Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus
© Vogt et al; licensee BioMed Central Ltd. 2005
Received: 30 September 2004
Accepted: 13 January 2005
Published: 13 January 2005
Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.
Here we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2s-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO7) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO7-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation.
The vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in A. fumigatus
Aspergillus fumigatus is a saprophytic filamentous fungus that has become the leading mould pathogen in leukemia treatment centers and transplantation units in developed countries, second only to Candida spp. as a cause of systemic mycosis . Despite some advances in therapy, currently available drugs for the treatment of aspergillosis continue to be hampered by problems with efficacy, toxicity, and the emergence of drug resistance. Moreover, a recent review of the Aspergillus case-fatality rate demonstrated that more than 50% of patients die with, or as a result of, aspergillosis, despite having received the reference standard of therapy . The continued expansion of the immunosuppressed population emphasizes the need for increased understanding of both the basic biology and virulence of this mould so that more effective antifungal therapies can be developed.
The completion of the annotated sequence of the A. fumigatus genome is expected to greatly facilitate efforts to determine the contribution of specific gene products to the virulence of this opportunistic pathogen. Unfortunately, the genetic tractability of A. fumigatus has lagged behind some other fungal systems, particularly in the area of conditional expression systems. Inducible promoter systems have proven to be instrumental for the elucidation of gene function in a number of species, most notably with essential genes. Experimental manipulation of gene expression in A. fumigatus is presently accomplished through the use of DNA cassettes that are introduced into the organism as transgenes [3–5], inserted into specific chromosomal loci [3, 6] or expressed from a multi-copy nonintegrating vector . An inducible expression system based upon the ethanol-inducible alcA promoter from A. nidulans has been successfully used in A. fumigatus . However, the conditions required to regulate the alcA promoter can have significant effects on the metabolism of the organism and thus remain a concern for many applications, particularly for in vivo studies.
The TetR/tetO system is biologically active in a number of eukaryotes [11–13], including yeasts [14–17], but has not yet been adapted to the filamentous fungi. In this report we demonstrate that the tetracycline-regulated promoter system can be used to manipulate gene expression in Aspergillus fumigatus using a simple co-transfection procedure.
Plasmids and strains
H237 (p444, p482) – isolate 1
H237 (p444, p482) – isolate 2
H237 (p480, p500)
Effects of doxycycline on the growth of A. fumigatus
Regulated expression of an essential gene by the 'tet-off' system
Higher levels of doxycycline were required to suppress the growth of the tTA-1 transformant on hygromycin medium (Fig. 4A). Northern blot analysis showed that the tTA-1 strain expressed about 5-fold more hph RNA than tTA-2 (Fig. 4B), which was consistent with the fact that tTA-1 grew faster than tTA-2 in the presence of the same concentration of hygromycin (Fig. 4A, compare tTA-1 and tTA-2, no doxycycline). The doublet shown in Fig. 4B was occasionally seen on Northern blots hybridized to the hph probe and is presumed to represent alternative splicing of the primary hph transcript. The higher levels of hph RNA in the tTA-1 strain could be due to a combination of increased tTA expression (which would be expected to be susceptible to doxycycline regulation) and/or basal expression from one or more integrated copies of the tetO7-hph reporter gene (which would not be affected by doxycycline). Since there was a clear dose-response effect of doxycycline on hph expression and hygromycin resistant growth in this strain (Fig. 4A and 4B), it is likely that the two strains differ primarily in the amount of tTA that they express. Although Northern blot analysis showed barely detectable levels of tTA in either strain (data not shown), undetectable levels of tTA have been reported in other applications of the tetracycline regulatory system and are thought to be due to the toxic effects of overexpression . Since very low levels of tTA protein are actually required to regulate a tetO promoter , even a small difference in tTA expression level that is beyond the limit of detection of a Northern blot could influence the amount of doxycycline required to suppress tTA activity in this transformant.
