role of the 14–3–3 protein in carbon metabolism of the pathogenic yeast candida albicans

YeastYeast 2004; 21: 685–702.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1079

Research Article

Role of the 14–3–3 protein in carbon metabolism ofthe pathogenic yeast Candida albicans

Ying-Kai Wang†, Biswadip Das, David H. Huber‡, Melanie Wellington§, M. Anaul Kabir, Fred Shermanand Elena Rustchenko*Department of Biochemistry and Biophysics, University of Rochester Medical School, Rochester, NY 14642, USA

*Correspondence to:Elena Rustchenko, Departmentof Biochemistry and Biophysics,Box 712, University of RochesterMedical School, Rochester, NY14642, USA.E-mail:elena [email protected]

†Current address: Bristol-MyersSquibb Company, MicrobiologyDepartment, 5 ResearchParkway, Wallingford, CT06492–7660, USA.

‡Current address: Department ofBiology, West Virginia StateCollege, Institute, WV25112, USA.

§Current address: Department ofMicrobiology and Immunology,University of Rochester MedicalSchool, Rochester, NY 14642,USA.

Received: 8 June 2003Accepted: 18 November 2003

AbstractWe previously demonstrated that the pathogenic yeast Candida albicans effectivelyadapts to utilize L-sorbose (Sou+) by a novel mechanism based on the loss of onecopy of chromosome 5, probably due to the reduction of copy number of a negativeregulator located on this chromosome. We report here another negative regulator of L-sorbose utilization, an orthologue of the Saccharomyces cerevisiae BMH1 gene, whichencodes the evolutionarily conserved protein 14–3–3. This essential gene is locatedon chromosome 1, does not have paralogues, and is supposedly a component of theregulatory network. Experiments involving disruption of one allele of BMH1 andoverexpression of BMH1 revealed that BMH1 represses the transcription of SOU1,which is responsible for the utilization of L-sorbose. Although the exact mechanismof the interaction between BMH1 and SOU1 is not known, it is clear that the controlis based on the ratio of gene copy number, and that BMH1 does not control theloss of chromosome 5, the major mechanism producing Sou+ mutants. We proposethat function of BMH1 as a negative regulator of SOU1 contributes to a generalcellular homeostasis. This is a first report on the role of the C. albicans essential geneBMH1 as a negative regulator of the utilization of secondary carbon source in yeast,which further substantiates the involvement of 14–3–3 proteins in diverse functions.Copyright 2004 John Wiley & Sons, Ltd.

Keywords: Candida albicans; 14–3–3; BMH1 ; negative regulation; sorbose utiliza-tion; SOU1

Introduction

A majority of secondary carbon sources are utilizedby some but not all strains of C. albicans (McGin-nis, 1980; Rustchenko et al., 1997). Using as exam-ples two sugars, L-sorbose and D-arabinose, wedemonstrated that C. albicans possesses a uniquemechanism to control the utilization of alternativecarbons, which is based on the copy number ofspecific and different chromosomes (Janbon et al.,1998; Rustchenko et al., 1994). The generality ofthis mechanism, as a means for C. albicans to copewith changing environment, was further revealed

by studying fluconazole and 5-fluoro-orotic acid(5-FOA) resistance (Perepnikhatka et al., 1999;M. Wellington and E. Rustchenko, unpublisheddata). In the case of L-sorbose utilization, wedemonstrated that the growth on L-sorbose mediumdepends on a structural gene, SOU1 (sorboseutilization), which resides on chromosome 4 (Jan-bon et al., 1998) and which encodes NADPH-dependent L-sorbose reductase (J. Greenberg, N.Price, R. Oliver, F. Sherman and E. Rustchenko,unpublished data). SOU1 is only weakly expressedin normal C. albicans strains that do not grow on L-sorbose, and, thus, these strains are considered to be

Copyright 2004 John Wiley & Sons, Ltd.

