tetR Promoter Research Articles

Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria

PurposeIn the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.ProceduresIn this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR.ResultsIn Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division.ConclusionsOverall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Tetanus Toxin Synthesis is Under the Control of A Complex Network of Regulatory Genes in Clostridium tetani.

Clostridium tetani produces a potent neurotoxin, the tetanus toxin (TeNT), which is responsible for an often-fatal neurological disease (tetanus) characterized by spastic paralysis. Prevention is efficiently acquired by vaccination with the TeNT toxoid, which is obtained by C. tetani fermentation and subsequent purification and chemical inactivation. C. tetani synthesizes TeNT in a regulated manner. Indeed, the TeNT gene (tent) is mainly expressed in the late exponential and early stationary growth phases. The gene tetR (tetanus regulatory gene), located immediately upstream of tent, encodes an alternative sigma factor which was previously identified as a positive regulator of tent. In addition, the genome of C. tetani encodes more than 127 putative regulators, including 30 two-component systems (TCSs). Here, we investigated the impact of 12 regulators on TeNT synthesis which were selected based on their homology with related regulatory elements involved in toxin production in other clostridial species. Among nine TCSs tested, three of them impact TeNT production, including two positive regulators that indirectly stimulate tent and tetR transcription. One negative regulator was identified that interacts with both tent and tetR promoters. Two other TCSs showed a moderate effect: one binds to the tent promoter and weakly increases the extracellular TeNT level, and another one has a weak inverse effect. In addition, CodY (control of dciA (decoyinine induced operon) Y) but not Spo0A (sporulation stage 0) or the DNA repair protein Mfd (mutation frequency decline) positively controls TeNT synthesis by interacting with the tent promoter. Moreover, we found that inorganic phosphate and carbonate are among the environmental factors that control TeNT production. Our data show that TeNT synthesis is under the control of a complex network of regulators that are largely distinct from those involved in the control of toxin production in Clostridium botulinum or Clostridium difficile.

Switching the Ligand Specificity of the Biosensor XylS from meta to para-Toluic Acid through Directed Evolution Exploiting a Dual Selection System.

The Pseudomonas putida transcriptional activator XylS induces transcription from the Pm promoter in the presence of several benzoic acid effectors, with m-toluic acid being the most effective and p-toluic acid being much less effective. To alter the effector specificity of XylS, we developed a dual selection system in Escherichia coli, which consists of (i) an artificial operon of an ampicillin resistance gene and tetR under Pm promoter control and (ii) a chloramphenicol resistance gene under tetR promoter control. This system enabled both positive selection to concentrate XylS mutants recognizing a desired ligand and negative selection to exclude undesired XylS mutants such as those recognizing undesired ligands and those that are active without effectors. Application of a random mutagenesis library of xylS to directed evolution that exploited this selection system yielded two XylS mutants that recognize p-toluic acid more effectively. Analysis of each missense mutation indicated three amino acid residues (N7, T74, and I205) important for p-toluic acid recognition. Then, a codon-randomized xylS library at these three residues was similarly screened, resulting in three XylS mutants with increased p-toluic acid-recognition specificity. Analysis of each amino acid substitution revealed that T74P attributes to both m-toluic acid sensitivity loss and subtle p-toluic acid sensitivity acquisition, and that N7R increases the overall ligand-sensitivity. Finally, the combination of these two mutations generated a desirable XylS mutant, which has a high p-toluic acid sensitivity and scarcely responds to m-toluic acid. These results demonstrate the effectiveness of the dual selection system in the directed evolution of biosensors.

Development of a Pseudomonas aeruginosa Agmatine Biosensor.

Agmatine, decarboxylated arginine, is an important intermediary in polyamine production for many prokaryotes, but serves higher functions in eukaryotes such as nitric oxide inhibition and roles in neurotransmission. Pseudomonas aeruginosa relies on the arginine decarboxylase and agmatine deiminase pathways to convert arginine into putrescine. One of the two known agmatine deiminase operons, aguBA, contains an agmatine sensitive TetR promoter controlled by AguR. We have discovered that this promoter element can produce a titratable induction of its gene products in response to agmatine, and utilized this discovery to make a luminescent agmatine biosensor in P. aeruginosa. The genome of the P. aeruginosa lab strain UCBPP-PA14 was altered to remove both its ability to synthesize or destroy agmatine, and insertion of the luminescent reporter construct allows it to produce light in proportion to the amount of exogenous agmatine applied from ~100 nM to 1mM. Furthermore it does not respond to related compounds including arginine or putrescine. To demonstrate potential applications the biosensor was used to detect agmatine in spent supernatants, to monitor the development of arginine decarboxylase over time, and to detect agmatine in the spinal cords of live mice.

