Nucleotide sequence of the gene, protein purification and characterization of the pSC101-encoded tetracycline resistance-gene-repressor

The nucleotide sequence of the pSC101-encoded tetracycline repressor gene (tetR) was confirmed. The deduced amino acid sequence is compared to that of other repressor proteins. To overproduce the repressor protein, tetR was placed under the control of bacteriophage lambda promoter pL. Tet repressor protein was purified to homogeneity and shown to bind specifically to two tet operators and also to tetracycline (Tc). The inducer function of Tc is demonstrated by the loss of the specific binding between the tet operator DNA and the Tet repressor-Tc complex.     

The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100

We determined the complete nucleotide sequence of the 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum LP-6 which encodes streptomycin, spectinomycin, and tetracycline resistance. The antibiotic resistance determinant of pTET3 comprises an intI1-like gene, which was truncated by the insertion sequence IS6100, and the novel aminoglycoside adenyltransferase gene cassette aadA9. The deduced AADA9 protein showed 61% identity and 71% similarity to AADA6 of integron In51 from Pseudomonas aeruginosa. In addition, pTET3 carries the novel repressor-regulated tetracycline resistance determinant Tet 33 which revealed amino acid sequence homology to group 1 tetracycline efflux systems. The highest level of similarity was observed to the tetracycline efflux protein TetA(Z) from the C. glutamicum plasmid pAG1 with 65% identical and 77% similar amino acids. Each antibiotic resistance region of pTET3 is flanked by identical copies of the widespread insertion sequence IS6100 initially identified in Mycobacterium fortuitum. Transposition assays with a cloned copy of IS6100 revealed that this element is transpositionally active in C. glutamicum. These data suggest a central role of IS6100 in the evolutionary history of pTET3 by mediating the cointegrative assembly of resistance gene-carrying DNA segments.     

Nucleotide sequence analysis of the class G tetracycline resistance determinant from Vibrio anguillarum.

The nucleotide sequence of the class G tetracycline resistance determinant previously isolated from Vibrio anguillarum has been determined. Two open reading frames of divergent polarity were identified. A resistance gene (tet A) encodes a protein of 393 amino acid residues (deduced molecular mass of 40.9 kDa), and a repressor gene (tet R) encodes a protein consisting of 210 amino acids with a calculated molecular mass of 23.6 kDa. Based on the deduced amino acid sequences, the proteins of tet A(G) and tet R(G) are about 60% homologous with those of RP1/Tn1721 (class A) and pSC101/pBR322 (class C), and about 50% homologous with Tn10 (class B). The relationship of the tet (G) sequence to five known tetracycline resistance determinants (class A to E) is discussed.     

Similarities between the regulatory sequences of the unrelated tetracycline genes of pBR322 and Tn10

The regulatory regions of the tetracycline genes present in pBR322 (pSC101) and in the transposon Tn10 are compared. They show a low degree of nucleotide sequence similarity but a high level of structure similarity. Furthermore, analyses of RNAs transcribed in the opposite direction of the pBR322 tet gene show that there are two mRNA initiation sites separated by 29 nucleotides. This suggests the existence of two promoters for the tet repressor gene in Tn10. These features reveal a strong resemblance of the mode of regulation between the tet operons of Tn10 and pSC101.     

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems

The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.     

Novel tetracycline resistance determinant isolated from an environmental strain of Serratia marcescens

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.     

Constitutive expression of tetracycline resistance mediated by a Tn10-like element in Haemophilus parainfluenzae results from a mutation in the repressor gene.

The Tn10-like constitutively expressed tetracycline resistance determinant from a Haemophilus parainfluenzae strain was cloned in Escherichia coli. Toxicity resulting from expression on multicopy plasmids necessitated its being cloned on a low-copy plasmid vector or in cells containing the Tn10-encoded repressor. Constitutive expression of tetracycline resistance was found to result from the synthesis of a truncated inactive repressor molecule. Instead of the 23-kilodalton repressor found in other Tn10-containing strains, this determinant encoded a 14.5-kilodalton molecule. The DNA sequence of the 700-base-pair region spanning the repressor gene and promoter-operator regions of the Haemophilus determinant was identical to that of the same region of Tn10, except for the absence of a single T X A base pair in the repressor gene. This deletion leads to premature termination of the protein. Antisera to the repressor suggested that the repressor was also absent in a second independently isolated H. parainfluenzae strain bearing a Tn10-like constitutive tetracycline resistance determinant.     

Regions associated with the stable maintenance of plasmid pSC101 and its tetracycline resistance

Summary Two regions tentatively called unsA and unsR were identified on pSC101. One, unsA, corresponds to less than 650 bp of the N-terminal in the tetracycline resistance structural gene and seems to inhibit stable maintenance of pSC101. The other, unsR, is defined within the 1 kb XhoI-EcoRI region located upstream of the tetracycline resistance structural gene and is a regulatory gene clearly distinct from tetR (Unger et al. 1984); it serves as a suppressor of the unsA function.     

Computer simulations and experimental studies of gel mobility patterns for weak and strong non-cooperative protein binding to two targets on the same DNA: application to binding of tet repressor variants to multiple and single tet operator sites

A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding. While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex. The doubly occupied complex remains stable under these conditions. This phenomenon is typical for protein binding to DNA fragments with two identical sites. It results from statistical disproportionation of the singly occupied complex in the gel. The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results. Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants. The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions. Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant. Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears. This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel. It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant. On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment. The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.     

Boundaries of the pSC101 minimal replicon are conditional.

The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

A suppressor of mutations in the region adjacent to iterons of pSC101 ori.

Some single-base changes in a 14-bp region (the downstream region) adjacent to three repeated sequences (iterons) in pSC101 ori are very deleterious for replication. We isolated a host suppressor mutation for one of these mutations and found that the suppressor suppressed all the mutations tested in the downstream region. The nucleotide sequence of the suppressor revealed that the suppressor gene was identical to dksA, which encodes a multicopy suppressor of the heat shock gene dnaK.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

Intracistronic complementation of the tetracycline resistance membrane protein of Tn10.

The structural gene region for tetracycline resistance on Tn10 consists of two complementation groups, tetA and tetB (M. S. Curiale and S. B. Levy, J. Bacteriol. 151:209-215, 1982). Using a series of deletion mutants, we have determined that the tetA region is 450 to 600 base pairs long and that the tetB region, which is adjacent to tetA, is 600 to 750 base pairs long. Point mutations in either tetA or tetB affected the amount and size of the inducible inner-membrane Tet protein synthesized in Escherichia coli maxicells. Moreover, deletions in these regions led to the synthesis of an appropriately smaller Tet protein. A single tetracycline-inducible RNA of about 1,200 bases was detected that was homologous with the tetracycline resistance structural gene region. These results indicate that the tetA and tetB complementation regions represent two parts of a single gene encoding two domains of the tetracycline resistance protein Tet.     

Altered tetracycline resistance in pSC101 recombinant plasmids

Summary Investigation of tetracycline resistance genetically determined by the plasmid pSC101 and several recombinants of pSC101 containing of EcoRI generated DNA fragments inserted at the EcoRI site has revealed significant differences in the phenotypic expression of that resistance. The altered phenotypes of the recombinant plasmids may be the result of the location of the EcoRI site of pSC101, which has been determined to be near the genetic elements involved with tetracycline resistance.     

Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.