INTRODUCTION

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The tetracyclines, which were discovered in the 1940s, are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site. Tetracyclines are broad-spectrum agents, exhibiting activity against a wide range of gram-positive and gram-negative bacteria, atypical organisms such as chlamydiae, mycoplasmas, and rickettsiae, and protozoan parasites. The favorable antimicrobial properties of these agents and the absence of major adverse side effects has led to their extensive use in the therapy of human and animal infections. They are also used prophylactically for the prevention of malaria caused by mefloquine-resistant Plasmodium falciparum. Furthermore, in some countries, including the United States, tetracyclines are added at subtherapeutic levels to animal feeds to act as growth promoters. Although the tetracyclines retain important roles in both human and veterinary medicine, the emergence of microbial resistance has limited their effectiveness. Undoubtedly the use of tetracyclines in clinical practice has been responsible for the selection of resistant organisms. Nevertheless, as we enter the new millennium, the use of tetracyclines and other antibiotics as animal growth promoters is becoming increasingly controversial because of concerns that this practice may be contributing to the emergence of resistance in human pathogens. The increasing incidence of bacterial resistance to tetracyclines has in turn resulted in efforts to establish the mechanisms by which genetic determinants of resistance are transferred between bacteria and the molecular basis of the resistance mechanisms themselves. The improved understanding of tetracycline resistance mechanisms achieved by this work has provided opportunities for the recent discovery of a new generation of tetracyclines, the glycylcyclines (see below). Further research, already under way, is also identifying approaches by which inhibitors of tetracycline resistance mechanisms might be developed for use in conjunction with earlier tetracyclines to restore their antimicrobial activity (185, 186).

The tetracyclines have been extensively reviewed both by the present authors (41, 43, 44, 100, 227-229) and others (56, 73, 263, 275). Nevertheless, in view of continuing interest in this group of antibiotics for both infectious and noninfectious diseases (95), we have decided to write a review that focuses on recent developments in the field.

Chlortetracycline and oxytetracycline (Tables 1 and 2), both discovered in the late 1940s, were the first members of the tetracycline group to be described. These molecules were products of Streptomyces aureofaciens and S. rimosus, respectively. Other tetracyclines were identified later, either as naturally occurring molecules, e.g., tetracycline from S. aureofaciens, S. rimosus, and S. viridofaciens and demethylchlortetracycline from S. aureofaciens, or as products of semisynthetic approaches, e.g., methacycline, doxycycline, and minocycline. Despite the success of the early tetracyclines, analogs were sought with improved water solubility either to allow parenteral administration or to enhance oral absorption. These approaches resulted in the development of the semisynthetic compounds rolitetracycline and lymecycline (Tables 1 and 2). The most recently discovered tetracyclines are the semisynthetic group referred to as glycylcyclines, e.g., 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, 9-(N,N-dimethylglycylamido)-minocycline, and 9-t-(butylglycylamido)-minocycline (Tables 1 and 2). These compounds possess a 9-glycylamido substitutent (Table 2). The antibiotics in Tables 1 and 2 can be referred to as first-generation (1948 to 1963), second-generation (1965 to 1972), and third-generation (glycylcycline) tetracyclines. The 9-t-butylglycylamido derivative of minocycline (tigilcycline; formerly known as GAR-936) commenced phase I studies in October 1999 and is currently undergoing phase II clinical trials (113). Some of the earlier compounds, e.g., clomocycline, are no longer marketed, and others, e.g., rolitetracycline, lymecycline, and chlortetracycline, are not available in all countries (73, 137).

The structural features that confer antibacterial activity to the tetracyclines are well established (56, 179, 250) and will only be briefly discussed. More recently, however, new aspects of structure-activity relationships have emerged. This has followed efforts to extend the therapeutic utility of this antibiotic class to encompass bacteria expressing resistance to first- and second-generation compounds through ribosomal protection- and efflux-based mechanisms. This aspect is considered in more detail below.

