Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters.

For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely. This was possibly due to the low level expression of the effector gene in ovaries. In the strains developed in this study, the early promoters from L. cuprina nullo or Cochliomyia macellaria CG14427 genes were used to drive the tetracycline transactivator (tTA) expression in the early embryo. In the absence of tetracycline, tTA activates expression of the proapoptotic gene Lshid which contains a female-specific intron. Consequently, only females produce active HID protein and die at the embryo stage. Crossing the tTA-expressing driver lines with an RFPex reporter line confirmed that there was no expression of the effector gene in the ovary. These new embryonic L. cuprina transgenic sexing strains hold great promise for genetic control programs and the system reported here might also be transferable to other major calliphorid livestock pests such as the New World screwworm, Cochliomyia hominivorax.

CtcS, a MarR family regulator, regulates chlortetracycline biosynthesis.

BACKGROUND: Chlortetracycline (CTC) is one of the commercially important tetracyclines (TCs) family product and is mainly produced by Streptomyces. CTC is still in a great demand due to its broad-spectrum activity against pathogens. Engineering transcriptional control allows the cell to allocate its valuable resources towards protein production and provides an important method for the build-up of desired metabolites. Despite extensive efforts concerning transcriptional regulation for increasing the productivities of TCs, the regulatory mechanisms of the CTC biosynthesis remain poorly understood. RESULTS: In this study, the possible regulatory function of CtcS, a potential member of MarR (multiple antibiotic resistance regulator) family of transcriptional regulators in S. aureofaciens F3, was demonstrated. Knockdown of ctcS altered the transcription of several biosynthesis-related genes and reduced the production of tetracycline (TC) and CTC, without obvious effect on morphological differentiation and cell growth. Especially, CtcS directly repressed the transcription of the adjacent divergent gene ctcR (which encodes a putative TC resistance efflux protein). A CtcS-binding site was identified within the promoter region of ctcR by DNase I footprinting and an inverted repeat (5'-CTTGTC-3') composed of two 6-nt half sites in the protected region was found. Moreover, both CTC and TC could attenuate the binding activity of CtcS with target DNA. CONCLUSION: ctcS regulated the production of TC and CTC in S. aureofaciens F3 and the overexpression of it could be used as a simple approach for the construction of engineering strain with higher productivity. Meanwhile, CtcS was characterized as a TC- and CTC-responsive MarR family regulator. This study provides a previously unrecognized function of CtcS and will benefit the research on the regulatory machinery of the MarR family regulators.

A Hoxb13-driven reverse tetracycline transactivator system for conditional gene expression in the prostate.

BACKGROUND: Genetically engineered mouse models play important roles in analyses of prostate development and pathobiology. While constitutive genetic gain- and loss-of-function models have contributed significantly to our understanding of molecular events driving these processes, the availability of a tightly regulated inducible expression system could extend the utility of transgenic approaches. Here, we describe the development of a Tet-regulatory system that employs Hoxb13 transcriptional control elements to direct reverse tetracycline transactivator (rtTA) expression in the prostate. METHODS: Using recombineering technology, the rtTA gene was placed under Hoxb13 cis-regulatory transcriptional control in the context of a 218-kb bacterial artificial chromosome. F(1) offspring carrying the Hoxb13-rtTA transgene were bred to a Tetracycline operator-Histone 2B-Green Fluorescent Protein (TetO-H2BGFP) responder line. Detailed reporter gene expression analyses, including doxycycline (Dox) induction and withdrawal kinetics, were performed in Hoxb13-rtTA|TetO-H2BGFP double transgenic adult mice and embryos. RESULTS: Dox-dependent GFP expression was observed exclusively in the prostate and distal colon epithelia of double transgenic mice. Reporter gene mRNA was detected in the prostate within 6 hr of Dox exposure, and was extinguished within 24 hr after Dox withdrawal. Furthermore, Dox-induced reporter gene expression persisted after castration. CONCLUSIONS: The Hoxb13-rtTA transgenic system provides a powerful tool for conditional Tet operator-driven transgene expression in the normal prostate and during disease progression. Used in conjunction with other prostate pathology models, these mice will enable precise, temporally controlled analyses of gene function and can provide opportunities for detailed analyses of molecular events underlying prostate diseases.

