INTRODUCTION

Top Abstract Introduction Materials and methods Results Discussion References

Genes encoding resistance to antibiotics have been identified in a wide spectrum of prokaryotic organisms, including gram-negative and gram-positive bacteria. Although most of the genes characterized to date have been isolated from clinical isolates, these determinants are believed to have originated in soil bacteria (38). More recently, genes encoding proteins that confer resistance to several different drugs, the multidrug systems, have been identified and characterized (37). Horizontal transfer of these determinants among diverse bacteria are challenging the effectiveness of antibiotics in infectious disease control (39, 48).

Genes conferring resistance to tetracycline are among the most abundant of the identified drug resistance elements (29). Although mechanisms such as target site protection and drug inactivation are known (7, 44), most tetracycline resistance determinants function through the efflux mechanism, pumping a drug-metal ion complex out of the bacterial cell by a process energized by the membrane proton gradient (37, 45).

It is a well-recognized but unpublished observation that Agrobacterium tumefaciens C58 and its derivatives give rise at a high frequency to spontaneous mutants resistant to tetracycline. Most genetic and molecular analyses concerning A. tumefaciens and its plasmids are conducted in the C58 chromosomal background. Since many broad-host-range cloning vectors encode resistance to tetracycline as the selectable marker, the propensity of this strain to mutate to tetracycline resistance poses problems to the genetic analysis of this bacterium and to its use as a plant transformation agent.

During a study of mutants affecting the induction of tra genes of pTiC58 in NT1 (51), a Ti plasmid-cured derivative of C58 (53), we observed that one mutant, NTM7, while remaining able to mutate to tetracycline resistance, expressed the phenotype at a much lower level than did its wild-type parent. NTM7 later was found to carry a Tn5 insertion in a locus encoding a protein homologous to the product of the Escherichia coli rnd gene (30). In E. coli, a miaA mutant is unable to express Tet(M)-mediated tetracycline resistance (8). Since both the rnd and miaA genes are involved in tRNA modification (12, 52), we reasoned that there may be a positively acting tetracycline resistance element in the genome of strain C58 which can function normally in the wild-type bacterium but not in the rnd mutant. A study was initiated to investigate the nature of such tetracycline-resistant mutants. We confirmed our hypothesis and report here the molecular cloning, nucleotide sequence, and genetic characterization of a tetracycline resistance gene unit from this A. tumefaciens strain.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Bacterial strains and growth conditions. The strains of E. coli and A. tumefaciens and the plasmids used in this study are listed in Table 1. E. coli strains were grown at 37°C in L broth (LB), on L agar plates (Difco Laboratories, Detroit, Mich.), or in A medium (33). Agrobacterium strains were grown at 28°C in LB or on nutrient agar (NA) (Difco Laboratories). AB medium (11) supplemented with 0.2% mannitol as the sole carbon source was used as the defined minimal medium for Agrobacterium strains. Biotin was added to a final concentration of 2 µg/ml for biovar 2 strains. Agar (Difco Laboratories) was added at 1.5% to make solid medium. Except when specified in the text, antibiotics were used at the following concentrations: for E. coli, kanamycin at 50 µg/ml, tetracycline at 10 µg/ml, and ampicillin at 100 µg/ml; for Agrobacterium spp., kanamycin at 50 µg/ml, carbenicillin at 50 or 100 µg/ml, and tetracycline at 2 µg/ml. X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside; Sigma Chemical Co., St. Louis, Mo.) was included in the medium at 40 µg/ml to monitor the production of -galactosidase.

DNA manipulations. Plasmids were isolated by an alkaline lysis method (40). Cloning followed standard recombinant DNA techniques (40). Restriction digestions were carried out in accordance with the instructions of the manufacturers, and digestion products were separated by electrophoresis in agarose gels with Tris-borate-EDTA buffer (40). DNA fragments were recovered from agarose gels by using GenElute spin columns (Supelco Inc., Bellefonte, Pa.). Plasmids were introduced into E. coli by CaCl₂-mediated transformation (40) and into A. tumefaciens by S17-1-mediated mating (43) or by electroporation (9).