Tetracycline molecules comprise a linear fused tetracyclic nucleus (rings designated A, B, C, and D [Table 2]) to which a variety of functional groups are attached. The simplest tetracycline to display detectable antibacterial activity is 6-deoxy-6-demethyltetracycline (Fig. 1) and so this structure may be regarded as the minimum pharmacophore (179). Features important for antibacterial activity among the tetracyclines are maintenance of the linear fused tetracycle, naturally occurring () stereochemical configurations at the 4a, 12a (A-B ring junction), and 4 (dimethylamino group) positions, and conservation of the keto-enol system (positions 11, 12, and 12a) in proximity to the phenolic D ring. The tetracyclines are strong chelating agents (20, 44) and both their antimicrobial and pharmacokinetic properties are influenced by chelation of metal ions (see below). Chelation sites include the -diketone system (positions 11 and 12) and the enol (positions 1 and 3) and carboxamide (position 2) groups of the A ring (20, 44). The newly discovered glycylcyclines, like other tetracycline derivatives, also form chelation complexes with divalent cations (278). Replacement of the C-2 carboxamide moiety with other groups has generally resulted in analogs with inferior antibacterial activity (179), probably because bacteria accumulate these molecules poorly (42). However, the addition of substituents to the amide nitrogen can impart significant water solubility, as in the case of rolitetracycline and lymecycline (Table 2). Dissociation of the prodrugs in vivo liberates free tetracycline (179, 250). Consistent with the above observations, substitutions at positions 1, 3, 4a, 10, 11, or 12 are invariably detrimental for antibacterial activity (179). Nevertheless, a number of other substitutions at different positions on the B, C, and D rings are tolerated, and molecules possessing these substituents have given rise to the tetracyclines in clinical use today, as well as the new glycylcycline molecules that are currently undergoing clinical trials (Table 2).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Structure of 6-deoxy-6-demethyltetracycline, the minimum tetracycline pharmacophore.

The extensive structure-activity studies referred to above revealed that with one exception, each of the rings in the linear fused tetracyclic nucleus must be six membered and purely carbocyclic for the molecules to retain antibacterial activity. For instance, the nortetracyclines, derivatives in which the B ring comprises a five-membered carbocycle, are essentially devoid of antibacterial activity (250). Nevertheless, 6-thiatetracycline, which possesses a sulfur atom at position 6 of the C ring, is an apparent exception to the rule that a purely carbocyclic six-membered ring structure is required for activity, since molecules in this series have potent antibacterial properties (43, 250). Nevertheless, it has now been established that the thiatetracyclines and a number of other tetracycline analogs, referred to collectively as atypical tetracyclines (43, 44), exhibit a different structure-activity relationship from the majority of tetracyclines. These molecules, which also include the anhydrotetracyclines, 4-epi-anhydrotetracyclines, and chelocardin, appear to directly perturb the bacterial cytoplasmic membrane, leading to a bactericidal response (43, 198, 199). This contrasts with the typical tetracyclines, which interact with the ribosome to inhibit bacterial protein synthesis and display a reversible bacteriostatic effect. The membrane-disrupting properties of the atypical tetracyclines are probably related to the relative planarity of the B, C, and D rings so that a lipophilic, nonionized molecule predominates. On interaction with the cell, the atypical tetracyclines are likely to be preferentially trapped in the hydrophobic environment of the cytoplasmic membrane, disrupting its function. These molecules are of no interest as therapeutic candidates because they cause adverse side effects in humans (250), which are probably related to their ability to interact nonspecifically with eukaryotic as well as prokaryotic cell membranes (43). A few other tetracycline molecules have been examined but have not moved on to further study (95, 185, 300).

There has been widespread emergence of efflux- and ribosome-based resistance to first- and second-generation tetracyclines (1, 2, 19, 38, 44, 70, 85, 103, 120, 122, 150, 165, 227-229, 307). To restore the potential of the tetracyclines as a class of useful broad-spectrum agents, a systematic search was undertaken during the early 1990s to discover new analogs that might possess activity against organisms resistant to older members of the class while retaining activity against tetracycline-susceptible organisms (295). This resulted in the discovery of the 9-glycinyltetracyclines (glycylcyclines) (15, 286, 287, 295) (Tables 1 and 2). Previous attempts to introduce substituents at position 9 of the molecule, e.g., 9-nitro, 9-amino, and 9-hydroxy, led to analogs with poor antibacterial activity (179, 250). However, during the 1990s, a team at Lederle Laboratories (now American Home Products) noted that 9-acylamido derivatives of minocycline exhibited antibacterial activities typical of earlier tetracyclines but without activity against tetracycline-resistant organisms (15). Nevertheless, when the acyl group was modified to include an N,N-dialkylamine moiety, e.g., as in the 6-demethyl-6-deoxytetracycline and minocycline derivatives (GAR-936) shown in Table 2, not only was antibacterial activity retained but also the compounds displayed activity against bacteria containing tet genes (Tables 3 to 5) responsible for both efflux of earlier tetracyclines [Tet(A)- to Tet(D) and Tet(K)] and ribosomal protection [Tet(M)] (15, 286, 287, 295). These findings were extended to the 9-t-butylglycylamido derivative of minocycline (Tables 1 and 2) (208). These data suggest that new structure-activity relationships may have been defined for activity against strains expressing efflux or ribosomal protection mechanisms that encompass the previous requirements for activity against tetracycline-susceptible strains but in addition require an N-alkyl glycylamido substitution at the 9 position of the molecule.