Regulated expression of an essential gene by the 'tet-on' system
A limitation of the tTA-regulated system is that it requires inhibition of transcription rather than activation. To address this, a 'reverse' tTA has been generated (rtTA) that requires interaction of the transactivator with tetracyclines before tetO binding can occur, a system that is referred to as 'tet-on' . Unfortunately, the mutations that reverse the response to doxycycline also reduce binding affinity for doxycycline ten-fold, thus requiring higher levels of doxycycline for maximal induction. Since there may be adverse effects associated with high doxycycline concentration in A. fumigatus under some conditions , we chose a derivative of rtTA that contains additional mutations that restore binding affinity for doxycycline . One particular variant, rtTA2S-M2, also contains a multimerized minimal VP16 activation domain to enhance transcriptional activity, and its sequence has been manipulated to optimize expression in eukaryotic cells .
Doxycycline-regulation is enhanced by low- iron medium
The tetracycline-inducible method of gene regulation has become one of the most popular tools to manipulate gene expression in eukaryotes . The efficacy of the system is attributed to the use of prokaryotic regulatory elements that respond to low concentrations of tetracyclines without affecting eukaryotic physiology, allowing control of gene expression without the concern for pleiotropic effects mediated by the effector. Although widely used in higher eukaryotes, including the model yeast S. cerevisiae , the system has not yet been reported in filamentous fungi. Candida albicans and C. glabrata are the only pathogenic fungi in which the system has been successfully applied thus far, however neither of these studies used the tetR-VP16 fusions upon which the tTA and rtTA systems are based [15–17].
In this study we show that both the tet-off (tTA) and tet-on (rtTA) systems can be used to regulate the expression of a hygromycin resistance reporter gene in A. fumigatus. Since the hph gene is essential in the presence of toxic levels of hygromycin, the ability to control hygromycin resistance by modulating the levels of hph transcription validates the system as a tool for analysis of essential genes in A. fumigatus. In the tTA system we found that individual transformants varied in the amount of doxycycline that was necessary to regulate expression of the tetO7-hph reporter gene. Since doxycycline prevents the tTA protein from binding to the tetO sequence, this is most likely due to variability in the amount of tTA protein that is expressed in each transformant. A limitation of the tTA approach described here is that the majority of the hygromycin-resistant transformants from the tTA/tetO7-hph co-transfection were not susceptible to regulation by doxycycline. This may be due in part to leaky expression of the tetO7-hph reporter, caused by enhancers in the proximity of the integration site [21, 25]. A second possibility is that the levels of tTA coming from the gpdA promoter used in this study were too high to be removed by non toxic concentrations of doxycycline. Since lower levels of tTA expression are more readily suppressed by doxycycline, it is conceivable that a weaker promoter used to drive tTA would increase the frequency with which doxycycline-regulatable transformants can be isolated. Lower levels of tTA expression could also be accomplished by using a shorter segment of the gpdA promoter used in this study.
The ability to quantitatively control expression from the tetO7-hph reporter gene was also observed in a strain expressing the reverse transactivator, rtTA. Concentrations of doxycycline from 2 μg/ml to 15 μg/ml gave a graded response of hygromycin resistance, indicating that A. fumigatus is responsive to concentrations of doxycycline that are similarly effective in S. cerevisiae  and C. albicans . Moreover, this level of sensitivity falls within the range of doxycycline concentrations that can be achieved in mouse tissues [15, 16], raising the possibility of using this system to modulate the expression of virulence-related genes in pathogenesis studies on A. fumigatus. Only 15% of the hygromycin-resistant colonies from an rtTA/tetO7-hph co-transfection showed doxycycline-dependent hygromycin resistance however, suggesting that some of the hygromycin resistance was due to leaky expression of the tetO7-hph gene. Leakage of tetO7-regulated genes has been described in other systems, and is attributed to enhancers located in the proximity of the integration site that increase expression of the tetO-linked gene [21, 25]. This type of problem will affect tetO7-controlled genes regardless of whether they are integrated randomly in the genome or targeted to specific loci.