Genomic bank construction. Cosmid pZL1 was constructed by cloning the cos site from pCP13 (15) as a 1.7-kb BglII fragment into pJB3 (6). Genomic DNA was digested with Sau3AI to give an average fragment size of 40 kb and ligated to pZL1 that had been digested with BamHI and treated with alkaline phosphatase. The ligation mixture was packaged into phage  by using the Packagene system (Promega Corporation, Madison, Wis.) and transduced into E. coli LE392.

Southern hybridization. Genomic DNA was prepared from overnight cultures of Agrobacterium spp. as described previously (18). After digestion with restriction endonuclease, DNA fragments were separated on 0.7% agarose gels and transferred to a nylon membrane. DNA probes were randomly labeled by using the Genius digoxigenin kit (Boehringer GmbH, Mannheim, Germany). Hybridization, washing, and detection followed protocols provided by the manufacturer. Hybridization and washing were performed under conditions of medium stringency.

Tn3HoHo1 mutagenesis. The insert in clone pDLE4.5 (Table 1) was mutagenized with Tn3HoHo1 as described previously (46). Sites and orientations of the insertions were determined by restriction mapping. Each mutant was tested for tetracycline resistance on LB medium containing this antibiotic.

DNA sequence analysis. DNA fragments containing the tet region were sequenced by use of the chain termination method (41) by the Genetic Engineering Facility at the University of Illinois. DNA sequences were analyzed by using DNA Strider (32). The BLAST (4) protocols were used to search the GenBank database for related sequences. The GAP subroutine of the GCG program (Genetics Computer Group, Madison, Wis.) was used to compare sequences for similarity.

Isolation of tetracycline-resistant mutants. Agrobacterium spp. were grown in LB liquid medium to saturation. A 200-µl volume of culture from each strain was spread onto NA medium containing tetracycline at 5 or 10 µg/ml. The capacity to give tetracycline-resistant mutants was assessed by the appearance of colonies on the plates after 72 h of incubation at 28°C.

Gene expression assays. E. coli and A. tumefaciens strains were grown in A and AB-mannitol media, respectively. Overnight cultures were washed and diluted 40-fold in the same respective media, and incubation was continued until the optical density at 600 nm (OD₆₀₀) reached about 0.7 for E. coli and 0.3 for A. tumefaciens. When needed, cultures were divided, and inducers (arabinose or tetracycline) were added to one subculture at the concentrations specified in Results. -Galactosidase activity was determined quantitatively as described previously (33). -Glucuronidase activity was measured as described previously (13, 24, 49). The activity of this enzyme was calculated by using the Miller formula (33), with OD₄₂₀ replaced by OD₄₁₅. All enzymatic activities were expressed as units per 10⁹ CFU.

Induction assays. The ability of tetracycline, its derivatives, and related compounds to induce the tet system of strain C58 (tet_C58 system) was assessed two ways. In the first, reporter strains were inoculated onto medium containing X-Gal and the compound being tested. The appearance of blue colonies signalled the induction of the reporter gene. In the second, reporter strains were grown in 30 ml of liquid medium with appropriate antibiotics to saturation and mixed with 50 ml of warm 0.8% agar with X-Gal, and 6-ml volumes were layered over a base of LB or AB medium in 100-mm-diameter petri dishes. Discs of Whatman 3MM paper (0.5-cm diameter), onto which a few crystals of the compound to be tested were sprinkled, were laid on the surface of the soft agar. Dissolution and diffusion of the chemical give a concentration gradient around the disc. The plates were incubated at 28°C for A. tumefaciens reporter strains and at 37°C for E. coli reporter strains and examined at intervals over a 3-day period. Susceptibility to a given compound was indicated by a zone of growth inhibition around the disc. Induction of the reporter by the compound was indicated by a blue zone around the disc or, in cases where the compound also was inhibitory, by a blue zone in the area of growth at the edge of the zone of inhibition.

