes.

Tetracycline-related transcriptional regulation of the CTnDOT mobilization region.

CTnDOT is a 65-kb conjugative transposon (CTn) in Bacteroides spp. that confers resistance to the antibiotics erythromycin and tetracycline (Tc). Conjugative transfer of CTnDOT is regulated upon exposure of cells to Tc. In the absence of Tc, no transfer is detectable; however, a cascade of regulatory events results in the conjugative transfer of CTnDOT upon Tc induction. Previous studies addressing regulation of CTnDOT conjugative transfer focused primarily on the 13-kb transfer (tra) operon, which encodes the proteins required for assembly of the mating apparatus. We report here that the mob operon that encodes the relaxase and coupling proteins required for mobilization of CTnDOT are regulated at the transcriptional level upon Tc induction. The Xis2d and Exc excision proteins are required for the upregulation of mob transcription upon Tc induction, and yet a deletion of xis2c has no effect. We also show preliminary evidence suggesting that the integrase, IntDOT, may play a regulatory role, as pLYL72 transfer is not detectable when intDOT is provided in trans.

The excision proteins of CTnDOT positively regulate the transfer operon.

The Bacteroides conjugative transposon, CTnDOT, is an integrated conjugative element (ICE), found in many human colonic Bacteroides spp. strains. It has a complex regulatory system for both excision from the chromosome and transfer and mobilization into a new host. It was previously shown that a cloned DNA segment encoding the xis2c, xis2d, orf3, and exc genes was required for tetracycline dependent activation of the P(tra) promoter. The Xis2c and Xis2d proteins are required for excision while the Exc protein stimulates excision. We report here that neither the Orf3 nor the Exc proteins are involved in activation of the P(tra) promoter. Deletion analysis and electromobility shift assays showed that the Xis2c and Xis2d proteins bind to the P(tra) promoter to activate the tra operon. Thus, the recombination directionality factors of CTnDOT excision also function as activator proteins of the P(tra) promoter.

The small RNA RteR inhibits transfer of the Bacteroides conjugative transposon CTnDOT.

CTnDOT is a 65-kb conjugative transposon present in Bacteroides spp. that confers resistance to erythromycin [erm(F)] and tetracycline [tet(Q)]. An interesting feature of CTnDOT is that both excision from the chromosome and transfer of CTnDOT are stimulated by exposure to tetracycline. However, when no tetracycline is present, transfer of CTnDOT is not detectable. Previous studies suggested that a region containing a small RNA, RteR, appeared to mediate repression of CTnDOT transfer; however, virtually nothing was known about RteR. We have demonstrated that RteR is a 90-nucleotide transcript that is not further processed. RteR inhibits conjugative transfer of CTnDOT by targeting the transfer region, a 13-kb operon that encodes the tra genes required to assemble the mating apparatus. We report here that RteR interacts with the region downstream of traA. Levels of the downstream tra mRNA are dramatically reduced when RteR is present. Further, RteR does not appear to decrease the half-life of the tra mRNA transcript, suggesting that RteR does not bind to the transcript to initiate RNase-dependent decay, similar to other trans-acting small RNAs. We predict that RteR may act to enhance termination of the tra operon within traB, which could account for the decreased abundance of the tra transcript downstream of traA and explain why the tra mRNA has the same half-life whether or not RteR is present. RteR is the only small RNA that has been characterized so far within the Bacteroidetes phylum.

Characterization of the Bacteroides CTnDOT regulatory protein RteC.

Excision of the Bacteroides conjugative transposon CTnDOT is stimulated by tetracycline. It was shown previously that a gene, rteC, is necessary for tetracycline-stimulated transcriptional regulation of the orf2c operon, which contains the excision genes. The protein encoded by this gene, RteC, did not have primary amino acid sequence homology to any known proteins in the databases. Accordingly, we sought structural homologs of RteC. A three-dimensional structure prediction by Robetta suggested that RteC might have two domains and that the C-terminal domain might have a winged helix motif. Based on the Robetta prediction, the human transcriptional factors E2F-4 and DP2 were identified as the most likely structural homologs of RteC. We made alanine substitutions within the putative DNA binding helix 3 region of RteC. Assays of orf2c::uidA activation by alanine mutants indicated that residues 174, 175, 178, 180, and 184 in helix 3 might contact the upstream region of P(E). The upstream region of orf2c contained two inverted-repeat half sites. Mutational analysis of these half sites showed that both half sites are important for activity. Thus, we have identified the DNA binding portion of RteC and the DNA site to which it binds.