This report establishes the utility of the tetracycline-regulated system as an approach to regulate gene expression in A. fumigatus. A limitation of the system was that only 10–15% of the transformants could be regulated by doxycycline, either when tTA or rtTA were used, emphasizing the need to screen for regulatable transformants. A recent approach to limit the problem of leakiness of a tetO-driven gene is the use of trans-silencer proteins comprised of fusions between tetR and a transcriptional silencing domain [26, 27]. It is conceivable that the incorporation of a synthetic A. fumigatus-derived trans-silencer protein into the co-transfection approach described in this study would improve the efficiency of the system.
All vectors are based on the pBluescript plasmid (Stratagene) and were linearized prior to transfection. PCR amplification of components were performed using standard amplification protocols using PfuTurbo DNA polymerase (Stratagene).
Hph Reporter Constructs (p482 and p500)
A segment containing seven copies of the tet operator sequence (tetO7) was PCR amplified from pUHD10-3  with the forward primer 5'-aagctt gcgtatcacgaggccctttc and the reverse primer 5'-aagctt ctcgacccgggtaccgag (added Hin dIII cloning sites are underlined) and cloned into the Hin dIII site of pBluescript. A 1.6 kb fragment containing a minimal gpdA promoter from A. nidulans (-175 relative to the ATG of the hph open reading frame), the hph gene encoding resistance to hygromycin, and the trpC terminator from A. nidulans, was then PCR amplified from pAN7-1  with forward primer 5'-gagctc cccatcttcagtatattcatc (added Sst I cloning site underlined) and reverse primer 5'-tctaga tcgcgtggagccaagagcgg (added Xba I cloning site underlined) and cloned downstream of tet07 into the Sst I and Xba I sites of the plasmid, creating p482. To minimize read-through from flanking sequences into tet07, a 280 bp segment of the terminator region of A. fumigatus cgrA  was inserted upstream of tet07 PCR to create p500. The cgrA terminator was PCR amplified from genomic DNA of A. fumigatus isolate H237 using the forward primer 5'aagctt acagcagaagaatctctc (added Hin dIII cloning site underlined) and reverse primer 5'ctcgag atgattcatgacgtatattc (added Xho I cloning site underlined), cloned into pCR2.1-Topo (Invitrogen), excised with Hin dIII, and inserted upstream of tetO7 in p482 to create p500.
tTA expression vectors (p473, p434, and p444)
A segment of the A. nidulans gpdA promoter was amplified from pAN7-1  (position -679 to -1, with +1 being the start of the hph open reading frame) using the forward primer 5'-aagctt cggagaatatggagctt (added Hin dIII cloning site underlined) and the reverse primer 5'-gaattc ggtgatgtctgctcaag (added Eco RI cloning site underlined) and cloned into pBluescript at the same sites. The tTA gene was then PCR amplified from pUHD15-1  with the forward primer 5'-gaattc tggcaatgtctagattagataaaag (added Eco RI cloning site underlined) and reverse primer 5'-atcatgtctggatcc tcgcg (internal Bam HI site underlined) and cloned into the Eco RI and Bam HI sites downstream of the gpdA (-679) promoter. A 280 bp segment of the terminator region of A. fumigatus cgrA  was then amplified from H237 genomic DNA using the forward primer 5'-actagt acagcagaagaatctctc (added Spe I site underlined) and reverse primer 5'-gcggccgc atgattcatgacgtatattc (added Not I site underlined) and inserted into the Spe I and Not I sites downstream of tTA. To introduce phleomycin selection into this construct, a phleomycin resistance cassette containing the A. nidulans gpdA promoter, the Streptoalloteichus hindustanus ble gene encoding resistance to phleomycin, and the S. cerevisiae CYC1 terminator was amplified from pBCphleo (Fungal Genetics Stock Center) using the forward primer 5'-cctcaggcggagaatatggagcttcatcg and the reverse primer 5'-cctcaggaattaaagccttcgagcgtccc. The PCR product was cloned into pCR-Blunt II-TOPO (Invitrogen), excised with Kpn I and Xho I and inserted into the PgpdA-tTA construct to create p444. The phleomycin cassette was excised from p444 with Hin dIII and re-ligated to create p473. To introduce hygromycin selection into p444, the phleomycin cassette was excised with Kpn I and Hin dIII and replaced with a hygromycin resistance cassette (containing the A. nidulans gpdA promoter, the hph gene encoding resistance to hygromycin, and the trpC terminator from A. nidulans) that was amplified from pAN7-1  with forward primer 5'-ggtacc cggagaatatggagcttc (added Kpn I cloning site underlined) and reverse primer 5'-aagctt gcttgagagttcaaggaag (added Hind III cloning site underlined) to make p434.
