1
Supplementary material
Plasmid constructions
MEP1 and MEP2 deletion and reinsertion constructs
A MEP2 deletion construct was generated in the following way: A KpnI-XhoI fragment
containing MEP2 upstream sequences from positions -610 to +31 with respect to the start
codon was amplified from genomic DNA of C. albicans strain SC5314 with the primer pair
MEP3 (5`-TAAATACggTACCCAAACGATTGGCTTGAATGTC-3`) and MEP4 (5`TTGAACACATCTCgAgCTGTGCCTGTTCC-3`) (the lowercase letters represent nucleotide
exchanges introduced to create the underlined KpnI and XhoI restriction sites). A BglII-SacI
MEP2 downstream fragment from positions +1357 to +1765 was amplified with the primers
MEP5
(5`-GAACCTATCaGaTCTACTACTATAAGCC-3`)
and
MEP6
(5`-
TTCTACTATGAGctCCTTCTATGGTAACC-3`). The MEP2 upstream and downstream
fragments were cloned on both sides of the URA3 flipper cassette of plasmid pSFUC1
(Morschhäuser et al., 1999) to result in pMEP2M2 in which the MEP2 coding region from
position +32 to +1356 (84 bp before the stop codon) is replaced by the URA3 flipper (see Fig.
S1A).
For reintegration of an intact MEP2 copy into one of the inactivated MEP2 alleles of mep2
single or mep1 mep2 double mutants, a PstI-SacI fragment containing MEP2 downstream
sequences from positions +1364 to +1765 was first amplified with the primers MEP8 (5`GAACCTATCCGTTCTgCagCTATAAGCC-3`) and MEP6 and cloned behind the URA3
selection marker of plasmid pCBF1M4 (Biswas et al., 2003) to result in pMEP2M3. A KpnIBglII fragment containing the complete MEP2 open reading frame and upstream sequences
from positions -610 to +1506 was then amplified with the primers MEP3 and MEP9 (5`CCATGAGTTGAGatctCAGTATCATTGTG-3`) and cloned in front of the ACT1
transcription termination sequence (TACT1) of pMEP2M3 to result in pMEP2K1 (see Fig.
S1B, top). The control construct pMEP2M4, which served for integration of only the URA3
selection marker, contains MEP2 upstream sequences from positions -610 to +28, amplified
with the primers MEP3 and MEP7 (5`-TTGAACACATCTCCAgaTcTGCCTGTTCC-3`), but
not the MEP2 open reading frame in front of the TACT1 sequence (see Fig. S1B, bottom).
2
A MEP1 deletion construct was generated as follows: A KpnI-XhoI fragment containing
MEP1 upstream sequences from positions -606 to -4 was amplified from genomic DNA of
C. albicans
strain
CAI4
with
the
TTGCGATGGGTaccAAAGCTTGAAACG-3`)
primer
and
pair
MEP11
MEP12
(5`(5`-
CTTCTGCTGACtcGAgGTGTATAGAC-3`). A BglII-SacI MEP1 downstream fragment
from positions +1583 to +2121 was amplified with the primers MEP13 (5`AGAGAAGCAgatcTgCAGTGGAATATCGTC-3`)
and
MEP14
(5`-
ATGAGTAAATGAGctcTTAGTATGCG-3`). The MEP1 upstream and downstream
fragments were substituted for the MEP2 sequences in pMEP2M2 to result in pMEP1M2 in
which the MEP1 coding region from position -3 to +1582 (20 bp before the stop codon) is
replaced by the URA3 flipper (see Fig. S2A).
For integration of the URA3 marker into one of the inactivated MEP1 alleles of mep1 single
or mep1 mep2 double mutants, an XhoI-BglII fragment containing MEP1 upstream
sequences from positions –634 to +11 was first amplified with the primers MEP15 (5`GTTTCACCTcgagGTTGTTGTTGTTG-3`)
and
MEP17
(5`-
TGTATTACTagaTCTGCTGACATGATG-3`) and cloned in front of the TACT1 sequence of
pSAP2-1K (Staib et al., 2002) to result in pMEP1M3. A PstI-SacI fragment containing MEP1
downstream sequences from positions +1583 to +2121 was then amplified with the primers
MEP13 (5`- AGAGAAGCAgatcTgCAGTGGAATATCGTC-3`) and MEP14 and cloned
behind the URA3 selection marker of plasmid pMEP1M3 to result in pMEP1M4 (see Fig.