An improved tetracycline-inducible expression vector for Staphylococcus aureus

The tetracycline-inducible expression vector pALC2073 allowed high level expression of the cloned sasG gene but repression by uninduced cells was leaky. The −10 box of the tetR promoter was mutated to the Bacillus subtitlis consensus, which resulted in complete repression of SasG protein expression. Anhydrotetracycline at 1.28 μg ml −1 gave the same high level of induction that was obtained with pALC2073 sasG using 160 ng ml −1 tetracycline, the highest concentration that could be used without inhibiting bacterial growth. This variant of pALC2073 thus offers almost complete repression when uninduced and high levels of expression when induced.

TheSSK2MAPKKK ofCandida albicansis required for oxidant adaptationin vitro

The Ssk2p (MAPKKK) of Candida albicans was deleted and functions assigned based on phenotyping studies. SSK2 deletion was first attempted using the UAU1 disruption method. All transformants lacking one copy of SSK2 appeared to be triploids, suggesting that the SSK2 is essential for the organism. To verify this observation, a strain was constructed in which one allele was deleted using the SAT1 flipper disruption method. The second allele was then placed under control of the on/off tetracycline-regulatable (TetR) promoter. The transcription of SSK2 was measured by reverse transcriptase-PCR and although the promoter was somewhat leaky, transcript was significantly reduced in an ssk2/TetR-SSK2 transformant (AT2) in the presence of doxycycline. Strains AT1 and AT2 constructed using the SAT1 flipper and TetR promoter method, respectively, were studied phenotypically in different growth media to determine the role of Ssk2p in morphogenesis. The mutants were also compared under on/off conditions in the presence of 1.5 M NaCl and various types of oxidants. Strain AT2 demonstrated resistance to 1.5 M NaCl in the absence of doxycycline but was inhibited by 8 mM hydrogen peroxide.

Dynamic and structural characterisation of multiple steps during complex formation between E. coli RNA polymerase and the tetR promoter from pSC101.

Kinetic, functional and structural studies of the recognition of the tetR promoter from pSC101 by E. coli RNA polymerase allowed the characterization of several steps in the specific complex formation and transcription initiation process. First, enzyme and DNA enter in a short life-time complex. An isomerization will convert this unstable complex into a closed stable one where RNA polymerase is tightly attached without establishing stable chemical contacts with the bases. In the next step, stable close contacts appear between both macromolecules involving mainly the downstream part of the promoter. A further isomerization will lead to an open complex where DNA is locally melted and the system is able to initiate transcription. This latter process is accompanied by changes in the upstream part of the promoter. Finally, in vitro transcription assays showed that the position of the major transcription start sites depends on temperature. From the reported results, it appears that the recognition event is a sequential process where different structural elements of the promoter, that can be located apart in the sequence, are involved in a concerted manner in each stage.

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.

Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant.

The construction and characterization of a promoter expression vector, lambda RS205 , is described. lambda RS205 can be used for the in vitro construction of transcriptional (operon) fusions to the lacZ gene of Escherichia coli K-12. The level of beta-galactosidase activity in lysogens of lambda RS205 fusion phages provides a quantitative measure of promoter function under single-copy conditions. The regulation of the Tn10 tetracycline resistance gene ( tetA ) and the Tn10 tet repressor gene (tetR) was examined by inserting DNA fragments that span the tetR- tetA promoter-operator region into lambda RS205 . Levels of beta-galactosidase in tetA -lacZ and tetR-lacZ fusion strains indicate that the tetA and tetR promoters are strong promoters; the tetA promoter is fourfold more active than the tetR promoter. Introduction of tetR+ plasmids into tetA -lacZ and tetR-lacZ fusion strains represses beta-galactosidase synthesis 15- to 60-fold and 6- to 15-fold, respectively. The concentration of tetracycline required to induce half-maximal beta-galactosidase synthesis in these tetR+ tet-lac strains depends on both the tetracycline resistance phenotype and the level of tetR repressor in the fusion strain. However, the induction of beta-galactosidase in isogenic tetA -lacZ and tetR-lacZ strains is coordinate. The data presented here support the current model of Tn10 tet gene organization and regulation and provide quantitative information about the regulation of tetA and tetR in vivo.

Overlapping divergent promoters control expression of Tn 10 tetracycline resistance

We have previously examined the genetic organization and regulation of the Tn10 tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for tetA, the Tn 10 tetracycline-resistance function, and for tetR, the Tn 10 tet represser, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both tetA and tetR is induced by tetracycline. Here we report the DNA sequence of the Tn 10 tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by S1 nuclease mapping of the 5′ ends of in vivo tet RNA. The tetA and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the Tn 10 tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby inhibiting transcription initiation at the tetA and tetR promoters. The Tn10 tet regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).