Figure 2 presents a summary of the features that confer optimum antibacterial activity to the tetracycline nucleus.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Stereochemical and substitution requirements for optimum antibacterial activity within the tetracycline series.

It is well established that tetracyclines inhibit bacterial protein synthesis by preventing the association of aminoacyl-tRNA with the bacterial ribosome (44, 263). Therefore, to interact with their targets these molecules need to traverse one or more membrane systems depending on whether the susceptible organism is gram positive or gram negative. Hence, a discussion of the mode of action of tetracyclines requires consideration of uptake and ribosomal binding mechanisms. Also pertinent to this discussion are explanations of the joint antibacterial-antiprotozoal activity of the tetracyclines and the microbial selectivity of the class as a whole. Most of these issues have been considered at length in recent years (44, 67, 78, 263), so the focus here will be on new information.

Tetracyclines traverse the outer membrane of gram-negative enteric bacteria through the OmpF and OmpC porin channels, as positively charged cation (probably magnesium)-tetracycline coordination complexes (44, 263). The cationic metal ion-antibiotic complex is attracted by the Donnan potential across the outer membrane, leading to accumulation in the periplasm, where the metal ion-tetracycline complex probably dissociates to liberate uncharged tetracycline, a weakly lipophilic molecule able to diffuse through the lipid bilayer regions of the inner (cytoplasmic) membrane. Similarly, the electroneutral, lipophilic form is assumed to be the species transferred across the cytoplasmic membrane of gram-positive bacteria. Uptake of tetracyclines across the cytoplasmic membrane is energy dependent and driven by the pH component of the proton motive force (192, 263). Within the cytoplasm, tetracycline molecules are likely to become chelated since the internal pH and divalent metal ion concentrations are higher than those outside the cell (263). Indeed, it is probable that the active drug species which binds to the ribosome is a magnesium-tetracycline complex (44, 144). Association of tetracyclines with the ribosome is reversible, providing an explanation of the bacteriostatic effects of these antibiotics (44).

Several studies have indicated a single, high-affinity binding site for tetracyclines in the ribosomal 30S subunit, with indications through photoaffinity labeling and chemical footprinting studies that protein S7 and 16S rRNA bases G693, A892, U1052, C1054, G1300, and G1338 contribute to the binding pocket (44, 180, 196, 263). However, Schnappinger and Hillen (263) have pointed out that these apparent sites for drug interaction in the ribosome may not necessarily reflect the actual binding site. Indeed, interpretation of the probing studies referred to above is complicated by the observation that binding of tetracycline (which measures approximately 8 by 12 Å) to the ribosome appears to cause wide-ranging structural change in 16 S rRNA (193). Furthermore, photoincorporation methods are subject to the limitation that upon irradiation, tetracycline photoproducts are generated which may react further with the ribosomes (196). Nevertheless, naturally occurring tetracycline-resistant propionibacteria contain a cytosine-to-guanine point mutation at position 1058 in 16S rRNA (251) (see below), which does at least suggest that the neighboring bases U1052 and C1054 identified by chemical footprinting (180) may have functional significance for the binding of tetracyclines to the 30S subunit.

The absence of major antieukaryotic activity explains the selective antimicrobial properties of the tetracyclines. At the molecular level, this results from relatively weak inhibition of protein synthesis supported by 80S ribosomes (302) and poor accumulation of the antibiotics by mammalian cells (78). However, tetracyclines inhibit protein syntheses in mitochondria (221) due to the presence of 70S ribosomes in these organelles. It has been recognized for some time that the spectrum of activity of tetracyclines encompasses various protozoan parasites such as P. falciparum, Entamoeba histolytica, Giardia lamblia, Leishmania major, Trichomonas vaginalis, and Toxoplasma gondii (28, 44, 67, 137, 214). The antiparasitic activity is explained in some cases by the finding that certain organisms, e.g., P. falciparum, contain mitochondria (67). However, a number of other protozoa which lack mitochondria nevertheless remain susceptible to tetracyclines. At present there is no satisfactory molecular explanation for these findings (67).