686 Y.-K. Wang et al.

Sou− (Janbon et al., 1998; this study). However, ifL-sorbose is the sole carbon source, normal strainsreadily produce sorbose-utilizing Sou+ mutantsthat lack one copy of chromosome 5 and have anincreased amount of the SOU1 transcript (Janbonet al., 1998, 1999; reviewed in Rustchenko, 2003).Causal relationship between the Sou+ phenotype,the increase of SOU1 expression and chromosome5 monosomy was proved by following chromo-some 5 alterations in 13 sequential series of Sou+and Sou− derivatives from two strains. The mono-somic (Sou+) and disomic (Sou−) derivatives forchromosome 5 consistently showed increase anddecrease, respectively, of SOU1 mRNA level (seeRustchenko, 2003, for more evidence). It has beensuggested that chromosome 5 contains a negativeregulator(s), CSU (control of sorbose utilization)that inhibits expression of SOU1 when presentin two copies. Previously, two negative regula-tors involved in another important phenotype ofC. albicans, fluconazole resistance, were reported(Alarco et al., 1994; Talibi and Raymond, 1999).In our search for CSU, we used a C. albicans totalgenomic library in order to clone a sequence carriedon chromosome 5, which would inhibit Sou+ cellgrowth on an L-sorbose plate. However, we foundseveral other candidate sequences distributed overdifferent chromosomes, which are currently underinvestigation. One of these candidate sequencesreported herein corresponded to an essential geneBMH1 carried on chromosome 1. The postulatedmajor negative regulator CSU, carried on chromo-some 5, will be described elsewhere.

An important aspect of the Sou+ mutants forma-tion on an L-sorbose plate is the dramatic increaseof mutational rates within the first few days aftercontact with L-sorbose (Janbon et al., 1999), a phe-nomenon called adaptive mutagenesis (reviewed inJanbon et al., 1999; Foster 1999; Lombardo et al.,1999; Rosenberg, 2001). The mechanisms respon-sible for the loss of one homologue of chromosome5, as well as for the increase of mutational rates, arenot established, although we earlier suggested thatchromosome non-disjunction can provide a loss ofone homologue (Janbon et al., 1998). In this paperwe addressed and dismissed the possibility that C.albicans BMH1 controls the frequency of Sou+mutant formation on a L-sorbose plate due to mono-somy of chromosome 5.

We found that BMH1 mRNA level controlsSOU1 mRNA level in a reverse fashion. Although

multiple functions are attributed to 14–3–3 pro-teins, their role in utilization of secondary carbonsources in yeast has not been previously reported.

Materials and methods

Strains, media, and growth conditions

C. albicans laboratory Ura− strain CAF4-2 (Fonziand Irwin, 1993) was used to obtain geneticallymodified strains. Loci BMH1 and LEU2 of strainCAF4-2 were chosen to generate Ura+ sequen-tial mutants, having one allele of BMH1 eitherdeleted, CA88 and CA91, and CA110 and CA114,or deleted and reconstituted, CA111 and CA115.In addition, we used the LEU2 locus to gen-erate the Ura+ Sou− control strains, CA61 andCA62. The sequential mutants were used, in turn,to obtain six Ura+ Sou+ mutants, CA183-CA188,on a L-sorbose plate, which acquired the growthdue to monosomy of chromosome 5. An excep-tional Ura−Sou+ mutant, Sor17, that was derivedfrom the strain CAF4-2, also on an L-sorboseplate, acquired a chromosome rearrangement dif-ferent from the usual monosomy of chromosome5 (E. Rustchenko, 2003). Sor17 is convenientlyused when a Sou+ mutant is needed for geneticmanipulations (Janbon et al., 1998) because, unlikemonosomic Sou+ mutants, the population of Sor17cells does not contain a portion of Sou− rever-tants having spontaneously produced duplication ofchromosome 5 when grown on rich medium (seeJanbon et al., 1998). Transforming strain CAF4-2 with different replicative plasmids, all of whichwere derived from low copy number pRC2312(Cannon et al., 1992), generated another group ofC. albicans strains, in which genes SOU1 andBMH1 were overexpressed either separately, forcontrol assays, or together. All C. albicans strainsare presented in Tables 1 and 2.

Saccharomyces cerevisiae strain B-6929 (MAT ahis3-�1 trp1-289 ura3-52) (Janbon et al., 1998)and Escherichia coli XL1-Blue strain (Gough andMurray, 1983) were used for the preparation ofplasmids and other manipulations.

Yeast extract/peptone/dextrose (YPD), syntheticdextrose (SD) and other media with various car-bon sources, L-sorbose, glycerol, lactate, galactoseor D-arabinose, have been described previously(Rustchenko et al., 1994, 1997; Sherman, 2002).

Copyright 2004 John Wiley & Sons, Ltd. Yeast 2004; 21: 685–702.