Nucleotide sequence accession number. DNA sequences of wild-type tet_C58 were deposited in the GenBank database under accession no. AF090987.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Cloning a tetracycline resistance determinant from A. tumefaciens C58. Plating strain C58 onto medium containing tetracycline at concentrations ranging from 0.5 to 20 µg/ml results in the appearance at a high frequency of mutants resistant to this antibiotic (Fig. 1A and data not shown). However, we observed that a C58 mutant with a Tn5 insertion in a gene homologous to rnd (30) yielded tetracycline-resistant mutants that grew much more slowly on medium containing the antibiotic (Fig. 1B). Since a miaA mutant of E. coli does not express Tet(M)-mediated tetracycline resistance (8) and both rnd and miaA genes encode products involved in tRNA modification, we reasoned that strain C58 contains an active tetracycline resistance element that functions in the rnd⁺ but not in the rnd mutant background. To test this, a genomic library of NT1TcR1, a tetracycline-resistant mutant of the otherwise-wild-type strain NT1, was constructed in the broad-host-range cosmid vector pZL1 (Table 1). This bank, in E. coli LE392, consists of 856 clones containing cosmids with an average insert size of 42 kb. The bank was screened on LB medium containing tetracycline, and one cosmid that confers resistance to this antibiotic on its E. coli host was isolated. This clone, named pZLT1, also conferred resistance to high levels of this antibiotic on several Agrobacterium strains, including 15955, R10, and K84 (data not shown).

View larger version (92K): [in this window] [in a new window]   FIG. 1.   A mutation in a tRNA processing gene in NTM7 (29) affects expression of resistance in spontaneous tetracycline-resistant mutants of NT1. Approximately 108 cells of strains NT1 (A) and NTM7 (B) were spread on NA plates supplemented with tetracycline at 5 µg/ml. The plates were incubated for 3 (A) or 6 (B) days at 28°C. Note that the tetracycline-resistant mutants of NTM7 arising on the plate grow considerably more slowly than do those of strain NT1 on medium containing the antibiotic.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Genetic organization of the tet locus from the tetracycline-resistant and wild-type genomes of A. tumefaciens C58. A restriction map and the locations of the independent Tn3HoHo1 insertions, identified in clone pDLE4.5, are shown. The response of each mutant to tetracycline was assessed as described in Materials and Methods and is expressed as resistant to the antibiotic (+) or susceptible to the antibiotic (). The horizontal crossbar capping each insertion site indicates the direction of transcription of the lacZ reporter of Tn3HoHo1 as determined by restriction mapping. -Galactosidase activity was assessed on medium containing X-Gal: the presence of blue colonies (+) or white colonies () is indicated. The IS426 element is indicated by the hatched portion of the bar. S, SmaI; E, EcoRI; K, KpnI; H, HindIII; Tc, tetracycline.

DNA sequence analysis. DNA fragments corresponding to the region defined by the Tn3HoHo1 insertion were subcloned and subjected to sequence analysis. BLAST searches revealed that one such EcoRI-HindIII fragment from plasmid pZLHE1.6 (Table 1) contained a portion of a tetR(A) homologue. As sequencing continued, however, this gene was abruptly disrupted by a totally different DNA sequence identical to the sequence of IS426, an IS element identified from A. tumefaciens T37 (50). Apparently, the 4.5-kb EcoRI fragment from the mutant contains an incomplete tet locus; insertion of IS426 introduced an EcoRI site which divided this region into two fragments when this enzyme was used for cloning. To obtain the complete sequence of the mutant version, the adjacent 4.9-kb EcoRI fragment was cloned into pBluescript SK(+) from pZLT1 to generate pZLE4.8 (Table 1). Analysis of the insert identified sequences corresponding to the remainder of IS426 followed by the 3' end of the tetR gene of strain C58 (tetR_C58).

View larger version (88K): [in this window] [in a new window]   FIG. 3.   Relatedness of TetA and TetR from strain C58 to the gene products of tetA and tetR from RP4. Alignments between the predicted amino acid sequence of TetAC58 and TetARP4 (A) and of TetRC58 and TetRRP4 (B) were performed by using the GAP algorithm from the GCG package as described in Materials and Methods. Identical amino acids are shown as white letters on a black background, and conserved amino acids are shown as black letters on a shaded background.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Organization of the intergenic regulatory regions from several related tet systems. (A) Locations of putative promoter and operator sequences. (B) Comparison of the DNA sequences of operators from the tet systems of C58, RP4, and Tn1721. Inverted repeat sequences are boxed. The transcriptional directions of the tetA and tetR genes are indicated by arrows. A-10 and A-35, the 10 and 35 sites for the tetA promoters; R-10 and R-35, the 10 and 35 sites for the tetR promoters; OC58, putative operator of tetC58; OL and OR, the left and right operators, respectively, of the tet systems of Tn1721 and RP4.