The tetracycline resistome.

Resistance to tetracycline emerged soon after its discovery six decades ago. Extensive clinical and non-clinical uses of this class of antibiotic over the years have combined to select for a large number of resistant determinants, collectively termed the tetracycline resistome. In order to impart resistance, microbes use different molecular mechanisms including target protection, active efflux, and enzymatic degradation. A deeper understanding of the structure, mechanism, and regulation of the genes and proteins associated with tetracycline resistance will contribute to the development of tetracycline derivatives that overcome resistance. Newer generations of tetracyclines derived from engineering of biosynthetic genetic programs, semi-synthesis, and in particular recent developments in their chemical synthesis, together with a growing understanding of resistance, will serve to retain this class of antibiotic to combat pathogens.

Heterologous Catalysis of the Final Steps of Tetracycline Biosynthesis by Saccharomyces cerevisiae.

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature's biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F420 reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that Fo, a synthetically accessible derivative of cofactor F420, can replace F420 in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.

Decoding and engineering tetracycline biosynthesis.

Tetracyclines have been important agents in combating infectious disease since their discovery in the mid-20th century. Following widespread use, tetracycline resistance mechanisms emerged and continue to create a need for new derivatives that are active against resistant bacterial strains. Semisynthesis has led to second and third generation tetracycline derivatives with enhanced antibiotic activity and pharmacological properties. Recent advancement in understanding of the tetracycline biosynthetic pathway may open the door to broaden the range of tetracycline derivatives and afford analogs that are difficult to access by synthetic chemistry.

The role of metabolic engineering in the production of secondary metabolites.

In the production of secondary metabolites yield and productivity are the most important design parameters. The focus is therefore to direct the carbon fluxes towards the product of interest, and this can be obtained through metabolic engineering whereby directed genetic changes are introduced into the production strain. In this process it is, however, important to analyze the metabolic network through measurement of the intracellular metabolites and the flux distributions. Besides playing an important role in the optimization of existing processes, metabolic engineering also offers the possibility to construct strains that produce novel metabolites, either through the recruitment of heterologous enzyme activities or through introduction of specific mutations in catalytic activities.

Activity of translation system and abundance of tmRNA during development of Streptomyces aureofaciens producing tetracycline.

Transition from exponential phase of growth to stationary phase in Streptomyces aureofaciens is characterized by a decrease in the rate of translation and induction of tetracycline (Ttc) biosynthesis. In exponential phase, no significant changes were found in the activity of ribosomes at binding of ternary complex Phe-tRNA.EF-Tu.GTP to the A-site on ribosomes. Overexpression of Ttc in stationary phase is accompanied by a decrease in the binding of the ternary complex Phe-tRNA.EF-Tu.GTP to the A-site of ribosome and a formation of an aggregate with Ttc by part of the ribosomes. Antibiotics that cause ribosome to stall or pause could increase the requirement for tmRNA in the process called trans-translation. We found differences in the level of tmRNA during the development of S. aureofaciens. Subinhibitory concentrations of Ttc, streptomycin and chloramphenicol induced an increase in the tmRNA level in cells from the exponential phase of growth. In vitro trans-translation system of S. aureofaciens was sensitive to Ttc at a concentration of > 15 micromol/L; the trans-translation system can thus be considered to contribute to resistance against Ttc produced only at sublethal concentrations. These experiments suggest that the main role of the rising tmRNA level at the beginning of the Ttc production is connected with ribosome rescue.

Ribosomal proteins of Streptomyces aureofaciens producing tetracycline.

Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P0 cores lacking proteins L7/L12. Reconstitution of the P0 cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.

Over expression of FGF7 enhances cell proliferation but fails to cause pathology in corneal epithelium of Kerapr-rtTA/FGF7 bitransgenic mice.

PURPOSE: Available evidence suggests that fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor, KGF) serves as a paracrine growth factor modulating corneal epithelial cell proliferation. In the present study, we used a binary inducible transgenic mouse model to examine the role of FGF7 on corneal epithelium proliferation. METHODS: A keratocyte specific 3.2 kb murine keratocan promoter (Kerapr) was used to prepare Kerapr-rtTA transgenic (Kr) mice that constitutively overexpress reverse tetracycline transcription activator (rtTA) by cornea stromal keratocytes. The Kr mice were crossed with tet-O-FGF7 mice to produce Kr/tet-O-FGF7 bitransgenic mice. Expression of human FGF7 (hFGF7) was induced by the administration of doxycycline via intraperitoneal injection and/or feeding mice doxycycline in drinking water and chow. Overexpression of hFGF7 was confirmed by RT-PCR and western blot. BrdU incorporation was used to determine cell proliferation. RESULTS: The rtTA mRNA and protein were constitutively expressed by the cornea with or without doxycycline induction, whereas hFGF7 was detected only in Kr/tet-O-FGF7 bitransgenic mice upon induction by doxycycline. Examination of induction kinetics in adult Kr/tet-O-FGF7 bitransgenic mice after a single intraperitoneal injection of doxycycline revealed that hFGF7 mRNA expression was detected 12 h after doxycycline administration, peaked at 36 h, was sustained up to 48 h, and declined thereafter. The elevated level of hFGF7 expression coincided with hyperproliferation of corneal epithelial cells. In bitransgenic mice, the number of BrdU labeled cells increased after 36 and 48 h of transgene induction compared to controls of noninduced bitransgenic or doxycycline treated single transgenic mice. The BrdU labeling index was 33+/-9.2 positive cells per corneal section for Kr/tet-O-FGF7 bitransgenic mice and 25+/-9.3 for tet-O-FGF7 single transgenic mice at 36 h post-doxycycline treatment. However, the excess FGF7 driven by doxycycline induction did not produce severe perturbation of corneal epithelium homeostasis. CONCLUSIONS: Our results demonstrate that the doxycycline inducible system is effective in regulating transgene expression in corneal stroma of Kr/tet-O-FGF7 bitransgenic mice. However, the development of pathology resulting from the overexpression of transgenes may depend on whether the amount of transgene product present is sufficient to alter the homeostasis of the targeted tissues.

Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli.

A generic vector, pASK75, was developed for the synthesis of foreign proteins in Escherichia coli under transcriptional control of the tetA promoter/operator. Tight regulation was achieved by placing the structural gene for the tet repressor, as a transcriptional fusion, downstream from the beta-lactamase-encoding gene (bla) on the same plasmid. Strong expression of the foreign gene was conveniently induced by adding anhydrotetracycline at a low concentration. Using the production of a recombinant murine immunoglobulin F(ab) fragment as an example, the system was shown to function independently of the host-strain background and to be extremely well repressed in the absence of the inducer. Thus, it represents an economic and independent alternative to IPTG-inducible promoter constructs. Additional features of pASK75 include a signal sequence and a multiple cloning site followed by a region encoding the Strep tag affinity peptide to facilitate purification of a bacterially produced protein.

Susceptibility of ribosomes of the tetracycline-producing strain of Streptomyces aureofaciens to tetracyclines.

Ribosomes from cells of Streptomyces aureofaciens producing tetracycline antibiotics (Tc-ribosomes) differ in electrophoretic mobility of ribosomal proteins S2, S10 and L19 from those of the same strain, where the production of tetracyclines was suppressed by changed cultivation conditions (C-ribosomes). Purified tight vacant couples C- and Tc-ribosomes are equally active in the translation of poly(U). Both types of S. aureofaciens ribosomes are more sensitive to tetracycline and chlortetracycline than ribosomes of Escherichia coli in the Phe-tRNA binding and the translation of poly(U).