S2B, bottom). To reintegrate an intact MEP1 copy in the same way, an XhoI-BglII fragment
containing the complete MEP1 open reading frame and upstream sequences from positions
-604
to
+1615
was
amplified
with
GCGATGGGTTctcgAGCTTGAAACGAG-3`)
the
and
primers
MEP18
MEP16
(5`(5`-
AAAAATATAgATCtACCTGACTAATC-3`) and cloned in front of the TACT1 sequence of
pMEP1M4 to result in pMEP1K1 (see Fig. S2B, top).
Plasmids containing lacZ reporter gene fusions
For the construction of lacZ reporter gene fusions, the N-terminal region of the Streptococcus
thermophilus lacZ ORF was amplified from plasmid pAU36 (Uhl and Johnson, 2001) with
the
primers
LACZ1
(5`-CAGCCctcgagaATGAACATGACTGAAAAAATTC-3`)
and
LACZ2 (5`-CCATGTACCGTGTGTTTCAAGG-3`), digested at the XhoI site (underlined)
introduced in front of the start codon (bold) and at an internal ClaI site, and ligated together
3
with a ClaI-BamHI fragment from pAU36 containing the remainder of the lacZ ORF into the
vector pBluescript to generate pLACZ2. A KpnI-XhoI fragment containing MEP2 upstream
sequences from positions -610 to -5 was amplified with the primers MEP3 and MEP24 (5`CCAGACActcgaGTTATTAACTATTCAGAG-3`) and cloned together with the XhoI-BamHI
fragment containing the lacZ gene from pLACZ2 into the KpnI/BglII digested pMEP2M4 to
obtain pMEP2LACZ2 in which the lacZ gene is expressed from the MEP2 promoter. To
place lacZ under control of the MEP1 promoter, the KpnI-XhoI MEP1 upstream fragment
from pMEP2M1 was ligated together with the XhoI-BamHI fragment containing the lacZ
gene from pLACZ2 into the KpnI/BglII digested pMEP1M4 to obtain pMEP1LACZ3.
Plasmids containing MEP1 and MEP2 under control of different promoters
To express MEP1 under control of the MEP2 promoter, the MEP1 coding region was
amplified with the primers MEP27 (5`-AGTCTATctcgAgaATGTCAGCAGAAGAAG-3`)
and MEP16. The PCR product was digested at the XhoI site (underlined) introduced in front
of the start codon (bold) and at an internal ApaI site and cloned together with an ApaI-NdeI
fragment from pMEP1K1 containing the remainder of the MEP1 ORF, TACT1, and URA3
sequences into the XhoI/NdeI digested pMEP2LACZ2 to generate pMEP1K2. To express
MEP1 from the C. albicans ADH1 promoter, an XhoI-EcoRI fragment from pMEP1K2
containing the MEP1 ORF, TACT1, and URA3 sequences was ligated into the SalI/EcoRI
digested pADH1G2 (Kusch et al., 2004), resulting in pMEP1E2. To place MEP2 under
control of the ADH1 promoter, the MEP2 coding region was amplified with the primers
MEP21 (5`-GTTAATctCgAgaATGTCTGGAAATTTCACTGG-3`) and MEP9. The PCR
product was digested at the XhoI and BglII sites introduced in front of the start and behind the
stop codon, respectively, and cloned together with a BamHI-EcoRI fragment from
pYPR127E2 (Kusch et al., 2004) containing TACT1 and URA3 sequences into the SalI/EcoRI
digested pADH1G2 to generate pMEP2E2. To express MEP2 under control of the MEP1
promoter, an XhoI-EcoRV fragment containing the N-terminal part of the PCR-amplified
MEP2 ORF was cloned together with an EcoRV-EcoRI fragment from pMEP2K1 containing
the remainder of the MEP2 ORF, TACT1, and URA3 sequences into the XhoI/EcoRI digested
pMEP1LACZ3 to generate pMEP2K2.
