Tet Promoter Research Articles

Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1

A putative esterase gene ( est) from Acinetobacter calcoaceticus RAG-1 has been cloned into Escherichia coli. Esterase-positive clones exhibited high levels of esterase activity even in intact cells. In addition, expression of the est gene conferred on E. coli the ability to grow on simple triglycerides such as triacetin (TAC). The original esterase-positive plasmid pRA17 carried a 2.2-kb insert from a partial MboI digest of RAG-1 DNA, which gave a single band with RAG-1 DNA following Southern hybridization. By subcloning and sequencing the est gene was found to contain a sequence of 870 bp which could be translated to yield a protein of M r 32700. In support of the sequencing results was the finding that when pRA17 was expressed in minicells, a unique peptide of M r 32500 was identified. This peptide was not found in minicells transformed with esterase-negative plasmids, such as pRA176, which contained a Tn 5 insertion in the est gene. The fact that the production of active esterase depended on the orientation of the est gene within the vector suggested that transcription proceeded from the tet promoter in pBR322.

The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting inEcherichia coliDNA topoisomerase I deletion mutant

Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed.

Transcription regulation in vitro by an E. coli promoter containing a DNA cruciform in the '-35' region.

A promoter with the potential to adopt a 50 basepair (bp) cruciform spanning from -19 to -69 has been constructed in the plasmid pBR322 tetracycline resistance gene (tet) by forming an inverted repeat from '-35' sequences. Compared to a control promoter, the sequence of this cruciform promoter differs only by a 22 bp insertion between -48 and -69, upstream from the usual location of promoter sequences. The cruciform is extruded in a supercoil-dependent manner, and transcription from this promoter in vitro by RNA polymerase decreases as the negative supercoil density of the plasmid DNA increases. In contrast, transcription from the control promoter increases with negative supercoiling. Thus, DNA secondary structure in the '-35' region can affect promoter-polymerase interaction. The tet promoter cruciform also influences expression of the pBR322 beta-lactamase gene (bla). This apparently results when extrusion of the cruciform reduces the superhelicity of the plasmid molecule to a level that is below the optimum for expression from the bla promoter, illustrating one mechanism for how DNA secondary structure may effect action-at-a-distance. Transcription from both promoters in vivo does not differ from controls, suggesting that this cruciform is not generated to a significant extent intracellularly, most probably as a result of the slow kinetics of extrusion.

Identification, cloning, and expression of bolA, an ftsZ-dependent morphogene of Escherichia coli.

A newly found morphogene of Escherichia coli, bolA, mapping at min 10 of the genetic map, was cloned in a 7.2-kilobase BamHI fragment and identified by its ability to produce osmotically stable spherical cells when overexpressed. This gene codes for a polypeptide of 13 kilodaltons. Overexpression of bolA+ was achieved in low-copy-number vectors with operon fusions to the tet and lac promoters, indicating a clockwise direction of transcription. While no modification of any of the penicillin-binding proteins was observed, morphological effects due to overexpression of bolA+ were shown to be dependent on the presence of an active ftsZ gene product. Our results suggest the existence of a mechanism mediated by FtsZ for modifying the conformation of nascent murein in the early steps of septum formation.

Transposon Tn5 target specificity: preference for insertion at G/C pairs.

The procaryotic transposon Tn5 inserts into many different sites within a single gene, but some sites (hotspots) are targeted repeatedly. Hotspots are not closely related in sequence, but most have G/C pairs at the ends of the nine base pairs duplicated by Tn5 insertion. In pBR322, the major hotspot coincides with the "-10 region" of the tet promoter. We mutated the G/C pairs at this hotspot and assayed for insertion into hotspot I, resistance to tetracycline, and plasmid supercoiling. We found that changing the G/C pairs to A/T pairs reduced the frequency of insertion into the hotspot by at least fivefold. The reduction in hotspot use caused by these G/C to A/T changes was not attributable to changes in plasmid supercoiling or tet promoter strength.

Mutations in the Tn 10 tet represser that interfere with induction : Location of the tetracycline-binding domain

