Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.     

Novel aerobic tetracycline resistance gene that chemically modifies tetracycline.

A tetracycline resistance gene that was found originally on the Bacteroides plasmid pBF4 confers resistance on Escherichia coli but only when cells are growing aerobically. When E. coli EM24 carrying this aerobic tetracycline resistance (*Tcr) gene is grown in medium containing tetracycline, the resulting spent medium is no longer toxic to tetracycline-sensitive (Tcs) E. coli EM24 (B.S. Speer and A.A. Salyers, J. Bacteriol. 170: 1423-1429, 1988). To determine whether the *Tcr gene product modified tetracycline, we characterized the material resulting from incubation of E. coli (*Tcr) with tetracycline. When [7-3H(N)]tetracycline was added to cultures of E. coli (*Tcr), at least 90% of the label was recovered in the extracellular fluid. Therefore, tetracycline was not being sequestered by the cells. The labeled material behaved similarly to tetracycline with respect to solubility in various organic solvents. However, the UV-visible light spectrum had a single peak at 258 nm, whereas the tetracycline spectrum had a peak at 364 nm. The labeled material also had a faster migration rate than did tetracycline on thin-layer plates in a solvent system of butanol-methanol-10% citric acid (4:1:2, vol/vol/vol) and was separable from tetracycline by reverse-phase high-pressure liquid chromatography, using an acetronitrile-0.1% trifluoroacetic acid solvent system. These results demonstrate that the *Tcr gene product chemically modifies tetracycline. The *Tcr gene is the first example of a chemically modifying tetracycline resistance mechanism.     

Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis

When Bacillus subtilis GSY908 (recE4-) (H. C. Spatz and T. A. Trautner, 1971, Mol. Gen. Genet. 113, 174-190) protoplasts were infected with Staphylococcus aureus plasmid pNS1 specifying tetracycline resistance (Tcr) (N. Noguchi et al., 1983, Gene 21, 105-112), which was modified such that it either could not replicate or did not carry a functional Tcr gene, a plasmid with a molecular weight of 3.1 X 10(6) (4.9 kb) was generated in Tcr phenotypes. This plasmid, named Tcr pNS1981, exhibited completely different restriction endonuclease cleavage patterns to pNS1 and showed only negligible sequence homology in hybridization experiments. Southern hybridization experiments revealed that pNS1981 arises by excision of a B. subtilis chromosomal DNA sequence. No sequence corresponding to pNS1 was detectable on the chromosome of pNS1981-maintaining B. subtilis. The production of pNS1981 was also observed in B. subtilis RM125 (r-Mm-Mrec+) (T. Uozumi et al., 1977, Mol. Gen. Genet. 152, 65-69.) with almost the same frequency as B. subtilis GSY908. Since the recipient B. subtilis Marburg 168 derivatives stated above are sensitive to Tc, the results indicate that information essential for Tcr is under negative regulatory control in the integrated state on the chromosome. Restriction endonuclease analysis suggested that pNS1981 is essentially the same as pBC16, formerly found in B. cereus (K. Bernhard, H. Schrempf, and W. Goebel, 1978, J. Bacteriol. 133, 897-903).     

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.     

Phenotypic and genotypic characterization of tetracycline- and oxytetracycline-resistant Aeromonas hydrophila from cultured channel catfish (Ictalurus punctatus) and their environments

Oxytetracycline-resistant (OTcr) and tetracycline-resistant (Tcr) Aeromonas hydrophila were isolated commonly from catfish intestinal contents and the water and sediment of catfish culture ponds, but less frequently in market catfish. Isolates demonstrated two resistance phenotypes, Tcr OTcr and Tcs OTcr, when plated directly on to MacConkey agar containing 30 micrograms of tetracycline or oxytetracycline per ml. Tcs OTcr isolates expressed Tcr after induction by 1 h of growth in tryptic soy broth containing 1 microgram of tetracycline per ml. Over 90% of the resistant aeromonads hybridized with DNA probes for class A or class E Tcr determinants; class E was twice as prevalent as class A. The distribution of class A and E Tcr determinants varied with the Tcr phenotypes. Prior to induction, 86% of isolates with class A determinants were Tcr as well as OTcr, while only 16% with class E determinant were Tcr. Three of 5 Tcr aeromonads without class A or class E determinants and 1 of 14 with class E determinants transferred Tcr to Escherichia coli.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.     