Plasmids containing GFP-tagged MEP1 and MEP2 genes
A C-terminal fusion of the Mep2 protein with the green fluorescent protein (GFP) was
constructed as follows. The MEP2 gene was amplified with the primers MEP21 and MEP32
4
(5`-AAAGAACTGgATccATTTTTAGCTTCTCC-3`), and the PCR product was digested at
an internal SalI site and at the BamHI site (underlined) introduced instead of the MEP2 stop
codon. The N-terminal part of the C. albicans-adapted GFP gene from pGFP41
(Morschhäuser
et
al.,
1998)
was
amplified
with
the
GATGggatccAGTAAAGGAGAAGAACTTTTCACTG-3`)
primers
and
GFP16
GFP4
(5`(5`-
TCTGGTAAAAGGACAGGGC-3`), and the PCR product was digested at the BamHI site
(underlined) introduced instead of the GFP start codon and at an internal NcoI site. The SalIBamHI fragment containing the C-terminal part of MEP2, the BamHI-NcoI fragment with the
N-terminal part of GFP, and an NcoI-PstI fragment from pGFP41 containing the remainder of
the GFP gene, TACT1, and the URA3 marker were inserted between the SalI and PstI sites of
pMEP2K1 to generate pMEP2G2 in which the GFP gene without its start codon is fused via
two additional codons from the BamHI site (encoding glycine and serine) to the last amino
acid codon of MEP2. To express the GFP-tagged MEP2 gene from the ADH1 promoter, a
PstI-EcoRI fragment from pMEP2G2 containing the C-terminal part of MEP2 fused to GFP,
TACT1, and URA3 sequences was substituted for the corresponding fragment in pMEP2E2,
resulting in pMEP2G4. Similarly, an EcoRV-EcoRI fragment from pMEP2G4 was
substituted for the corresponding fragment in pMEP2K2 to generate pMEP2G5 in which the
MEP2-GFP fusion is placed under control of the MEP1 promoter.
To generate a Mep1p-GFP fusion, the MEP1 gene was amplified with the primers MEP27
and MEP37 (5`-ACCTGACggATCcACTGGGACGATATTCC-3`). The PCR product was
digested at an internal SalI site and at the BamHI site introduced instead of the MEP1 stop
codon and ligated together with a BamHI-EcoRI fragment from pMEP2G2 containing GFP,
TACT1, and URA3 sequences between the SalI and EcoRI sites of pMEP1K1 to generate
pMEP1G1. To express the MEP1-GFP fusion from the MEP2 promoter, an ApaI-EcoRI
fragment from pMEP1G1 containing the C-terminal part of MEP1 fused to GFP, TACT1, and
URA3 sequences was substituted for the corresponding fragment in pMEP1K2, yielding
pMEP1G2. Similarly, a ClaI-EcoRI fragment from pMEP1G1 was substituted for the
corresponding fragment in pMEP1E2, yielding pMEP1G3 in which the MEP1-GFP fusion is
placed under control of the ADH1 promoter.
Plasmids containing truncated MEP1 and MEP2 genes
Constructs carrying C-terminally truncated versions of MEP1 and MEP2 were generated as
follows. MEP2 was amplified with primer MEP3, which binds in the MEP2 upstream region
5
(see above) and one of the following primers, each of which introduces a stop codon (reverse
sequence in bold) followed by a BglII site (underlined): primer MEP19 (5`AATGGGAgatctTaCATGGCAAGCAAAATAATGG-3`) introduces a stop codon behind
Met406,
primer
introduces
a
MEP22
stop
(5`-TGGGTTGagaTCtaATCGTCATCAGCATAATAGG-3`)
codon
behind
Asp440,
primer
MEP25
(5`-
TTGGGCCAgaTCtGTTCaCAACATTTCCTCG-3`) introduces a stop codon behind Leu423,
and primer MEP26 (5`-TCGTGGAGAtcttaTCTGAGAATGGGATTCTG-3`) introduces a
stop codon behind Arg413. The truncated MEP2 genes were substituted for the full length
gene in pMEP2K1 to generate plasmids pMEP2C1, pMEP2C2, pMEP2C3, and
pMEP24, carrying MEP2C406, MEP2C440, MEP2C423, and MEP2C413, respectively.
Similarly, MEP1 was amplified with primer MEP18, which binds in the MEP1 upstream
region
(see
above)
and
either
primer
MEP20
(5`-
AGGGAagaTcTTaAATGACAAAACATAATATGG-3`), which introduces a stop codon
behind
Ile404,
or
primer
MEP23
(5`-
AGAAATCagaTCTaCACTTCAACGTAATCATAAGC-3`), which introduces a stop codon
behind Val438. The truncated MEP1 genes were substituted for the full length gene in
pMEP1K1 to generate plasmids pMEP1C1, carrying MEP1C404, and pMEP1C2, carrying
MEP1C438.