Tetracycline induces transcription of the Tn 10 tetracycline resistance gene ( tetA) by binding to the tet represser, thereby reducing the repressor's affinity for two operator sites that overlap the tet promoters. We characterized mutations in the tet repressor ( tetR s mutations) that interfere with induction of tetA expression. The mutations were isolated on multicopy Tn 10 tet plasmids by selecting for resistance to the inducer 5a,6-anhydrotetracycline. Under these conditions, maximal induction of tetA expression inhibits the growth of Escherichia coli K-12. DNA sequence analysis of 25 spontaneous tetR s mutations identified amino acid changes at 13 different positions clustered near the middle of the 207 amino acid residue sequence of tet repressor. This region (residues 64 to 107) is distinct from the bihelical DNA-binding motif of tet repressor (residues 26 to 47). The capacity of tetR s repressors to bind tet operator DNA and to respond to inducer was examined in vivo in tetA-lacZ fusion strains. In three cases, the capacity of tetR s repressors to bind tetracycline was examined in vitro using cell extracts enriched in repressor. Mutations 64Y (His64→Tyr) and 82H (Asn82→His) reduce the repressor's affinity for tetracycline more than 1000-fold and more than 100-fold, respectively, suggesting that His64 and Asn82 may be part of the inducerbinding site or directly involved in maintaining its conformation. Mutation 1031 (Thr103→Ile) reduces the repressor's affinity for tetracycline less than tenfold, yet it interferes with induction to a greater extent than either 64Y or 82H, suggesting that 1031 may also reduce the repressor's capacity to undergo a conformational change required for induction. The properties of tetR s mutants suggest that the region of amino acid residues 64 to 107 is involved in inducer binding and in signalling between the inducer-binding and operator-binding domains of the repressor.

Differential regulation of the Tn10-encoded tetracycline resistance genes tetA and tetR by the tandem tet operators O1 and O2.

The Tn10-encoded tet transcriptional control sequence consists of bidirectional, overlapping promoters which are superimposed by a tandem operator arrangement. Three mutations have been constructed by oligonucleotide-directed mutagenesis which reduce binding of Tet repressor to either one or both of the tandem tet operators 1000-fold as determined by DNAseI footprinting in vitro. The affinity of Tet repressor for mutant tet operators is not affected by the presence of an already occupied neighbouring wild-type operator, indicating little or no cooperativity. The regulation of the divergently oriented tet promoters PA and PR by the tet operators O1 and O2 and Tet repressor provided in trans is determined using transcriptional fusions of the promoters to lacZ and galK indicator genes located with different polarity on the same plasmid. The results demonstrate that expression of the resistance gene tetA is regulated by Tet repressor bound to either O1 or O2. Expression of the repressor gene tetR is only marginally reduced when Tet repressor is bound to O2. This result is discussed with respect to the double promoter structure found for PR. Occupation of O1 with Tet repressor turns off transcription from PR completely. The implications of these findings on the establishment of tetracycline resistance upon induction are discussed.

Leakage of Cellulomonas fimi cellulases from Escherichia coli

Transcription of the cenA gene of Cellulomonas fimi, encoding endoglucanase A (EngA), from the lac promoter of pUC18 rather than from the tet promoter of pBR322 increased the level of expression of the gene in Escherichia coli some 800-fold. Most of the EngA activity was exported to the periplasm, even though the EngA leader peptide in the construct was 9 amino acids longer than usual. The increased level of expression of cenA resulted in the temperature-dependent leakage of periplasmic proteins, including EngA and β-lactamase, into the culture medium. In contrast to C. fimi, which processes pro-EngA at a single site, E. coli processed it at two distinct sites 3 amino acids apart. Increasing the level of expression of cex, encoding an exoglucanase (Exg) of C. fimi, to a comparable extent also led to the temperature-dependent leakage of Exg into the culture medium.

Leakage ofCellulomonas fimicellulases fromEscherichia coli

Transcription of the cenA gene of Cellulomonas fimi, encoding endoglucanase A (EngA), from the lac promoter of pUC18 rather than from the tet promoter of pBR322 increased the level of expression of the gene in Escherichia coli some 800-fold. Most of the EngA activity was exported to the periplasm, even though the EngA leader peptide in the construct was 9 amino acids longer than usual. The increased level of expression of cenA resulted in the temperature-dependent leakage of periplasmic proteins, including EngA and β-lactamase, into the culture medium. In contrast to C. fimi, which processes pro-EngA at a single site, E. coli processed it at two distinct sites 3 amino acids apart. Increasing the level of expression of cex, encoding an exoglucanase (Exg) of C. fimi, to a comparable extent also led to the temperature-dependent leakage of Exg into the culture medium.

Transcription initiation at the tet promoter and effect of mutations.

We have identified the startpoint for transcription in vitro of the tetracycline resistance gene (tet) of pBR322 and several deletion and insertion mutations which alter tet promoter structure. Tetracycline resistance in host bacteria correlates qualitatively with the efficiency of DNA fragments from these plasmids to promote tet transcription in vitro. Only in active promoters could we find by computer analysis promoter structures in which the -10 and -35 sequences and the relative spacing of the two regions agree with consensus sequence determinants. These data support the current model of the E. coli promoter sequence. Two promoter mutants gave heterogeneous 5' termini with additional A residues not encoded by the DNA sequence.