CTn12256, a chimeric Bacteroides conjugative transposon that consists of two independently active mobile elements.

A Bacteroides conjugative transposon (CTn), CTn12256, which has an estimated size of more than 150 kbp appeared to contain most or all of a previously discovered 65 kbp CTn, CTnDOT. To determine whether the integrated CTnDOT was still intact and to identify the element into which CTnDOT had integrated, large segments of CTn12256 were cloned and sequenced. Results of this analysis revealed that an intact CTnDOT type CTn had integrated into another CTn that was most closely related to a putative CTn found in the genome sequence of Bacteroides fragilis YCH46 (CTn3Bf). The CTnDOT portion of CTn12256 (CTnDOT2) proved to be capable of excising from CTn12256 and to transfer itself independently of CTn12256. The CTn3Bf region rarely transferred independently of CTnDOT2, and the transferred CTn3Bf contained a large deletion flanking the site of CTnDOT2 insertion and was no longer transmissible. Nonetheless, genetic analysis showed that the CTn3Bf portion controls transfer of CTn12256.

Differences between the normal vaginal bacterial community of baboons and that of humans.

Humans and baboons (Papio spp.) share considerable anatomical and physiological similarities in their reproductive tracts. Given the similarities, it is reasonable to expect that the normal vaginal microbial composition (microbiota) of baboons would be similar to that of humans. We have used a 16S rRNA phylogenetic approach to assess the composition of the baboon vaginal microbiota in a set of nine animals from a captive facility and six from the wild. Results show that although Gram-positive bacteria dominate in baboons as they do in humans, there are major differences between the vaginal microbiota of baboons and that of humans. In contrast to humans, the species of Gram-positive bacteria (Firmicutes) were taxa other than Lactobacillus species. In addition, some groups of Gram-negative bacteria that are not normally abundant in humans were found in the baboon samples. A further level of difference was also seen even within the same bacterial phylogenetic group, as baboon strains tended to be more phylogenetically distinct from human strains than human strains were with each other. Finally, results of our analysis suggests that co-evolution of microbes and their hosts cannot account for the major differences between the microbiota of baboons and that of humans because divergences between the major bacterial genera were too ancient to have occurred since primates evolved. Instead, the primate vaginal tracts appear to have acquired discrete subsets of bacteria from the vast diversity of bacteria available in the environment and established a community responsive to and compatible with host species physiology.

Baboon vaginal microbiota: an overlooked aspect of primate physiology.

The bacterial population of the primate vaginal canal is an infant primate's first exposure to the microbial population inhabiting the outside world. Yet, little is known about this population and the effect it might have on the development and survival of the infant primate. As a first step toward characterizing the vaginal microbiota of a nonhuman primate, we used denaturing gradient gel electrophoresis to evaluate variations in the vaginal microbiota of a group of 35 baboons (Papio hamadryas), which were housed in a facility where they shared the same diet and the same environmental conditions. We found that, despite the uniform environment, there were appreciable differences in the composition of the microbiota from one individual to another. Our results also indicate that a simple swab test is sufficient for sampling the vaginal microbiota in the field, a finding that should help make more detailed characterization of the microbiota of wild primates feasible in the future.

An unexpected effect of tetracycline concentration: growth phase-associated excision of the Bacteroides mobilizable transposon NBU1.

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 microg/ml) to a subinhibitory level (0.05 microg/ml). Independence of growth phase also occurred when rteA and rteB were placed under the control of a heterologous maltose-inducible promoter, P(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.

Tetracycline-associated transcriptional regulation of transfer genes of the Bacteroides conjugative transposon CTnDOT.

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.

CTnDOT integrase performs ordered homology-dependent and homology-independent strand exchanges.

Although the integrase (IntDOT) of the Bacteroides conjugative transposon CTnDOT has been classified as a member of the tyrosine recombinase family, the reaction it catalyzes appears to differ in some features from reactions catalyzed by other tyrosine recombinases. We tested the ability of IntDOT to cleave and ligate activated attDOT substrates in the presence of mismatches. Unlike other tyrosine recombinases, the results revealed that IntDOT is able to perform ligation reactions even when all the bases within the crossover region are mispaired. We also show that there is a strong bias in the order of strand exchanges during integrative recombination. The top strands are exchanged first in reactions that appear to require 2 bp of homology between the partner sites adjacent to the sites of cleavage. The bottom strands are exchanged next in reactions that do not require homology between the partner sites. This mode of coordination of strand exchanges is unique among tyrosine recombinases.

