Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414, USA1
Author for correspondence: M. Andrew Uhl. Tel:+1 415 502 0859. Fax: +1 415 476 8201. e-mail: uhl{at}socrates.ucsf.edu
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ABSTRACT |
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Keywords: ß-galactosidase, yeast, fungal pathogen, gene regulation
a Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.
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INTRODUCTION |
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Studies of transcriptional regulation in C. albicans have been limited by several properties of the organism; e.g. it is diploid with a little-understood sexual cycle, making conventional genetic analysis impossible (Odds, 1988 ). Additionally, expression of foreign genes, including reporter genes, in C. albicans has been complicated by the alternative codon usage of the organism. The codon CTG is read as a serine in C. albicans but as a leucine in other organisms (Ohama et al., 1993
). Escherichia coli lacZ, which has been successfully adapted as a reporter gene in many organisms, contains 51 CTG codons (Gilbert & Maxam, 1973
); therefore successful expression of a functional enzyme seems unlikely without extensive alteration. Other common reporter genes, such as uidA (ß-glucuronidase) also contain several CTG codons (Blanco et al., 1985
). We were particularly interested in developing a ß-galactosidase as a reporter for C. albicans because of the extensive variety of substrates available for this enzyme. In addition, C. albicans is not known to have an endogenous ß-galactosidase and cannot utilize lactose as a carbon source (Kwon-Chung, 1992
).
Here we report the use of lacZ from Streptococcus thermophilus as a versatile reporter gene for C. albicans. We show that ß-galactosidase activity can be readily detected by a variety of methods when lacZ is expressed from several C. albicans promoters.
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METHODS |
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The Strep. thermophilus lacZ gene was amplified from pRH116 using the primers LacZ5' (GTGTCCCGGGTCCATGAACATGACTGAAAAAATTCAA) and LacZ3' (TCCACGAGGATCCCTAATTTAGTGGTTCAATCATGAAG) and cloned into the various expression constructs to create the following plasmids: MAL2lacZ (pAU22), HWP1lacZ (pAU95) and ACT1lacZ (pAU36). Alteration of the CTG codon to TTA in lacZ was done by PCR-based mutagenesis using the primer pairs LacZ-1631 (ATGGTGTCGTGAGTTTGA) and LacZm3' (CAGTTGCTTCTCTTAAGACACAAGCAACTTCGTAAATTTG), and LacZm5' (GTTGCTTGTGTCTTAAGAGAAGCAACTGAATGGGCTCC) and LacZ3'. The resulting PCR fragments were mixed and served as a template for amplification with the primers LacZ-1631 and LacZ3'; this was cloned into pAU22 to give pAU106. Presence of the mutation was screened for by creation of an AflII site and confirmed by sequencing. For expression of Strep. thermophilus lacZ in Saccharomyces cerevisiae, lacZ was amplified with Pfu DNA polymerase (Stratagene) using linearized pAU22 as a template and the primers LacZ5' and MAL2-3'Bln (CCTGCCTAGGAGACATACGCTTTGCAGGTGGTGTT). The resulting product was digested with XmaI and BlnI and cloned into XmaI/XbaI-cut pDK20 (a gift from Doug Kellogg, Sinsheimer Laboratories, Dept of Biology, University of California, Santa Cruz).
C. albicans methods.
C. albicans strain CAI4 (ura3::imm434/ura3::imm434) served as the parent strain for all manipulations (Fonzi & Irwin, 1993 ). Linearized DNA was transformed into CAI4 by the modified lithium acetate method (Hill et al., 1991
; Gietz et al., 1995
), and transformants were selected on medium lacking uridine (Guthrie & Fink, 1991
). Standard recipes were utilized for all media, and carbon sources such as maltose, glucose, galactose or lactose were added at a final concentration of 2% (v/v) (Guthrie & Fink, 1991
). For expression of lacZ in Sacch. cerevisiae, strain W303 (ade2-1 trp1-1 cau1-100 leu2-3,112 his3-11 ura3 psi+) was utilized (Guthrie & Fink, 1991
). Plasmids were introduced into W303 by lithium acetate transformation (Hill et al., 1991
; Gietz et al., 1995
).
ß-Galactosidase assays.