Construction and characterization of multicopy expression-vectors in Streptomyces spp.

A strong transcriptional signal previously cloned from the Streptomyces griseus genome in S. lividans was subcloned and its nucleotide sequence was determined. Upstream of the transcriptional start point which was determined by high-resolution S1 nuclease mapping, -35 (5'-TTGCCG-3') and -10 (5'-TAGCGT-3') sequences, separated by 18 nucleotides, were present. By replacing the tet promoter of pBR322 with the Streptomyces promoter, no expression of the tet gene was observed in Escherichia coli cells. The result suggests that notwithstanding a similarity to the E. coli -35 and -10 sequences, the Streptomyces promoter is not functional in E. coli. The strong promoter was inserted in multi-copy and wide host range plasmids pIJ702 and pKS11, resulting in the pSEV series of expression-vectors with several unique restriction endonuclease cleavage sites downstream of the promoter for cloning of foreign genes. The extremely heat-stable malate dehydrogenase of Thermus flavus, when its coding sequence with a ribosome-binding site was located downstream of the strong promoter in pSEV2, was produced in large quantities in S. lividans throughout growth. When an extracellular cellulase from Bacillus subtilis was expressed in a cellulase-negative S. lividans strain, virtually all of the cellulase activity was found in the culture supernatant.

A transcriptional terminator sequence in the prokaryotic transposable element IS1.

The prokaryotic transposable element IS1 is known to exert a strong polar effect upon integration into an operon. To elucidate this polar effect, we constructed a plasmid which has an IS1 integrated between the 5' half of the tet gene for tetracycline resistance and the cat structural gene for chloramphenicol resistance. The cat gene is expressed by the tet promoter and the presence of IS1 in orientation I, in which the IS1 transposase genes insA and insB are in the same orientation as the cat gene, reduced the cat expression. By introducing deletions or insertions within the IS1 sequence, we were able to map a rho-dependent terminator TIS1A between the insA and insB genes. Translational interruption between these ins genes is important for TIS1A to be an active terminator.

Cotranscription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes. Fine-structure mapping of the lsp internal promoter.

We have applied the technique of Northern blotting hybridization analysis to examine the transcription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes. RNA samples from the wild-type strain CS412 and CS412 containing the plasmids pMT521 and pSYC890 were examined with ileS- and lsp-specific 32P-labeled probes. pMT521 is a pBR322 derivative into which a chromosomal DNA fragment containing the ileS and lsp genes has been cloned downstream of the pBR322 tet promoter. pSYC890 is a derivative of pMT521 which lacks the tet promoter, but nevertheless is active in lsp mRNA transcription due to the presence of a weak lsp internal promoter located in the distal portion of the upstream ileS DNA, RNA from CS412(pMT521) exhibited a high level of a 5500-nucleotide ileS-lsp cotranscript that hybridized to both probes. RNA from CS412(pSYC890) contained a high level of low molecular weight lsp-specific transcripts from the lsp internal promoter. However, only high molecular weight (5000-6500 nucleotides) cotranscripts were detected in RNA from CS412 by Northern blotting analysis. A trace level of lsp-specific mRNA was detected in CS412 by S1 nuclease mapping. The 5'-end of this transcript was mapped using RNA from CS412(pSYC890). The mRNA begins 192/193 base pairs upstream of the lsp translation initiation codon at a site within ileS DNA that codes for the aspartate residue located 61 amino acids from the carboxyl-terminal of ILES. In conclusion, most of the transcription of lsp in wild-type E. coli originates from upstream of ileS and yields ileS-lsp cotranscripts.

The expression of the Escherichia coli lacZ gene in Streptomyces.

The Escherichia coli lacZ gene was stably introduced into phi C31-based phage cloning vectors in Streptomyces lividans. However, lacZ could not be stably introduced into S. lividans on the plasmid vectors pIJ702 or pIJ41. Studies of the expression of lacZ in S. lividans and S. coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous beta-galactosidase activity was low or absent. Plaques and lysogens of phi C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained beta-galactosidase activity. Activities were markedly higher in S. lividans than in S. coelicolor. Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function. Several phi C31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived. The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S. fradiae. When phi C31 DNA fragments were inserted into KC659, at least one recombinant phage gave high beta-galactosidase activity in lytic infections, but not in lysogens. The potential usefulness of lacZ fusions in Streptomyces is discussed.