14–3–3 protein in carbon metabolism of C. albicans 687

Table 1. C. albicans strains used to study the role of BMH1 copy number on SOU1 expression

Strain Description Phenotype

Original strainCAF4-2 BMH1/BMH1 ura3-�::imm434/ura3-�::imm434 LEU2/LEU2 SOU1/SOU1 Sou− Ura−

Strains with artificial genomic alterations sequentially derived from CAF4-2CA61, CA62 Control strains, same as CAF4-2, except LEU2/LEU2::p[LEU2 URA3] Sou− Ura+CA88, CA91 Same as CAF4-2, except BMH1/bmh1-�::hisG-URA3-hisG Sou− Ura+CA94, CA98 Same as CA88, CA91 except BMH1/bmh1-D::hisG and monosomic for chromosome 5 Sou+ Ura−CA110, CA114 Same as CA94, CA98 except LEU2/LEU2::p[LEU2 URA3] and disomic for chromosome 5 Sou− Ura+CA111, CA115 Same as CA94, CA98 except LEU2/LEU2::p[LEU2 URA3 BMH1] and disomic for

chromosome 5Sou− Ura+

Sou+ mutants obtained on L-sorbose plate in this workCA183-CA187 Same as CA88, CA91 except monosomic for chromosome 5 Sou+ Ura+CA188 Same as CA111 except monosomic for chromosome 5 Sou+ Ura+

Sou+ mutant obtained on L-sorbose plate and used as a recipient strain to clone BMH1Sor17 CAF4-2 with a chromosomal rearrangement different than monosomy of chromosome 5 Sou+ Ura−

CAF4-2 strain transformed with different replicative plasmidsCA182 pRC2312, Control strain Sou− Ura+CA102CA103

pCA88 Overexpression of SOU1pCA89 Overexpression of SOU1

}Different orientation of SOU1 Sou+ Ura+

CA181 pCA121 Overexpression of BMH1 Sou− Ura+CA106CA107

pCA102 Overexpression of BMH1 and SOU1pCA103 Overexpression of BMH1 and SOU1

}Different orientation of BMH1 Sou− Ura+

Table 2. Abbreviated genotypes and some phenotypes

Copy number FrequencyAbbreviated Position Chromo- of Sou+

Strain genotype of URA3 SOU1 BMH1 some 5 Phenotype mutants

CA61,CA62

SOU1/SOU1 BMH1/BMH1 LEU2 2 2 2 Sou− Ura+ 3 × 10−4

CA88,CA91

SOU1/SOU1 BMH1/bmh1 BMH1 2 1 2 Sou− Ura+ 4 × 10−3

CA94,CA98

SOU1/SOU1 BMH1/bmh1 NA 2 1 1 Sou+ Ura− NA

CA110,CA114

SOU1/SOU BMH1/bmh1 LEU2 2 1 2 Sou− Ura+ ND

CA111,CA115

SOU1/SOU1 BMH1/bmh BMH1 LEU2 2 2 2 Sou− Ura+ 2 × 10−4

CA183-CA187

SOU1/SOU1 BMH1/bmh1 BMH1 2 1 1 Sou+ Ura+ NA

CA182 SOU1/SOU1 BMH1/BMH1 Plasmid 2 2 2 Sou− Ura+ NACA103 SOU1/SOU1 p[SOU1]N BMH1/BMH1 Plasmid >2 2 2 Sou+ Ura+ NACA181 SOU1/SOU1 BMH1/BMH1 p[BMH1]N Plasmid 2 >2 2 Sou− Ura+ NACA106,CA107

SOU1/SOU1 p[SOU1]N BMH1/BMH1 p[BMH1]N Plasmid >2 >2 2 Sou− Ura+ NA

NA, not applicable; ND, not determined.

Plates containing 5-FOA medium were used toselect for the eviction of one copy of ectopic URA3that resulted in Ura− strains (Boeke et al., 1984).Spider medium (Liu et al., 1994) was used to

induce hyphal growth. Medium 199 (Gibco BRL,Gaithersburg, MD) and 20% bovine serum (Kohlerand Fink, 1996) were used to induce germ tube for-mation. Uridine (25 µg/ml) was added according



to Fonzi and Irwin (1993) when needed. Strainswere grown at 37 ◦C. Storage and maintenanceconditions of C. albicans, designed not to inducechromosomal instability, were described previously(Perepnikhatka et al., 1999).