Nucleotide sequence analysis of tetracycline resistance gene tetO from Streptococcus mutans DL5.

Streptococcus mutans DL5, isolated from the dental plaque of a pig, was resistant to high levels of streptomycin (Sm, 20 mg/ml), erythromycin (Em, 1 mg/ml), and tetracycline (Tc, greater than 100 micrograms/ml), but contained no detectable plasmid DNA. The Smr and Emr determinants were cloned from cellular DNA on the self-replicating 5-kilobase-pair (kbp) EcoRI fragment of pAM beta 1 and the 4.2-kbp cryptic plasmid pVA380-1, respectively, by transformation of Streptococcus sanguis Challis. Helper plasmid cloning, with a Challis host containing pVA380-1, was required to clone the Tcr determinant of strain DL5 on this vector. A single-colony isolate of the original Tcr clone contained a hybrid plasmid, pDL421, composed of 2.6 kbp of vector DNA and 11.4 kbp of S. mutans DNA. Plasmid pDL421 did not hybridize to plasmids containing the streptococcal Tcr determinants tetL, tetM, and tetN. A shortened derivative of this hybrid plasmid, pDL422, missing a 4.9-kbp HincII fragment from the S. mutans DNA but still encoding Tcr, was obtained by subcloning in S. sanguis Challis. The Tcr gene was located in a 1,917-base-pair open reading frame (ORF) corresponding to a 72-kilodalton protein. The ORF exhibited 99.4% sequence identity with the 1,917-base-pair tetO gene from a strain of Campylobacter coli (W. Sougakoff, B. Papadopoulou, P. Nordmann, and P. Courvalin, FEMS Microbiol. Lett. 44:153-160, 1987). A 1.67-kbp NdeI fragment, internal to the ORF from strain DL5, as well as pDL421 hybridized under stringent conditions to DNA from 10 of 10 Tcr strains of C. coli and Campylobacter jejuni from human and animal sources, but not to DNA from Tcs isolates of these two species.     

Chromosomal-DNA amplification in Bacillus subtilis.

Tetracycline-resistant (Tetr) mutants RAD1, RAD2, RAD6, and RAD7 were isolated from Bacillus subtilis BC92 after protoplasting, polyethylene glycol treatment, and regeneration on a medium containing tetracycline. The Tetr phenotype in RAD1, RAD2, and RAD6 was very stable with less than 5% loss of resistance after 30 generations of growth in the absence of selection. Of the four isolates, three contained amplified chromosomal DNA closely associated with the Tetr phenotype. The intensity of restriction fragments present in HindIII and EcoRI digests of chromosomal DNA from RAD1, RAD6, and RAD7 indicated the presence of tandemly duplicated DNA. Disparity in the size and number of amplified fragments suggested that the tandemly duplicated DNA is different in all three isolates. The sizes of the duplicated DNA present in RAD1, RAD6, and RAD7 were estimated to be 10, 19, and 20 kilobases, respectively. No amplified DNA was detected in RAD2. Results of transductional-mapping studies with PBS1 showed that the tetracycline resistance (tet) loci of RAD1, RAD2, and RAD6 all mapped near the origin of chromosomal replication and close to the guaA locus. Amplified DNA characteristic of RAD1 and RAD6 was cotransduced with the tet locus. Cotransfer of amplified DNA with the guaA locus or other nearby loci in the absence of tet was not observed. In every case, loss of Tetr was accompanied by loss of amplified DNA. A possible explanation for the occurrence of the amplified DNA is presented.     

Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transfer.

The 55-kilobase plasmid, pLS20, of Bacillus subtilis (natto) 3335 promotes transfer of the tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis. Frequency of pBC16 transfer ranged from 2.3 x 10(-6) to 2.8 x 10(-3). Evidence for a plasmid-encoded conjugationlike mechanism of genetic exchange includes (i) pLS20+ strains, but not pLS20- strains, functioned as donors of pBC16; (ii) plasmid transfer was insensitive to the presence of DNase; and (iii) cell-free filtrates of donor cultures did not convert recipient cells to Tcr. Cotransfer of pLS20 and pBC16 in intraspecies matings and in matings with a restriction-deficient B. subtilis strain indicated that pLS20 was self-transmissible. In addition to mobilizing pBC16, pLS20 mediated transfer of the B. subtilis (natto) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. The fertility plasmid did not carry a selectable marker. To facilitate direct selection for pLS20 transfer, plasmid derivatives which carried the erythromycin resistance transposon Tn917 were generated. Development of this method of genetic exchange will facilitate the introduction of plasmid DNA into nontransformable species by use of transformable fertile B. subtilis or B. subtilis (natto) strains as intermediates.     

Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.     

Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids

Ten plasmids were isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant thermophilic bacteria. Of the 10 plasmids tested, 2 could transform Bacillus subtilis, yielding resistance to specific antibiotics. Plasmid pTB20 (2.8 X 10(6) daltons, approximately 24 copies per chromosome) specifies resistance to tetracycline (Tcr), whereas pTB19 (17.2 X 10(6) daltons, approximately 1 copy per chromosome) renders the host resistant to both kanamycin and tetracycline (KMrTcr). Three plasmids were not self-transmissible. The restriction endonuclease cleavage maps of the two plasmids, pTB19 and pTB20, were constructed. pTB19 and pTB20, both of which were originally isolated from thermophilic bacilli, were tested for stability in B. subtilis. Digestion of pTB19 followed by ligation yielded deletion plasmids pTB512 (Kmr), pTB52 (Tcr), and pTB53 (KmrTcr). Determinants of Kmr, Tcr, and DNA replication were associated with EcoRI fragments R1b (4.2 X 10(6) daltons), R3 (2.8 X 10(6) daltons), and R1a (4.2 X 10(6) daltons), respectively. Restriction endonuclease cleavage maps of pTB51, pTB52, and pTB53 were constructed. Tetracycline resistance of pTB20 was confirmed to be in the EcoRI fragment (1.85 X 10(6) daltons).     

A site-specific DNA inversion in Bacteroides plasmid pBF4 is influenced by the presence of the conjugal tetracycline resistance element.

pBF4 is a 42-kb R plasmid from Bacteroides fragilis which transfers clindamycin resistance (Clr) independently of the chromosomal tetracycline resistance (Tcr) transfer element. We have found that this plasmid exists in two nonequimolar conformations, A and B. These forms differ by an inversion of approximately 11.5 kb which does not involve the repeated DNA sequences previously mapped on the plasmid. The presence of chromosomal tetracycline resistance conjugal elements influences the relative amounts of the two conformations: induction with tetracycline shifts the dominant form from B to A.     

Identification of a circular intermediate in the transfer and transposition of Tn4555, a mobilizable transposon from Bacteroides spp.