The effect of nalidixic acid on expression from related [formula omitted] promoters

The effect of the DNA gyrase inhibitor, nalidixic acid, on expression from E. coli promoters was studied using the pKO-1, galactokinase expression vector system. Expression from a series of related hybrid promoters, tet promoter variants and the trp promoter flanked by oligonucleotide blocks was measured after incubation with nalidixic acid. Expression from the pBR322 tet promoter and tet promoter mutants within the −10 region was reduced after the drug treatment. The lac UV5, trp , and tettrp promoters were essentially unaffected while the trplac and the trptet promoters were stimulated. Studies of the trp promoter flanked by upstream or downstream oligonucleotide blocks revealed similar responses to the trp promoter parent control plasmids.

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

A hybrid plasmid, pJS37, was made by combining pLSl, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJ S 140 (6.0 kb), from which pC 194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4(3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLSl. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.

In vivo and in vitro effect of mutations in tetA promoter from pSC101: insertion of poly(dA.dT) stretch in the spacer region does not inactivate the promoter

Two mutants, mapping at the HindIII site (between the consensus sequences) of the pSC101 tetA promoter, were studied: MA2 corresponds to a 4 bp deletion between positions -12 and -15; B30 bears a 44 bp insertion C(TA)21 G at the HindIII site. Both mutants were assayed in vivo (ability of the plasmid to confer resistance to tetracycline, plasmid-directed protein synthesis, S1-mapping of mRNA) and in vitro (abortive initiation assay). Compared to w.t., MA2 is a poor promoter in vivo; RNA polymerase binding, complex activation and rate of initial oligonucleotide synthesis are strongly reduced in vitro; this is in keeping with the known effects of altering the consensus elements in E. coli promoters. In contrast, B30 shows in vivo a promoter activity only slightly reduced in comparison to that of the w.t. tetA promoter; both in vivo and in vitro, the transcription start site is outside and downstream the (TA)21 stretch, 5-7 bp upstream that found in the w.t. To adjust the behaviour of B30 and the claimed consensus distance between the E. coli promoter consensus sequences, some structural modification in the (TA)21 stretch -either spontaneous or induced by RNA polymerase- can be hypothesized. Unless the (TA)21 stretch itself plays the role of a relatively good promoter, the results suggest that promoter-specific elements may be distributed along the DNA sequences over distances longer, but seldom less, than the 17 +/- 2 bp consensus distance.

Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetP A is 7 to 11 times more active than the two tetR promoters ( tetP R1 and tetP R2) combined. We characterized ten mutations in tetP A, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetP A activity in vivo. Unexpectedly, tetP A mutations reduce by two- to fourfold the combined activity in vivo of tetP R1and tetP R2, in spite of their locations outside the −35 and −10 regions of tetP R1 and tetP R2. For two tetP A mutations, the negative effect on tetP R activity was also demonstrated in tetR − tetR R- lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetP A mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.

A physical domain of herpes simplex virus ICP8 is expressed and active in Escherichia coli.

In this report, we describe a series of procedures to assay the function of fusion genes in Escherichia coli and the specific application to the carboxy-terminal third of the herpes simplex virus type 1 (HSV-1) DNA-binding protein ICP8. E. coli cells containing the cloned HSV-1 BamHI G fragment with the HSV-1 BamHI-G-V site, map unit 0.388, nearest the tet promoter in pBR322 synthesized an active product containing a portion of ICP8. The new product induced phenotypic alterations in recipient hosts that were measurable and stable yet limited to the stability of the plasmid. The corresponding cloned DNA from the characterized HSV-1 DNA-binding protein mutant tsHA1 exhibited a predictable temperature-sensitive phenotype. Screening procedures based on the loss of induction of the parental plasmid-induced phenotype in E. coli cells allowed us to select additional mutations. One of these, which conferred a phenotype different from that of tsHA1, was transferred to the viral genome by marker transfer techniques. We suggest that any mutant could be isolated in any sequence, provided that the wild-type coding sequences induce alterations in E. coli cells. The observed alterations should have relevance in determining the mode of action of the protein in its normal environment.

Orientation and expression of the cloned hemolysin gene of Pseudomonas aeruginosa

The structural gene for Pseudomonas aeruginosa hemolysin, carried on recombinant plasmid pSL2 and cloned in Escherichia coli, was analyzed by insertional and deletional mutagenesis. Expression of the hemolysin was blocked by insertion of transposon Tn5 into different locations. Two of the mutants allowed detectable synthesis of truncated hemolysin polypeptides of two different sizes and thus defined the structural gene. The location of the hemolysin gene in the recombinant plasmid, and the direction of transcription, were further established by nuclease BAL 31 digestion, and by construction of gene fusions between hemolysin and β-galactosidase. Evidently, the tet promoter contributed to the majority of the expression of cloned hemolysin gene, but the Pseudomonas promoter was present in the cloned DNA and was functional in E. coli since inactivation of the tet promoter either by Tn5 insertion or by deletion decreased synthesis of the 80-kDal hemolysin but did not fully abolish it.