C. albicans ß-galactosidase assays were performed as described by Ausubel et al. (1992) for Sacch. cerevisiae with minor modifications. For liquid assays, 1 ml cells was resuspended in an equal volume of Z buffer (Ausubel et al., 1992
) and placed on ice. The OD600 was determined for each sample. Then 10100 µl of cells was added to Z buffer to a final volume of 1 ml, and the cells were permeabilized with 15 µl 0·1% SDS and 30 µl chloroform. There was no appreciable change in activity when alternative methods such as toluene or glass beads were used for permeabilizing cells. Cells were equilibrated at 37 °C for 5 min, then 0·2 ml ONPG (4 mg ml-1) was added and the cells were mixed and incubated at 37 °C. Reactions were stopped by addition of 0·5 ml 1 M Na2CO3, spun for 5 min at 10000 g and the A420 and A550 were read. Units of activity were determined by the standard equation given by Ausubel et al. (1992)
.
Filter assays were performed as described by Ausubel et al. (1992) . Patched colonies of C. albicans were replica-plated onto solid medium with an overlay of a circular Whatman filter and grown overnight. Filters were frozen in liquid nitrogen and incubated in 3 ml Z buffer with 20 µl 3% X-Gal and incubated at 37 °C.
Visual screens for C. albicans were carried out by patching colonies onto X-Gal plates (Ausubel et al., 1992 ). The standard formulation for X-Gal plates was utilized for most applications. A slightly modified recipe proved more sensitive for detection of ß-galactosidase, although strains grew more slowly on this medium (X-Gal Modified Medium, XMM). XMM contained 1·7 g Yeast Nitrogen Base (without amino acids or ammonium sulfate), 20 g glucose, 5 g ammonium sulfate and 20 g agar in 930 ml H2O. After autoclaving, 70 ml 1 M potassium phosphate pH 7·0 and 2 ml of a 20 mg ml-1 X-Gal solution were added.
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RESULTS AND DISCUSSION |
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Expression of the ACT1lacZ construct was constant during all growth conditions, varying less than 1·5-fold during all growth conditions (Table 1). The ACT1 gene is known to be downregulated by starvation and during growth-phase changes; these conditions were not tested for the ACT1lacZ fusion (Delbruck & Ernst, 1993
; Swoboda et al., 1994
). In contrast, the HWP1lacZ construct was strongly induced by growth under filamentous conditions, including YEP-glucose+10% fetal calf serum at 37 °C. Compared to growth in YEP-glucose, the HWP1lacZ construct was induced approximately 250-fold by growth in YEP-glucose+10% fetal calf serum. This is consistent with measurements of RNA levels of HWP1 in cells grown in YEP-glucose or YEP-glucose+10% fetal calf serum at 37 °C (data not shown). HWP1lacZ was also induced to a lesser degree by substitution of maltose for glucose in the growth medium (see Table 1
and Fig. 2
), and utilization of maltose as a carbon source has been reported to be an inducer of filamentous growth in C. albicans (Odds, 1988
).
In order to optimize enzymic assays for the Strep. thermophilus ß-galactosidase, we performed assays at various temperatures and pH values. The temperature optimum of Strep. thermophilus LacZ was found to lie between 45 and 50 °C; enzymic activity fell sharply at temperatures higher than 50 °C. Activity of the enzyme was 2·2-fold higher at 45 °C than at 30 °C. The pH optimum of the enzyme was between 6·8 and 7·2.
Expression of lacZ does not allow growth on lactose as a sole carbon source
Since expression of ACT1lacZ was independent of carbon source, this allowed a test of whether C. albicans expressing ß-galactosidase could utilize lactose as a sole carbon source. We grew C. albicans SC5314 (wild-type) and C. albicans CA361 (ACT1::lacZ) on minimal medium with glucose (SG), minimal medium with lactose (SL) and minimal medium without any carbon source (S). No growth of SC5314 or CA361 was observed on SL or S medium, while both strains grew on SG medium (data not shown). A similar phenotype has been noticed for Sacch. cerevisiae strains expressing ß-galactosidase, and expression of a lactose permease was necessary before lactose could be utilized as a carbon source (Sreekrishna & Dickson, 1985 ).