Expression of tet_C58 is not induced by tetracycline or related compounds. In class A and class B tet systems, TetR represses expression of tetA and tetR, and this repression is relieved by subinhibitory levels of the antibiotic (3, 21, 23). Tetracycline does not induce the Agrobacterium tet system; as noted above, when a large number cells are spread onto medium containing this antibiotic, only a very small fraction of the cells grow to produce colonies. Moreover, of those such colonies tested, all now are constitutively resistant to tetracycline (data not shown). To study the regulation of the tetA and tetR genes of Agrobacterium spp., a 66-bp fragment of the intergenic region, which comprises sequence between the putative ribosome binding sites for the tetA_C58 and tetR_C58 genes, was cloned into the divergent promoter detection vector pRG970b (13) to generate pZLOP1. In this construct, tetA_C58 and tetR_C58 are transcriptionally fused to the lacZ and uidA genes, respectively, of the vector. In addition, the tetR_C58 gene was cloned as a NcoI fragment from pZLT2 into the expression vector pBAD22 (19) to generate pBetR. This construct allows us to regulate the expression of tetR_C58 with arabinose. To express tetR_C58 in Agrobacterium spp., this gene was excised from pBetR as an EcoRI-BamHI fragment and cloned into pDBL4 (Table 1), a broad-host-range expression vector containing the arabinose promoter and downstream transcriptional terminator from pBAD22 cloned into pBBRMCS-2 (27), to construct pDBL4-tetR. Expression of tetR_C58 in these two constructs is inducible in A. tumefaciens and E. coli by addition of arabinose to the culture medium at a final concentration of 0.4%.

Cross-operator recognition by TetR_C58 and TetR_RP4. By sequence analysis, the putative 15-bp operator of tet_C58 is identical to those of other tet systems (Fig. 4B). Furthermore, the sequence from residues 26 to 47 of TetR_C58 is virtually identical to that of the N-terminal helix-turn-helix domain of several related TetR proteins, including TetR_RP4, TetR_Tn10, and TetR_Tn1721 (Fig. 3B and data not shown). Given these similarities, we determined whether TetR from A. tumefaciens and from RP4 will recognize the operators of the noncognate homologues. Strains DH5(pRK415K, pBetR) and DH5(pRK415K, pZLOP1) were constructed for this study. Plasmid pRK415K carries the RP4 tetracycline resistance gene unit, and expression of this determinant is inducible by tetracycline (14, 25). If TetR_C58 can repress the expression of tetA_RP4, upon induction of tetR_C58, strain DH5(pRK415K, pBetR) should fail to grow on medium containing tetracycline. As shown in Table 3, this strain grows well on LB medium with tetracycline at a concentration of 10 µg/ml. However, when the expression of tetR_C58 on pBetR was induced by arabinose, this strain was unable to grow on the same medium. These results indicate that TetR_C58 can repress at the tetA_RP4 promoter and that this repression is not responsive to tetracycline. To determine if TetR_RP4 can repress tetA_C58, the expression of the tetA_C58::lacZ reporter was examined in strain DH5(pRK415K, pZLOP1). Results from this test indicated that TetR_RP4 from pRK415K indeed repressed tetA_C58 expression, and like the repressive effect on its cognate tetA, the repression was responsive to tetracycline (Table 3). Thus, TetR_RP4 can recognize and repress expression from the promoter of the A. tumefaciens tet system. Furthermore, unlike the tet_C58 system, this repression of tetA_C58 by TetR_RP4 is relieved by tetracycline.