SV40 pseudovirions as highly efficient vectors for gene transfer and their potential application in cancer therapy.

Among viral and non-viral gene delivery systems, SV40-based vectors show great promise in the cancer gene therapy field. SV40 vectors very efficiently deliver genes such as anti-viral agents, DNA vaccine, genes for chemoprotection (such as ABC transporters genes), suicide genes and antiangiogenic genes. The recombinant SV40 vectors can infect a wide variety of cells-dividing cells as well as non-cycling ones. Most of the SV40-based vectors can incorporate larger transgenes than the capacity of the SV40 wild-type, which is 5.2 kb; Moreover, in vitro packaged vectors demonstrate efficient delivery of plasmids with a molecular weight of up to 17.7 kb. SV40-based vectors carry some SV40 viral sequences, but the SV40 in vitro-packaged vectors are free of any SV40 wild-type viral DNA sequences. These vectors are prepared with nuclear extracts of SF9 insect cells containing the main viral capsid protein of the SV40 wild-type virus, VP1. This review summarizes different strategies in which SV40 vectors are used to deliver genes in vitro, to living mice, and to tumors growing in nude mice.

Characterization of a XhoI isoschizomer in Streptomyces aureofaciens after actinophage infection.

After infection of tetracycline producing strains of S. aureofaciens with actinophages mu 1/6 and B1 some phage resistant colonies were obtained in each experiment. These colonies expressed a new restriction-modification (RM) system of type II, which was different from the common RM system (SauLPI) of these strains recognizing the sequence GCCGGC. This new RM system was not detected before in parental strains. The new endonuclease was purified from a phage resistant strain of S. aureofaciens B96, using two step column chromatography to the grade without non-specific nucleolytic activity. SauLPII endonuclease recognized and cleaved the palindromic hexanucleotide sequence 5'-C/TCGAG-3', thus it was a true isoschizomer of XhoI.

Comparison of the performances of stirred tank and airlift tower loop reactors.

Following a consideration of the prerequisites for reactor comparison and the fundamental differences between stirred tank and airlift tower loop reactors, their performances are compared for the production of secondary metabolites: penicillin V by Penicillium chrysogenum, cephalosporin C by Cephalosporium acremonium, and tetracycline by Streptomyces aureofaciens. In stirred tank reactors, cell mass concentrations, volumetric productivities, and specific power inputs are higher than in airlift tower loop reactors. In the latter, efficiencies of oxygen transfer are higher, and specific productivities with regard to power input, substrate and oxygen consumptions, and yield coefficients of product formation with regard to substrate and oxygen consumptions are considerably higher than in stirred tank reactors. The prerequisites for improved performance are discussed.

Toward higher order control modalities in mammalian cells-independent adjustment of two different gene activities.

Heterologous higher order control modalities will be important tools for targeted multigene interventions in next-generation gene therapy, tissue engineering, and sophisticated gene-function studies. In this study, we present the design and rigorous quantitative analysis of a variety of different dual-regulated gene transcription control configurations combining streptogramin- and tetracycline-responsive expression systems in a one-vector format. Quantitative assessment of dual-regulated expression performance in various mammalian and human cell lines is based on two compatible secreted reporter genes, SEAP, the human placental secreted alkaline phosphatase, and the recently developed SAMY, the secreted alpha-amylase. Assembly of streptogramin- and tetracycline-responsive transgene control units in consecutive (--> -->), divergent (<-- -->), and convergent (--> <--) orientation showed excellent regulation characteristics in most genetic arrangements exemplified by neglectable interference and high transgene induction ratios in all four control settings (ON/ON, OFF/ON, ON/OFF, OFF/OFF). The overall regulation performance of divergent dual-regulated expression configurations could be substantially increased when placing noncoding stuffer fragments or insulator modules between the divergently oriented antibiotic-responsive promoters. Dual-regulated expression technology pioneers artificial higher order gene control networks that will likely enable new opportunities in multigene metabolic engineering and generate significant therapeutic impact.