Plasmids containing hybrid MEP genes
MEP1/MEP2 hybrid genes were generated in the following way. To construct pMEP21H2, a
fragment containing the N-terminal part and upstream sequences of MEP2 from positions
-606 to +1254 was amplified with primer MEP3, which introduces an upstream KpnI site (see
above), and the phosphorylated primer MEP33p (5`-TTCGTGGAGACGGATTCTGAG-3`).
In addition, a fragment containing the C-terminal part of MEP1 from positions +1249 to
+1615
was
amplified
with
the
phosphorylated
primer
MEP34p
(5`-
AATGGAGAAGAAGCTGGTGTTG-3`) and primer MEP16, which introduces a BglII site
behind the MEP1 stop codon (see above). The MEP2 and MEP1 fragments were digested at
the KpnI and BglII sites, respectively, fused at their blunt ends, and substituted for the MEP2
gene in pMEP2K1. The MEP2-MEP1 hybrid gene contained in pMEP21H2 encodes a fusion
protein in which the first 418 amino acids of Mep2p are fused to the last 118 amino acids of
Mep1p (amino acids 417 to 534). For pMEP12H2, the C-terminal part of MEP2 was
amplified
with
the
primers
MEP35
(5`-
6
CCTTAGAATCGATTATGACGAGGAAATGTTGGGAACCG-3`)
and
MEP9.
Primer
MEP35 consists of MEP1 sequences from positions +1233 to +1248 followed by MEP2
sequences from positions +1255 to +1276; primer MEP9 binds to the end of the MEP2 ORF
(see above). The PCR product was digested at the ClaI site (underlined) within the MEP1 part
and at the BglII site introduced behind the MEP2 stop codon and substituted for the
corresponding C-terminal part of MEP1 in pMEP1K1. The MEP1-MEP2 hybrid gene
contained in pMEP12H2 is expressed from the MEP1 promoter and encodes a fusion protein
in which the first 416 amino acids of Mep1p are fused to the last 62 amino acids of Mep2p
(amino acids 418 to 480). The MEP1-MEP2 hybrid ORF was also expressed from the MEP2
promoter by substituting the ClaI-BglII fragment from pMEP12H2 for the corresponding
fragment in pMEP1K2 to generate pMEP12H3. In addition, a MEP1-MEP2 hybrid gene in
which the truncated C-terminal part of MEP2C440 (amino acids 418 to 440) was fused to the
N-terminal part of MEP1 (amino acids 1-416) was expressed from the MEP2 promoter. The
corresponding plasmid pMEP12H4 was obtained by amplifying a part of the MEP1-MEP2
hybrid gene of pMEP12H2 with the primers MEP27 and MEP22, digesting the PCR product
at an internal SalI site and at the BglII site introduced behind Asp440 (see above), and
substituting the hybrid fragment for the MEP1 C-terminal part in pMEP12H3.
Plasmids containing dominant active RAS1G13V and GPA2Q354L alleles
To express the dominant active RAS1G13V allele under control of the ACT1 promoter, an XbaISalI fragment containing ACT1 sequences from positions -490 to -10 was PCR-amplified
from
genomic
DNA
of
strain
SC5314
CAGCGTCAAAtCTAGAGAATAATAAAG-3`)
with
the
and
primers
ACT31
ACT20
(5`(5`-
TTTGtcgacTTATATTTTTTTAATATTAATATCGAG-3`). The C. albicans RAS1 ORF was
then
amplified
with
the
primer
RAS1
(5`-
CATATgtcgACCATGTTGAGAGAATATAAATTAGTTGTTGTTGGAGGTGtTGGTGTT
GG-3`), which introduces a SalI site in front of the start codon and substitutes a valinespecific GTT codon (bold) for the original glycine-specific GGT codon at positions +37 to
+39, and primer RAS2 (5`-TTTAGagatCTCAAACAATAACACAACATCC-3`), which
introduces a BglII site behind the stop codon. The ACT1 upstream fragment and the RAS1G13V
fragment were digested with XbaI/SalI and SalI/BglII, respectively, and cloned together into
the XbaI/BglII-digested pSAP2-1K (Staib et al., 2002) to generate pRAS1E1.