rtTA expression vectors
The tTA gene was excised from p473 with Eco RI and Bam HI and replaced with an Eco RI-Bam HI fragment containing the rtTA2s-M2 variant of rTA from pUHrT62-1 (generous gift from C. Berens, Erlangen, FRG) to create p474. To introduce phleomycin resistance into p474, the phleomycin resistance cassette was excised from p444 with Kpn I and Hind III and cloned into the same sites in p474 to create p480. To introduce hygromycin resistance into p474, the hygromycin resistance cassette described in p434 was excised from an unrelated plasmid as a Hin dIII fragment and cloned into the Hin dIII site of p474 to make p502.
Strains and culture conditions
The A. fumigatus strains used in this study are listed in Table-1. The wild-type strain, H237, is a clinical isolate. Conidia were harvested from strains grown on Aspergillus minimal medium plates . This minimal medium contains 4.5 μM FePO4·4H20. For low-iron minimal medium, the FePO4·4H20 concentration was reduced to 0.45 μM.
Plasmids were introduced into A. fumigatus protoplasts as previously described . Following transformation, protoplasts were plated onto 20 ml of osmotically stabilized minimal medium containing 100 μg/ml doxycycline (for transformations involving rtTA-expressing plasmids) or no added doxycycline (for transformations involving tTA-expressing plasmids). After incubating at room temperature overnight, each plate was overlaid with 10 ml of minimal medium top agar containing 0.5% agar, 1M sorbitol, and 8 mg hygromycin B (Invivogen, San Diego, CA). Doxycycline was also incorporated into the top agar overlay (100 μg/ml) for experiments involving rtTA-expressing plasmids. Colonies arising on these primary plates were transferred onto secondary selection plates containing the same selective agents, and conidia from the secondary plates were replated onto selective medium at low density to isolate colonies derived from single conidia. All subsequent experiments were performed on monoconidial isolates. For co-transfection experiments, 5 μg of the linearized tetO7-hph reporter construct was co-transfected with 5 μg of the linearized tTA plasmid (p444), or 50 μg of the linearized rtTA plasmid (p474).
For experiments addressing the effects of doxycycline on hygromycin sensitivity, ten thousand conidia were spotted onto the surface of Aspergillus minimal medium agar containing hygromycin and doxycycline at the concentrations specified in the Figure legends. The plates were then incubated at 37°C, and colony diameter was measured with time. Radial growth rates were calculated from the exponential part of the resulting growth curves.
Northern blot analysis
For analysis of hph gene expression, RNA was isolated from overnight cultures in minimal medium supplemented with the indicated concentrations of doxycycline by crushing in liquid nitrogen and extracting RNA from the crushed mycelium with phenol/chloroform. Twenty micrograms of total RNA were fractionated by formaldehyde gel electrophoresis as previously described , transferred to positively charged nylon membranes (MSI, Inc., Westborough, MA, USA) and hybridized to a 32P-labeled hph DNA probe under stringent conditions in 50% (v/v) formamide/5XSSC (1X SSC is 0.15 M NaCl/0.015 M Na3·citrate, pH 7.6)/2X Denhardt's solution/10% (w/v) dextran sulfate/1% (w/v) sodium dodecyl sulfate (SDS). The hph probe was an 800 bp Eco RI-Bam HI fragment from pAN7-1  containing a segment of the hph open reading frame. Hybridization intensity was quantified with a Phosphorimager (Molecular Dynamics) and normalized for differences in gel loading by quantitating the relative levels of SYBR-green II-stained rRNA (Molecular Probes, Inc., Eugene, OR, USA).