Nature of the tetracycline-resistant mutants. Our data indicate that a tet unit in the genome of strain C58 accounts for the appearance of tetracycline-resistant mutants when this strain is grown in the presence of this antibiotic. In the particular mutant used in this study, the tetracycline resistance phenotype arose from the transposition of IS426 into tetR_C58. Since tetracycline does not induce the expression of tetA_C58, we were interested in determining whether all tetracycline-resistant mutants were caused by the same type of mutation. To examine this, genomic DNA from eight independent tetracycline-resistant mutants was probed with a DNA fragment containing the tet_C58 genes. These eight mutants grouped into four types based on the size of the hybridizing fragment (data not shown). One class, represented by three mutants, contained a fragment indistinguishable in size from that of the wild type. In the second, two of the eight mutants gave a fragment indistinguishable in size from that of NT1TcR1, the original mutant from which the tet locus was cloned. The remaining three mutants contained two restriction fragment length polymorphism (RFLP) patterns different from that of the wild type and of NT1TcR1 (data not shown).

Surveying for the presence of the tet locus in the genomes of Agrobacterium strains. We examined a collection of wild-type isolates of Agrobacterium spp. for the tet locus by Southern analysis using a probe containing part of tetA and most of tetR. We also examined some commonly used derivatives of strain C58. Of those not derived from C58, only strain T37 contains DNA with detectable relatedness to the tet locus from strain C58 (Table 4). Moreover, the probe hybridized to an EcoRI fragment of the same size in the two strains (data not shown). Consistent with these results, of the 26 strains tested, only T37 and those derived from C58 gave rise to tetracycline-resistant mutants (Table 4). The probe hybridized with all derivatives of strain C58 tested, including strain UIA5, which lacks both pTiC58 and the 450-kb catabolic plasmid pAtC58 (Table 4). Consistent with this, UIA5 gives rise to mutants resistant to tetracycline.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

We report here the characterization of a tet system from A. tumefaciens C58. This active resistance system accounts for the long-known observation that C58 and its derivatives give rise to tetracycline-resistant mutants at a high frequency.

This resistance determinant is structurally similar to that of tet systems found in plasmids and transposons from E. coli and other gram-negative bacteria, with the highest relatedness being to the class A tetracycline resistance gene system from Tn1721. The tet_C58 system confers resistance to tetracycline and derivatives such as chlortetracycline and oxytetracycline but not to minocycline. This pattern of resistance suggests that tet_C58 does not belong to the Tet(S) class of tetracycline resistance genes, which confers resistance to minocycline as well as to tetracycline, chlortetracycline, and oxytetracycline (10).

Like other class A systems, tet_C58 is organized as two genes, a regulatory gene and a gene coding for an efflux pump, transcribed divergently from an intergenic region. Potential promoter elements for both genes are overlapped by a single putative operator composed of a 15-bp perfect IR element. Our genetic analysis indicates that this intergenic region contains all of the cis-acting information required for the regulated expression of these two genes. The DNA sequence of the putative operator is identical to that of operators from other tet systems. However, the promoter region of tet_C58 contains only one repeat while the promoter regions of the tet determinants of RP4 and Tn1721 each contain two copies of the operators (Fig. 4B).

Heterologous repressor-operator recognition occurs between different classes of tet systems, even those with dissimilar operators (26). The high degree of sequence similarity between operators from the tet_C58 and tet_RP4 systems suggested that TetR proteins from these two systems could recognize the noncognate operator. This proved to be the case: TetR of RP4 repressed the tet system of C58 and TetR of C58 repressed tetracycline resistance conferred by the RP4 tet system. The capacity of TetR_RP4 to repress expression of the tetA_C58::lacZ fusion suggests that the 15-bp IR is the operator and indicates that a single copy of this element is sufficient for regulation of both genes.