7
To obtain the dominant active GPA2Q354L allele, the N-terminal part of the GPA2 ORF was
PCR-amplified from genomic DNA of C. albicans strain CAI4 with the primer GPA1 (5`AACTTgtcgACCATGGGTTCTTGTGCTTCG-3`), which introduces a SalI site in front of
the
start
codon,
and
the
phosphorylated
primer
GPA2
(5`-
ACCAACATCAAATAAATTCATATTTAATCC-3`). The remainder of the GPA2 ORF was
amplified
with
the
phosphorylated
primer
GPA3
(5`-
GGTttAAGGTCAGAAAGAAAAAAATGGATC-3`), which substitutes the leucine-specific
TTA codon (bold) for the original glutamine-specific CAA codon at positions +1060 to
+1062, and primer GPA4 (5`-TGCATagatCTATAAAATACCACTATCTTTAAGAG-3`)
which introduces a BglII site at the stop codon. The PCR products were digested with SalI
and BglII, respectively, fused at their blunt ends, and substituted for the RASG13V fragment in
pRAS1E1 to generate pGPA2E1 in which the GPA2Q354L allele is placed under control of the
ACT1 promoter.
Plasmids for heterologous expression of MEP1 and MEP2 in S. cerevisiae
To express C. albicans MEP1 and MEP2 in S. cerevisiae, the XhoI-BglII fragment from
pMEP1K1 containing MEP1 or the KpnI-BglII fragment from pMEP2K1 containing MEP2
were cloned into the 2µ vector pYEplac195 (Gietz and Sugino, 1988) to generate plasmids
pYEpCaMEP1 and pYEpCaMEP2, respectively.
Generation of C. albicans mep1 and mep2 mutants
To obtain a mep2 mutant, strain CAI4 was transformed with the insert from plasmid
pMEP2M2 in which most of the MEP2 ORF was replaced by the URA3 flipper cassette (see
Fig. S1A). In the parental strain CAI4 the two MEP2 alleles can be distinguished by an EcoRI
restriction site polymorphism (Fig. S1A and C, lane 1), and two independent transformants
were selected in which the deletion cassette was integrated either into the MEP2-1 allele
(strain MEP2M1A, Fig. S1C, lane 2) or the MEP2-2 allele (strain MEP2M1B, Fig. S1C, lane
3). The cassette was excised from these strains by FLP-mediated recombination, resulting in
strains MEP1M2A and B (Fig. S1C, lanes 4 and 5). The remaining wild-type alleles were
deleted in a second round of insertion/excision of the URA3 flipper, which generated strains
MEP2M3A and B (Fig. S1C, lanes 6 and 7) and their ura3-negative derivatives MEP2M4A
and B (Fig. S1C, lanes 8 and 9). An intact MEP2 copy was then reinserted with the help of
the URA3 selection marker into the genome of the two homozygousmep2 mutants (see
Fig. S1B, top). We selected two independent complemented strains in which reinsertion had
8
occurred into the mep2-2 allele of strain MEP2M4A or into the mep2-1 allele of strain
MEP2M4B, generating strains MEP2MK1A and B, respectively (Fig. S1C, lanes 12 and 13).
To obtain appropriate control strains, the URA3 marker alone was inserted in the identical
way (see Fig. S1B, bottom), thereby generating the prototrophic, homozygous mep2 mutants
MEP2M5A and B (Fig. S1C, lanes 10 and 11).