List of abbreviations
reverse tetracycline transactivator
- tetO :
TetR binding sequence
- hph :
hygromycin resistance gene
- ble :
phleomycin resistance gene
We thank Jay card for assistance with the figures. This work was supported by National Institutes of Health Grant R03AI53184 to DSA.
- Ho PL, Yuen KY: Aspergillosis in bone marrow transplant recipients. Crit Rev Oncol Hematol. 2000, 34: 55-69.View ArticlePubMedGoogle Scholar
- Lin SJ, Schranz J, Teutsch SM: Aspergillosis case-fatality rate: systematic review of the literature. Clin Infect Dis. 2001, 32: 358-366. 10.1086/318483.View ArticlePubMedGoogle Scholar
- Bhabhra R, Miley MD, Mylonakis E, Boettner D, Fortwendel JR, Panepinto J, Postow M, Rhodes JC, Askew DS: Disruption of the Aspergillus fumigatus gene encoding nucleolar protein CgrA impairs thermotolerant growth and reduces virulence. Infect Immun. 2004, 72: 4731-4740. 10.1128/IAI.72.8.4731-4740.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Fortwendel JR, Panepinto JC, Seitz AE, Askew DS, Rhodes JC: Aspergillus fumigatus rasA and rasB regulate the timing and morphology of asexual development. Fungal Genet Biol. 2004, 41: 129-139. 10.1016/j.fgb.2003.10.004.View ArticlePubMedGoogle Scholar
- Wasylnka JA, Moore MM: Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein. Infect Immun. 2002, 70: 3156-3163. 10.1128/IAI.70.6.3156-3163.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Langfelder K, Philippe B, Jahn B, Latge JP, Brakhage AA: Differential expression of the Aspergillus fumigatus pksP gene detected in vitro and in vivo with green fluorescent protein. Infect Immun. 2001, 69: 6411-6418. 10.1128/IAI.69.10.6411-6418.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu W, May GS, Lionakis MS, Lewis RE, Kontoyiannis DP: Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine: genetic approach to studying gene dose-dependent resistance to antifungals in A. fumigatus. Antimicrob Agents Chemother. 2004, 48: 2490-2496. 10.1128/AAC.48.7.2490-2496.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Romero B, Turner G, Olivas I, Laborda F, De Lucas JR: The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen. Fungal Genet Biol. 2003, 40: 103-114. 10.1016/S1087-1845(03)00090-2.View ArticlePubMedGoogle Scholar
- Gossen M, Bonin AL, Bujard H: Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 1993, 18: 471-475. 10.1016/0968-0004(93)90009-C.View ArticlePubMedGoogle Scholar
- Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H: Transcriptional activation by tetracyclines in mammalian cells. Science. 1995, 268: 1766-1769.View ArticlePubMedGoogle Scholar
- Weinmann P, Gossen M, Hillen W, Bujard H, Gatz C: A chimeric transactivator allows tetracycline-responsive gene expression in whole plants. Plant J. 1994, 5: 559-569.View ArticlePubMedGoogle Scholar
- Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992, 89: 5547-5551.PubMed CentralView ArticlePubMedGoogle Scholar
- Stebbins MJ, Yin JC: Adaptable doxycycline-regulated gene expression systems for Drosophila. Gene. 2001, 270: 103-111. 10.1016/S0378-1119(01)00447-4.View ArticlePubMedGoogle Scholar
- Nagahashi S, Nakayama H, Hamada K, Yang H, Arisawa M, Kitada K: Regulation by tetracycline of gene expression in Saccharomyces cerevisiae. Mol Gen Genet. 1997, 255: 372-375. 10.1007/s004380050508.View ArticlePubMedGoogle Scholar