Qualitative assays of ß-galactosidase activity
Growth of cells on medium containing X-Gal provides a useful method of screening large numbers of cells for the expression of ß-galactosidase activity under particular sets of conditions. When cells were plated on the appropriate medium containing X-Gal, activity was easily detectable from C. albicans strains expressing MAL2lacZ (CAU221), HWP1lacZ (CAU951) and ACT1lacZ (CAU361) (Fig. 2). After 2 d, colonies expressing the HWP1lacZ fusions turned blue on X-Gal+10% fetal calf serum, but remained white on X-Gal+glucose or X-Gal+maltose (not shown). However, after 5 d, strains with the HWP1lacZ fusion began to turn blue on all media (Fig. 2
). This result was expected since HWP1 expression is highly induced under a variety of conditions that promote filamentous growth, including starvation, a condition that arises in colonies upon prolonged growth (see below). Colonies of the ACT1lacZ fusion strain turned visibly blue on all media as soon as 2 d, and developed further after 5 d (Fig. 3
). Colonies of the MAL2lacZ fusion strain turned blue on X-Gal+maltose medium in 45 d, but remained white on X-Gal+glucose medium (Fig. 2
). The longer developing time of the MAL2lacZ fusion strains compared with the other strains is consistent with the lower level of expression as detected by the liquid assays (Table 1
).
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Although growth on X-Gal medium is a useful monitor of lacZ expression, it can require several days for the colour to develop. For more rapid detection of ß-galactosidase activity, colonies grown on solid medium can be transferred to paper filters, permeabilized by freezing in liquid nitrogen and incubated with X-Gal (Ausubel et al., 1992 ). Results with filter assays matched results of the liquid ß-galactosidase assays and plate assays (not shown). Positive reactions were obtained after only a few hours incubation at 37 °C for C. albicans strains with integrated MAL2lacZ, HWP1lacZ or ACT1lacZ fusions.
Strep. thermophilus lacZ in Sacch. cerevisiae
Since it is often useful to compare regulatory pathways in C. albicans and Sacch. cerevisiae (Gimeno et al., 1992 ), we tested whether Strep. thermophilus lacZ also functioned in Sacch. cerevisiae. Sacch. cerevisiae strains that contain the Strep. thermophilus lacZ fused to the GAL1,10 promoter and integrated in the genome readily exhibit regulated ß-galactosidase activity as determined by filter assays (Guthrie & Fink, 1991
). When grown on YEP-galactose, 88% of the transformants produced ß-galactosidase activity within 1 h, while no activity was observed when transformants were grown on YEP-glucose. No activity was detected from Sacch. cerevisiae stains containing the GAL1,10 vector alone. Thus, Strep. thermophilus lacZ can be shuttled between C. albicans and Sacch. cerevisiae and used as a reporter gene in both yeasts.
The results presented in this paper show that lacZ from Strep. thermophilus is a useful reporter gene for the human pathogen C. albicans. Other reporter genes have been developed for C. albicans, including the sea pansy luciferase (Srikantha et al., 1996 ), the K. lactis ß-galactosidase LAC4 (Leuker et al., 1992
), the URA3 gene from C. albicans (Myers et al., 1995
) and the yeast-enhanced green fluorescent protein (yEGFP; Cormack et al., 1997
). These reporter genes have been used effectively for studies of transcriptional regulation in C. albicans (Wirsching et al., 2000
; Srikantha et al., 1996
, 1997
; Stoldt et al., 1997
; Leuker et al., 1997
). Strep. thermophilus LacZ combines the advantages of sensitivity, simplicity of qualitative assays, and ease of colorimetric visual screens in growing colonies of C. albicans.
E. coli lacZ has proved to be a highly versatile reporter gene in Sacch. cerevisiae and has been used to study many aspects of signal-transduction pathways, gene regulation and other cellular processes (Guarente & Ptashne, 1981 ; Rose et al., 1981
; Guarente, 1983
; Burns et al., 1994
). We believe that the Strep. thermophilus lacZ reporter gene can be used for many of these same purposes in C. albicans.
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ACKNOWLEDGEMENTS |
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Received 12 December 2000;
accepted 15 January 2001.