The MEP1 gene was deleted in an analogous procedure to generate mep1 single as well as
mep1 mep2 double mutants; the construction of the mep1 mep2 double mutants and
complemented strains is documented in Fig. S2C and D. The two independent mep2
mutants MEP2M4A and B were transformed with the insert from plasmid pMEP1M2 in
which most of the MEP1 ORF was replaced by the URA3 flipper cassette (see Fig. S2A). The
two MEP1 alleles in strain CAI4 and its derivatives can be distinguished by a ClaI restriction
site polymorphism (see Fig. S2A and C), and two transformants were selected in which the
deletion cassette was integrated either into the MEP1-1 allele of strain MEP2M4A or into the
MEP1-2 allele of strain MEP2M4B, resulting in strains MEP12M1A and B, respectively
(Fig. S2C, lanes 4 and 5). The cassette was excised from these strains by FLP-mediated
recombination, resulting in strains MEP12M2A and B (Fig. S2C, lanes 6 and 7). Deletion of
the remaining MEP1 wild-type alleles in a second round of insertion/excision of the URA3
flipper generated strains MEP12M3A and B (Fig. S2C, lanes 8 and 9) and their ura3-negative
derivatives MEP12M4A and B (Fig. S2C, lanes 10 and 11). The homozygous mep1 mep2
double mutants then served as host strains for reintroduction of an intact MEP1 copy with the
help of the URA3 marker (Fig. S2B, top) into either of the two inactivated mep1 alleles,
resulting in strains MEP12MK1A and B (Fig. S2C, lanes 16 and 17), or of an intact MEP2
copy into either of the two inactivated mep2 alleles, resulting in strains MEP12MK2A and B
(Fig. S2D, lanes 18 and 19). To obtain appropriate control strains, the URA3 marker alone
was inserted in the identical way (see Fig. S1B, bottom, and Fig. S2B, bottom) into either of
the four possible alleles, thereby generating the prototrophic, homozygous mep1 mep2
double mutants MEP12M5A and B and MEP12M6A and B (Fig. S2C and D, lanes 12 to 15).
Two independent series of mep1 single mutants and complemented strains were constructed
from the parental strain CAI4 in an identical fashion (data not shown).
9
Complementation of a S. cerevisiae mep1 mep2 mep3 triple mutant
To test whether MEP1 and MEP2 from C. albicans could complement the growth and
filamentation defect of a S. cerevisiae mep1 mep2 mep3 triple mutant, strain MLY131a/
(Lorenz and Heitman, 1998) was transformed with plasmids pYEpCaMEP1 or
pYEpCaMEP2 containing the C. albicans MEP1 or MEP2 genes, respectively. As controls,
the strain was transformed with plasmids pML100, pML151, or pML113 containing the
S. cerevisiae MEP1, MEP2, or MEP3 genes, respectively (Lorenz and Heitman, 1998), or
with the empty vector pYEplac195. In each case, four independent transformants were tested
for their ability to grow and filament under low ammonium conditions. For this purpose, YPD
overnight cultures of the strains were appropriately diluted, spread on SLAD agar plates, and
incubated at 30 °C for 7 days. Like the three MEP1 genes from S. cerevisiae, MEP1 and
MEP2 from C. albicans rescued the growth defect of the triple mutant at limiting ammonium
concentrations. In addition, MEP2, but not MEP1, restored filamentous growth (Fig. S3).
References
Biswas, K., Rieger, K.J., and Morschhäuser, J. (2003) Functional characterization of CaCBF1, the Candida
albicans homolog of centromere binding factor 1. Gene 323: 43-55.
Gietz, R.D., and Sugino, A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro
mutagenized yeast genes lacking six-base pair restriction sites. Gene 74: 527-534.
Kusch, H., Biswas, K., Schwanfelder, S., Engelmann, S., Rogers, P.D., Hecker, M., and Morschhäuser, J. (2004)
A proteomic approach to understanding the development of multidrug-resistant Candida albicans strains. Mol
Genet Genomics 271: 554-565.
Lorenz, M.C., and Heitman, J. (1998) The MEP2 ammonium permease regulates pseudohyphal differentiation in
Saccharomyces cerevisiae. EMBO J 17: 1236-1247.
Morschhäuser, J., Michel, S., and Hacker, J. (1998) Expression of a chromosomally integrated, single-copy GFP
gene in Candida albicans, and its use as a reporter of gene regulation. Mol Gen Genet 257: 412-420.
Morschhäuser, J., Michel, S., and Staib, P. (1999) Sequential gene disruption in Candida albicans by FLPmediated site-specific recombination. Mol Microbiol 32: 547-556.
Staib, P., Kretschmar, M., Nichterlein, T., Hof, H., and Morschhäuser, J. (2002) Host versus in vitro signals and
intrastrain allelic differences in the expression of a Candida albicans virulence gene. Mol Microbiol 44: 13511366.
Uhl, M.A., and Johnson, A.D. (2001) Development of Streptococcus thermophilus lacZ as a reporter gene for
Candida albicans. Microbiology 147: 1189-1195.