- Nakayama H, Mio T, Nagahashi S, Kokado M, Arisawa M, Aoki Y: Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans. Infect Immun. 2000, 68: 6712-6719. 10.1128/IAI.68.12.6712-6719.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakayama H, Izuta M, Nagahashi S, Sihta EY, Sato Y, Yamazaki T, Arisawa M, Kitada K: A controllable gene-expression system for the pathogenic fungus Candida glabrata. Microbiology. 1998, 144 ( Pt 9): 2407-2415.View ArticleGoogle Scholar
- Stoyan T, Gloeckner G, Diekmann S, Carbon J: Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata. Mol Cell Biol. 2001, 21: 4875-4888. 10.1128/MCB.21.15.4875-4888.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Degenkolb J, Takahashi M, Ellestad GA, Hillen W: Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor. Antimicrob Agents Chemother. 1991, 35: 1591-1595.PubMed CentralView ArticlePubMedGoogle Scholar
- Gari E, Piedrafita L, Aldea M, Herrero E: A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast. 1997, 13: 837-848. 10.1002/(SICI)1097-0061(199707)13:9<837::AID-YEA145>3.0.CO;2-T.View ArticlePubMedGoogle Scholar
- Moy TI, Boettner D, Rhodes JC, Silver PA, Askew DS: Identification of a role for Saccharomyces cerevisiae Cgr1p in pre-rRNA processing and 60S ribosome subunit synthesis. Microbiology. 2002, 148: 1081-1090.View ArticlePubMedGoogle Scholar
- Freundlieb S, Baron U, Bonin AL, Gossen M, Bujard H: Use of tetracycline-controlled gene expression systems to study mammalian cell cycle. Methods Enzymol. 1997, 283: 159-173.View ArticlePubMedGoogle Scholar
- Hughes CE, Harris C, Peterson LR, Gerding DN: Enhancement of the in vitro activity of amphotericin B against Aspergillus spp. by tetracycline analogs. Antimicrob Agents Chemother. 1984, 26: 837-840.PubMed CentralView ArticlePubMedGoogle Scholar
- Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W: Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci U S A. 2000, 97: 7963-7968. 10.1073/pnas.130192197.PubMed CentralView ArticlePubMedGoogle Scholar
- Avery AM, Goddard HJ, Sumner ER, Avery SV: Iron blocks the accumulation and activity of tetracyclines in bacteria. Antimicrob Agents Chemother. 2004, 48: 1892-1894. 10.1128/AAC.48.5.1892-1894.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Berens C, Hillen W: Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. Eur J Biochem. 2003, 270: 3109-3121. 10.1046/j.1432-1033.2003.03694.x.View ArticlePubMedGoogle Scholar
- Deuschle U, Meyer WK, Thiesen HJ: Tetracycline-reversible silencing of eukaryotic promoters. Mol Cell Biol. 1995, 15: 1907-1914.PubMed CentralView ArticlePubMedGoogle Scholar
- Belli G, Gari E, Piedrafita L, Aldea M, Herrero E: An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. Nucleic Acids Res. 1998, 26: 942-947. 10.1093/nar/26.4.942.PubMed CentralView ArticlePubMedGoogle Scholar
- Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA: Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene. 1987, 56: 117-124. 10.1016/0378-1119(87)90164-8.View ArticlePubMedGoogle Scholar
- Boettner, Huebner N, Rhodes JC, Askew DS: Molecular cloning of Aspergillus fumigatus CgrA, the ortholog of a conserved fungal nucleolar protein. Med Mycol. 2001, 39: 517-521.View ArticlePubMedGoogle Scholar
- Cove DJ: The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta. 1966, 113: 51-56.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.