The expression of almost all tetracycline resistance gene systems examined to date is under strict control (29, 45). Among these, repression of the Tet(B) class by TetR is the best understood (21, 45). In the absence of tetracycline, the repressors bind to operators overlapping the promoters of both the tetA and tetR genes (21). Binding of tetracycline to TetR(B) induces a conformational change that leads to release from the operators and concomitant expression of tetA (22, 34). Tetracycline-inducible TetR-mediated regulation of other tet systems is believed to function by a similar mechanism (21). Two lines of evidence indicate that tetracycline does not induce the Agrobacterium tet system. First, when cells were spread onto medium containing tetracycline at concentrations ranging from 10 to 1 µg/ml, only a small portion of the cells grew into colonies (data not shown). Moreover, those that did grow now expressed constitutive resistance to tetracycline. If tetracycline functioned as the inducer for tet_C58, virtually all of the cells should be resistant to this antibiotic. Furthermore, E. coli and Agrobacterium strains harboring pZLE8.5 (Table 1), which carries the wild-type tet_C58 locus, are sensitive to tetracycline, even at concentrations to which tet_RP4 confers resistance (data not shown). Second, while the tetA_C58::lacZ and the tetR_C58::uidA reporter fusions were expressed constitutively in cells lacking TetR_C58, the expression of both fusions was depressed considerably in cells harboring tetR_C58. Furthermore, neither reporter was inducible by tetracycline. Repression of tetA_RP4 by TetR_C58 is not relieved by tetracycline. On the other hand, repression of tet_C58 by TetR_RP4 does respond to tetracycline. These two sets of results indicate that the failure of the tet system of C58 to respond to tetracycline is a function of TetR and not of the tet operator. Mutants of the related repressor from Tn10 that do not respond to tetracycline have been isolated and characterized. These noninducible mutants repress the expression of tetA_Tn10::lacZ fusions by binding to the operators even in the presence of tetracycline (5, 20, 34). We propose that TetR_C58 represents such a noninducible form of TetR. Clearly, this repressor can bind DNA. Why it no longer reacts to tetracycline remains to be determined. We considered the possibility that tetracycline is not the true ligand for TetR_C58. However, none of the 25 compounds we tested induced the system. Certainly, this was not an exhaustive survey. But on balance, that the locus of strain C58 can be regulated by TetR_RP4 in response to tetracycline strongly suggests that this gene unit once was responsive to this antibiotic.

The interchangeability with respect to operator recognition exhibited by the two TetR proteins could explain another observation concerning tetracycline resistance in strain C58. Derivatives of this A. tumefaciens strain harboring RP4 and vectors carrying tet_RP4 express only low level resistance to tetracycline. We propose that TetR_C58 is partially dominant over TetR_RP4. Thus, since TetR_C58 does not respond to tetracycline, the tet_RP4 determinant is not fully expressed in its A. tumefaciens host.

Expression of tetracycline resistance in C58 apparently occurs only following a mutation affecting regulation. These mutations can be of several types; in the particular mutant used in this study, the tetracycline resistance phenotype resulted from the insertion of IS426 in tetR_C58. In genetic analysis carried out with C58 and its derivatives, this element is commonly associated with phenotypes in which the selection gives derepressed mutants (17). However, diverse RFLP patterns in Southern analysis of eight independent mutants indicated that other types of mutations can result in the tetracycline resistance phenotype. While mutants with a pattern indistinguishable from that of NT1TcR1 may have arisen from IS426 transposition, those with a pattern similar to that of the wild type could represent point mutations or very small deletions or insertions in the tetR_C58 gene. They also could represent operator-constitutive mutations that no longer allow TetR_C58 to bind at the promoter.

Among the agrobacteria we examined, only strain T37 contains DNA with detectable homology to the tet locus from C58. Furthermore, only C58 and T37 give rise to tetracycline-resistant mutants (Table 4). These two strains were isolated from different parts of the United States (42). Moreover, although strain C58 induces unorganized tumors, strain T37 causes teratomas. Both strains contain a nopaline/agrocinopine-type Ti plasmid, but RFLP analysis indicates that these two elements have diverged considerably from each other (42). Our survey also shows that generation of tetracycline-resistant mutants is associated with the presence of a tet_C58 homologue in the genome of the particular isolate; those lacking this element do not yield mutants at a detectable level. The presence of the tet locus in strain UIA5, a derivative of C58 lacking both pTiC58 and pAtC58, indicates that this resistance determinant is associated with one of the two chromosomes of strain C58 (1). Interestingly, this is not the only antibiotic resistance determinant in strain C58. This strain, which also mutates to resistance to chloramphenicol at a high frequency, contains a gene coding for an atypical chloramphenicol acetyltransferase (47). Whether this cat gene is present in strain T37 or any other isolate of Agrobacterium spp. is not known.