RESISTANCE TO TETRACYCLINES

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Introduction

Prior to the mid-1950s, the majority of commensal and pathogenic bacteria were susceptible to tetracyclines (144), as illustrated by the finding that among 433 different members of the Enterobacteriaceae collected between 1917 and 1954, only 2% were resistant to tetracycline (106). Studies of naturally occurring environmental bacteria, representative of populations existing before the widespread use of tetracyclines by humans (52), also support the view that the emergence of resistance is a relatively modern event that has followed the introduction of these agents for clinical, veterinary, and agricultural use. Indeed, resistance to tetracyclines has now emerged in many commensal and pathogenic bacteria due to genetic acquisition of tet genes. Subsequent parts of this section describe the genetic and biochemical mechanisms of tetracycline resistance, the regulation of resistance gene expression, and the distribution of tet genes in pathogenic and commensal bacteria. The impact of resistance on the use of tetracyclines for human medicine is examined later in the review.

Nomenclature of resistance determinants. Mendez et al. (176) in 1980 first examined the genetic heterogeneity of tetracycline resistance determinants from plasmids from members of the Enterobacteriaceae and Pseudomonadaceae. They used restriction enzyme analysis, DNA-DNA hybridization, and expression of resistance to tetracycline and various analogs to categorize the tetracycline-resistant (Tc^r) plasmids. Currently, two genes are considered related (i.e., of the same class) and given the same gene designation if they have 80% of their amino acid sequences in common. Two genes are considered different from each other if they have 79% amino acid sequence identity (150). This comparison can now be done using GenBank sequence information, since with few exceptions [tet(I) and otr(C)], representatives of all tet and otr genes have been sequenced and are available (150, 151) (Table 3).

Efflux proteins. The efflux proteins are the best studied of the Tet proteins. The genes encoding then belong to the major facilitator superfamily (MFS), whose products include over 300 individual proteins (205). All the tet efflux genes code for membrane-associated proteins which export tetracycline from the cell. Export of tetracycline reduces the intracellular drug concentration and thus protects the ribosomes within the cell. Efflux genes are found in both gram-positive and gram-negative species (Table 4 and 5). Most of these efflux proteins confer resistance to tetracycline but not to minocycline or glycylcyclines. In contrast, the gram-negative tet(B) gene codes for an efflux protein which confers resistance to both tetracycline and minocycline but not glycylcyclines (44, 295). However, laboratory-derived mutations in tet(A) or tet(B) have led to glycylcycline resistance, suggesting that bacterial resistance to this group of drugs may develop over time and with clinical use (90; M. Tuckman, P. J. Petersen, and S. Projan, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C98, p. 97, 1998).

Ribosomal protection proteins. Nine ribosomal protection proteins are listed in Table 3. These are cytoplasmic proteins that protect the ribosomes from the action of tetracycline and confer resistance to doxycycline and minocycline. They confer a wider spectrum of resistance to tetracyclines than is seen with bacteria that carry tetracycline efflux proteins, with the exception of Tet(B). The ribosomal protection proteins have homology to elongation factors EF-Tu and EF-G (259, 292). The greatest homology is seen at the N-terminal area, which contains the GTP-binding domain. The Tet(M), Tet(O), and OtrA proteins reduce the susceptibility of ribosomes to the action of tetracyclines. The Streptomyces Otr(A) protein has greatest overall amino acid similarity to elongation factors. The mechanism of ribosomal protection works in vivo and in vitro, unlike the action of efflux proteins; which require intact membranes to function. Binding of the Tet(M) protein is not affected by tetracycline but is inhibited by thiostrepton, which also inhibits the binding of the EF-G protein (53). EF-G and the Tet(M) proteins compete for binding on the ribosomes, with Tet(M) having a higher affinity than EF-G. This suggests that these two proteins may have overlapping binding sites and that Tet(M) must be released from the ribosome to allow EF-G to bind (53).