Human intestinal bacteria as reservoirs for antibiotic resistance genes.

Human intestinal bacteria have many roles in human health, most of which are beneficial or neutral for the host. In this review, we explore a more sinister side of intestinal bacteria; their role as traffickers in antibiotic resistance genes. Evidence is accumulating to support the hypothesis that intestinal bacteria not only exchange resistance genes among themselves but might also interact with bacteria that are passing through the colon, causing these bacteria to acquire and transmit antibiotic resistance genes.

Ecology and physiology of infectious bacteria--implications for biotechnology.

Escalating incidents of life-threatening infections by antibiotic-resistant bacteria in recent years have provided strong impetus to discover new antibiotics and alternative treatment modalities. The need to couple information about bacterial physiology and ecology with innovative technologies will become ever more critical in the search for new antibiotics and for other therapies, including probiotics, improved vaccines, alternative antimicrobials and antitoxins.

Regulation of CTnDOT conjugative transfer is a complex and highly coordinated series of events.

UNLABELLED: CTnDOT is a 65-kb conjugative transposon that is found in Bacteroides spp., which are one of the more abundant members within the lower human gastrointestinal tract. CTnDOT encodes resistance to the antibiotics erythromycin and tetracycline (Tc). An interesting feature of CTnDOT is that exposure to low levels of Tc induces a cascade of events that ultimately results in CTnDOT conjugative transfer. However, Tc is apparently not a switch that activates transfer but rather a signal that appears to override a series of negative regulators that inhibit premature excision and transfer of CTnDOT. In this minireview, we summarize over 20 years of research that focused on elucidating the highly coordinated regulation of excision, mobilization, and transfer of CTnDOT. IMPORTANCE: Bacteroides spp. are abundant commensals in the human colon, but they are also considered opportunistic pathogens, as they can cause life-threatening infections if they should escape the colon. Bacteroides spp. are the most common cause of anaerobic infections and are rather difficult to treat due to the prevalence of antibiotic resistance within this genus. Today over 80% of Bacteroides are resistant to tetracycline (Tc), and a study looking at both clinical and community isolates demonstrated that this resistance was specifically due to the conjugative transposon CTnDOT.

Antibiotic resistance genes in the vaginal microbiota of primates not normally exposed to antibiotics.

Previous studies of resistance gene ecology have focused primarily on populations such as hospital patients and farm animals that are regularly exposed to antibiotics. Also, these studies have tended to focus on numerically minor populations such as enterics or enterococci. We report here a cultivation-independent approach that allowed us to assess the presence of antibiotic resistance genes in the numerically predominant populations of the vaginal microbiota of two populations of primates that are seldom or never exposed to antibiotics: baboons and mangabeys. Most of these animals were part of a captive colony in Texas that is used for scientific studies of female physiology and physical anthropology topics. Samples from some wild baboons were also tested. Vaginal swab samples, obtained in connection with a study designed to define the normal microbiota of the female vaginal canal, were tested for the presence of two types of antibiotic resistance genes: tetracycline resistance (tet) genes and erythromycin resistance (erm) genes. These genes are frequently found in human isolates of the two types of bacteria that were a substantial part of the normal microbiota of primates (Firmicutes and Bacteroidetes). Since cultivation was not feasible, polymerase chain reaction and DNA sequencing were used to detect and characterize these resistance genes. The tet(M) and tet(W) genes were found most commonly, and the tet(Q) gene was found in over a third of the samples from baboons. The ermB and ermF genes were found only in a minority of the samples. The ermG gene was not found in any of the specimens tested. Polymerase chain reaction analysis showed that at least some tet(M) and tet(Q) genes were genetically linked to DNA from known conjugative transposons (CTns), Tn916 and CTnDOT. Our results raise questions about the extent to which extensive exposure to antibiotics is the only pressure necessary to maintain resistance genes in natural settings.

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.

Possible origins of CTnBST, a conjugative transposon found recently in a human colonic Bacteroides strain.

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.

Integration site selection by the Bacteroides conjugative transposon CTnBST.

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.

Integration and excision of a newly discovered bacteroides conjugative transposon, CTnBST.

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 erythromycin 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.