HPLC as a rapid means of monitoring erythromycin and tetracycline fermentation processes.

Reverse phase high performance liquid chromatography (HPLC) was used as a rapid means of monitoring the erythromycin and the tetracycline fermentation processes. The sample preparation process for tetracycline in the fermentation broth includes simple dilution and filtration through a Millipore filter prior to injection into the HPLC column. Fermentation growth samples showed no interference, and excellent separation for selective determination of tetracycline, 4-epitetracycline, anhydrotetracycline, chlortetracycline, and 4-epianhydrotetracycline was obtained. The relative standard deviation for the HPLC analysis for tetracycline is about one percent and the correlation coefficient between the HPLC and the spectrophotometric assay methods is better than 0.994. The sample preparation procedure for erythromycin determination in fermentation broth requires solvent cleanup and extraction processes. The chromatographic analysis takes approximately 25 minutes, and the HPLC method is capable of separating and quantifying erythromycins A, B, C, and various epimers and degradation compounds. The correlation coefficient between the HPLC and the microbiological assay method is 0.970.

Use of carbon-13 in biosynthetic studies: origin of the malonyl coenzyme A incorporated into tetracycline by Streptomyces aureofaciens.

The proton noise decoupled 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride prepared from Streptomyces aureofaciens cultures supplemented with [1-13C]acetate and [2-13C]acetate showed enrichment of nine alternating ring carbons. In addition, a small enrichment of the carboxamide carbon by [1-13C]acetate was observed. The labelling patterns clearly demonstrated the polyketide origin of the tetracyclic nucleus. The 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride derived from [1,2-13C]acetate showed all 18 ring carbons as doublets with coupling constants appropriate for the incorporation of nine intact two-carbon precursors, confirming that head-to-tail condensation of C2 units had occurred. Absence of bond scission within the C2 units and a low level of uncoupled 13C in the carboxamide substituent indicated that when the organism is supplemented with acetate, malonyl coenzyme A used for tetracycline biosynthesis is formed by direct carboxylation of acetyl coenzyme A.

Response surface analysis of chlortetracycline and tetracycline production with K-carrageenan immobilized Streptomyces aureofaciens.

A full-factorial experimental design at three levels with two independent variables, carrageenan concentration (1.0, 1.5, and 2.0%) and potassium chloride concentration (0.3, 0.7, and 1.1 M) was studied in order to analyze the effect of both factors on the antibiotic production of K-carrageenan-immobilized mycelia of Streptomyces aureofaciens. The response surfaces obtained have indicated that both carrageenan and potassium chloride concentrations have a pronounced effect on the yield of chlortetracycline (CTC) and tetracycline (TC) produced by S. aureofaciens. By exclusively varying the immobilization conditions, the tetracycline production can be enhanced more than eight times (12.3 mg g-1 biomass for immobilized cells vs. 1.5 mg g-1 biomass for free cells) in comparison with free-cell mycelial cultures.

Characterization of an asporogenous mutant of Streptomyces aureofaciens with increased resistance to several aminoglycoside antibiotics.

An asporogenous spontaneous mutant of Streptomyces aureofaciens named ASR1 was selected on streptomycin gradient plates. The mutant is very stable and differs in ultrastructure and morphology, it is prototrophic but it lost the ability to grow well on soybean extract medium and produces one-tenth tetracyclines of the parent. The ASR1 mutant has a 3-4-fold increased resistance to streptomycin and is cross-resistant to other aminoglycosides. Comparison of the protein profiles from both strains on SDS gels revealed a very low expression of a 29.5 kDa protein in the ASR1 mutant which is overexpressed in both vegetative cells and spores of the parental strain.