Enzymatic inactivation of tetracycline. The tet(X) gene (281) encodes the only example of tetracycline resistance due to enzymatic alteration of tetracycline. Two closely related anaerobic Bacteroides transposons containing the tet(X) gene have been described (281). The tet(X) gene was found because it is linked to erm(F), which codes for a rRNA methylase gene. The erm(F) gene was cloned into E. coli, and the clones were found to confer tetracycline resistance in E. coli when grown aerobically. The tet(X) gene product is a 44-kDa cytoplasmic protein that chemically modifies tetracycline in the presence of both oxygen and NADPH. Sequence analysis indicates that this protein has amino acid homology with other NADPH-requiring oxidoreductases and should not be able to function in the natural anaerobic Bacteroides host (281). It has not been found outside Bacteroides. However, to date no surveys have been conducted to assess the distribution of the tet(X) gene. Thus, even though the transposon carrying tet(X) and linked erm(F) is thought to be of gram-positive aerobic or facultative origin, a putative ancestor has not been identified.

Other/unknown mechanisms of resistance. The tet(U) gene confers low-level tetracycline resistance (220). This gene encodes a 11.8-kDa protein containing 105 amino acids, which is smaller than the efflux proteins (45 kDa) and the ribosomal proteins (72 kDa) (see above). There is 21% similarity over the 105 amino acids between the Tet(U) and Tet(M) proteins, beginning close to the carboxy terminus of the latter. These similarities do not include the consensus GTP-binding sequences, which are thought to play a role in resistance in the Tet(M) and related proteins. However, the sequence is not really similar to either the efflux or ribosomal protection genes, and the mechanism is thus listed as unknown in Table 3.

Efflux genes. The gram-negative efflux determinants consist of two genes, one coding for an efflux protein and one coding for a repressor protein. Both genes are regulated by tetracycline. The two genes are oriented divergently and share a central regulatory region with overlapping promoters and operators (99). In the absence of tetracycline, the repressor protein occurs as a homodimer, which binds two -helix-turn--helix motifs to the two tandemly orientated tet operators (99, 130). This blocks transcription of the structural genes for both the repressor and the efflux protein. Induction in the system occurs when a tetracycline-Mg²⁺ complex enters the cell and binds to the repressor protein. Drug binding changes the conformation of the repressor so that it can no longer bind to the DNA operator region. Only nanomolar concentrations of tetracycline are needed for binding to the repressor protein. This system is the most sensitive effector-inducible transcriptional regulation system yet described. After the repressor binds the tetracycline-Mg²⁺ complex, transcription of the efflux structural and repressor genes occurs. This is a relatively rapid process (99, 144). The tet gene in Tn10 is differentially regulated so that the repressor protein is synthesized before the efflux protein is expressed. The repressor protein will rebind to the DNA only when there is insufficient tetracycline (smaller than nanomolar amounts) present in the cell. This type of regulation most probably occurs with all the gram-negative efflux genes, tet(A), tet(C), tet(D), tet(E), tet(G), and tet(H), and probably also for the tet(I) gene. Crystallography has shown that the three -helices at the N-terminal region of the repressor protein form the DNA-binding domain in the repressor molecule and that conformational changes in the repressor protein occur in the presence of tetracycline complexed with Mg²⁺ (130, 203). The tetracycline-binding pocket and the interaction between tetracycline and the repressor protein have also been characterized (99). The structural basis of tet(B) regulation is summarized in reference 203.

Ribosomal protection. The expression of both Tet(M) and Tet(O) proteins appears to be regulated. Wang and Taylor (306) have suggested that the 400-bp region directly upstream from the coding region of the tet(O) gene was needed for full expression of the gene; however, the function of the region is not understood (292). Burdett (29) reported that the amount of Tet(M) protein increased when streptococci carrying the determinant were exposed to tetracycline. Similarly, Nesin et al. (188) found that preexposure to subinhibitory concentrations of tetracycline in S. aureus strains carrying the tet(M) gene resulted in both an increase in tetracycline resistance and an increase in the level of mRNA transcripts for tet(M). Su et al. (285) reported a stem-loop structure in the upstream region of the structural gene from Tn916, and both short and long transcripts were found by Northern blot analyses, similar to descriptions for attenuation of mRNA transcription of gram-positive proteins. Based on the DNA sequences from the upstream region in the Ureaplasma urealyticum tet(M) sequence, a similar stem-loop structure to that described for Tn916 would be possible (228). However, we have not looked at the transcripts from this gene.