Transmissible cefoxitin (FX) resistance in Bacteroides vulgatus CLA341 was associated with the 12.5-kb, mobilizable transposon, Tn4555, which encoded the beta-lactamase gene cfxA. Transfer occurred by a conjugation-like mechanism, was stimulated by growth of donor cells with tetracycline (TC), and required the presence of a Bacteroides chromosomal Tcr element. Transconjugants resistant to either FX, TC, or both drugs were obtained, but only Fxr Tcr isolates could act as donors of Fxr in subsequent matings. Transfer of Fxr could be restored in Fxr Tcs strains by the introduction of a conjugal Tcr element from Bacteroides fragilis V479-1. A covalently closed circular DNA form of Tn4555 was observed in donor cells by Southern hybridization, and the levels of this circular transposon increased significantly in cells grown with TC. Both the cfxA gene and the Tn4555 mobilization region hybridized to the circular DNA, suggesting that this was a structurally intact transposon unit. Circular transposon DNA purified by CsCl-ethidium bromide density gradient centrifugation was used to transform Tcs B. fragilis 638, and Fxr transformants were obtained. Both the circular form and the integrated Tn4555 were observed in transformants, but the circular form was present at less than one copy per chromosomal equivalent. Examination of genomic DNA from Fxr transformants and transconjugants revealed that Tn4555 could insert at a wide variety of chromosomal sites. Multiple transposon insertions were present in many of the transconjugants, indicating that there was no specific barrier to the introduction of a second transposon copy.     

Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.     

Tetracycline-dependent appearance of plasmidlike forms in Bacteroides uniformis 0061 mediated by conjugal Bacteroides tetracycline resistance elements.

Some human colonic Bacteroides strains carry conjugal tetracycline resistance (Tcr) elements, which are thought to be chromosomal. We have found that some of these Tcr elements can mediate the appearance of plasmidlike forms in Bacteroides uniformis 0061. When B. uniformis 0061, containing a conjugal Tcr element designated Tcr ERL, was grown in medium containing tetracycline (1 microgram/ml), two circular DNA forms were found in the alkaline plasmid preparations: NBU1 (10.3 +/- 0.5 kilobases) and NBU2 (11.5 +/- 0.5 kilobases). Restriction analysis of NBU1 and NBU2 showed that they were not identical, although Southern blot analysis indicated that they did contain some region(s) of homology. Results of Southern blot analysis also demonstrated that both NBU1 and NBU2 were normally integrated in the chromosome of B. uniformis or in some undetected large plasmid. Although we were unable to determine the exact structure and location of the integrated forms of NBU1 and NBU2 in B. uniformis, they appear to be in close proximity to each other. Neither NBU1 or NBU2 could be detected as a plasmidlike form in cells exposed to UV light, thymidine starvation, mitomycin C, or autoclaved chlortetracycline (50 micrograms/ml). Four conjugal Tcr elements other than the Tcr ERL element were able to mediate the appearance of NBU1 alone, and two Tcr elements did not mediate the excision of either NBU1 or NBU2. Three strains from different Bacteroides species contained some DNA sequences which had homology to NBU1 and NBU2.     

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector.

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.     

Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans

A chromosomal tetracycline resistance (Tcr) determinant previously cloned from Streptococcus mutans into Streptococcus sanguis (Tobian and Macrina, J. Bacteriol. 152:215-222, 1982) was characterized by using restriction endonuclease mapping, deletion analysis, and Southern blot hybridization. Deletion analysis allowed localization of the Tcr determinant to a 2.8-kilobase region of the originally cloned 10.4-kilobase sequence. This cloned determinant hybridized to a representative of the tetM class of streptococcal Tcr determinants but not to representatives of the tetL and tetN classes and, like other tetM determinants, mediated high-level resistance to tetracycline and low-level resistance to minocycline. A portion (approximately 3 kilobases) of the isolated streptococcal fragment was subcloned into Escherichia coli, where it conferred resistance to tetracycline and minocycline. Two proteins with apparent molecular weights of 33,000 and 35,000, encoded by the S. mutans DNA, were synthesized in E. coli minicells. Insertion of DNA into a unique SstI site of the cloned S. mutans fragment resulted in inactivation of Tcr expression in E. coli and S. sanguis, as well as loss of production of both the 33,000- and 35,000-dalton proteins in E. coli minicells. Incubation of minicells in subinhibitory concentrations of tetracycline did not result in changes in the levels of synthesis of either protein. Our data suggest that at least one of these proteins is involved in the expression of Tcr.