Overview for pathogenic and opportunistic organisms. Most work on bacterial resistance has either been conducted with pathogenic bacteria, which usually cause disease when present, or opportunistic bacteria, which occasionally cause disease (145, 146, 157, 160, 223, 229, 230, 232-244). Opportunistic bacteria are often part of the host's normal flora and can cause disease when they leave their normal sites. The majority of tet genes in bacteria have been associated with mobile plasmids, transposons, conjugative transposons, and integrons (gene cassettes) (176, 216, 224-228, 230, 232). These mobile units have enabled the tet genes to move from species to species and into a wide range of genera by conjugation (Tables 4 and 5). The gram-negative tet genes, first described in the Enterobacteriaceae and Pseudomonadaceae, are now also found in Neisseria, Haemophilus, Mannheimia, Treponema, and Vibrio (Table 4) (121, 132, 144, 149, 168, 182, 232, 233, 271). The tet(B) gene has the widest host range of the gram-negative tet genes and has been identified in 20 gram-negative genera (Table 4), while tet(M) is found in 26 genera including gram-negative and gram-positive bacteria (Tables 4 and 5). We have speculated that some genes, such as tet(E), may have a more limited host range because they are located on nonmobile plasmids, which reduces opportunities for transfer to other species and genera (61, 280). Some tet genes in some species or genera may confer low levels of resistance, which would be unlikely to protect the bacterial cell exposed to tetracyclines in clinical or environmental settings.

Overview for commensal microorganisms. The commensal flora consists of microorganisms which are present in and on surfaces of a host and are not thought to cause disease. These organisms are often beneficial to the host, providing nutrients and inhibiting the growth of potential pathogens by preventing them from becoming established (10). Not surprisingly, these bacteria have the same tet genes, plasmids, transposons, conjugative transposons, and integrons as their disease-producing counterparts among the opportunistic and pathogenic bacteria. Many oral viridans streptococci (74, 93, 201) have acquired tet(M), tet(O), tet(L), or tet(K), as have the pathogenic streptococcal species S. pneumoniae and S. pyogenes. Most people today carry Tc^r viridans streptococci in their mouth regardless of use of tetracycline therapy or age, while Tc^r S. pneumoniae and S. pyogenes are significantly less common in most populations (162). This differs from isolates recovered before the introduction of tetracycline therapy, when the majority of bacteria were susceptible to tetracycline (12).

Overview. The tet genes are found in a variety of bacteria isolated from humans, animals, and the environment (Tables 4 and 5). The majority of the tet genes are associated with either conjugative or mobilizable elements, which may partially explain their wide distribution among bacterial species (115, 176, 216). The gram-negative tet efflux genes are found on transposons inserted into a diverse group of plasmids from a variety of incompatibility groups (115, 176). Gram-positive efflux genes are associated with small plasmids (124, 265, 266). The ribosomal protection genes tet(S) and tet(O) can be found on conjugative plasmids, or in the chromosome, where they are not self-mobile (36, 37, 162). The tet(M) and tet(Q) genes are generally associated with conjugative chromosomal elements, which code for their own transfer (48, 152, 256, 257). These conjugative transposons transfer mobilizable plasmids to other isolates and species and even unlinked genomic DNA (26, 48, 152, 166, 184, 272). Genes in the tet(Q) operon have been identified which mediate excision and circularization of discrete nonadjacent segments of chromosomal DNA in Bacteroides. The transfer origin (oriT) region of one of the Bacteroides conjugative transposons has been located near the middle of the conjugative transposon (152). Bacteroides conjugative transposons range from 65 to over 150 kb; most elements carry both tet(Q) and erm(F) and belong to a family of elements with the prototype being Tc^r Em^r DOT (257). A 16-kb region of the transposon is required and sufficient for conjugal transfer of the element and for mobilization of both coresident plasmids and unlinked integrated elements. DNA transfer is tetracycline regulated and mediated by at least three regulatory genes including a putative sensor (rteA), a putative regulator (rteB), and a third gene, rteC, which seems to stimulate transfer in an unknown fashion (152). This differs from the mechanism proposed for tetracycline regulation of conjugation of the Tn916 family of elements, where Manganelli et al. (166) suggest that tetracycline increases the number of circular intermediates present in the cell, which leads to more transconjugants. A gene can be induced to increase conjugal transfer by the presence of low doses of tetracycline. This has been illustrated in vitro with tet(M) and gram-positive cocci and rods (69, 222, 272) and in the gram-positive Listeria spp. (69). Transfer of the tet(Q) gene is also inducible by tetracyclines in Bacteriodes spp. (256, 257).