Plasmids

The integrative pRC3915 and low-copy replica-tive pRC2312 plasmids, both containing URA3 andLEU2 genes (Cannon et al., 1992), the plasmidpCUB-6 containing the hisG-URA3-hisG construct(Fonzi and Irwin, 1993), and the plasmid pUC19(Yanisch et al., 1985) have been described previ-ously. Other plasmids, denoted by the prefix pCA,were constructed for this study from the above-mentioned plasmids, as described below.

Plasmid constructions

pCA79 contains an original 5.9 kbp genomic frag-ment encompassing BMH1, which is described inFigure 1A. This plasmid was retrieved from a totalgenomic library constructed by insertion of frag-ments at the BamHI site of pRC2312 (Janbonet al., 1998). An EcoRI fragment of 3.0 kbp car-rying the BMH1 gene was recovered from pCA79and inserted into the SmaI site of pRC3915 andpRC2312, thus creating, respectively, pCA116, anintegrative plasmid, and pCA121, a replicativeplasmid, both containing BMH1. Special replica-tive plasmids containing two genes, BMH1 andSOU1, whose interaction is analysed herein andwhich differ in the orientation of BMH1, wereconstructed and designated pCA102 and pCA103.First, a 1.5 kbp SmaI/Sal I fragment carrying theSOU1 gene was removed from pCA2 (Janbonet al., 1998) and inserted in the SmaI site ofpRC2312, thus creating pCA88 and pCA89, whichdiffered in the orientation of the insert. The ori-entation of the SOU1 gene did not effect over-expression of this gene. Subsequently, a 3.0 kbpEcoRI fragment carrying the BMH1 gene wasremoved from pCA79 and inserted in the SmaIsite of pCA89. pCA119 contains a URA3 blasterdesigned to disrupt BMH1 genes. To construct thisplasmid, the 3.0 kbp EcoRI/EcoRI fragment con-taining BMH1 from pCA79 was first inserted inpUC19. The resulting plasmid was then digestedwith Bcl I to remove a 540 bp internal region of

1 kb

2.3 kb

5.9 kb

3.0 kb

Eco

RI

Eco

RI

Eco

RI

Pst

I

Pst

I

Sm

lI

Sm

lI

Bcl

I

BcI

lH

indI

II

URA 3

hisG hisG

P2 P3

P4P1

Cla

I

Bcl

I

Bcl

I

BMH1

Probe

M 1 2 3 M 1 2 3 M 1 2 3

4.0

3.0

2.0

1.5

1.0

0.5

P1/P3 P1/P2 P1/P4

A

B

kb

Figure 1. Disruption of a single copy of C. albicans BMH1gene. (A) Physical map of the 5.9 kbp genomic fragmentcarrying the BMH1 gene (black arrow). Also shown arethe following: the BMH1 probe used in Southern andNorthern blot analysis; primers P1–P4 used to generatePCR fragments; and the EcoRI fragment of pCA119 used todisrupt BMH1. (B) Identification of BMH1/bmh1 mutants byPCR. The results are shown with the parental strain CAF4-2(BMH1/BMH1) (lane 1); with strain CA91 (BMH1/bmh1hisG-URA3-hisG) (lane 2) and strain CA98 (BMH1/bmh1 hisG)(lane 3). M denotes the 1 kbp ladder marker. Combinationsof primers (see Figure 1A) are indicated at the top of the gel.P1/P3 revealed the presence of the hisG-URA3-hisG sequencein strain CA91 as a 2.1 kb fragment, but not in CAF4-2 orin CA98. P1/P2 revealed the hisG segment in both CA91and CA98, as a 0.8 kbp fragment, but not in CAF4-2. P1/P4revealed both the intact allele of BMH1 (1.2 kbp) in all thestrains and the replacement of BMH1 gene by hisG segmentin CA98 (1.7 kbp). The 4.2 kbp band containing the lengthof hisG-URA3-hisG sequence in CA91 is absent, probablybecause it was too long to be amplified

the BMH1 open reading frame (ORF), and subse-quently the hisG-URA3-hisG cassette was ligatedto the remaining portion of the plasmid.



Cloning procedures

C. albicans Ura− Sou+ mutant Sor17, which doesnot frequently revert to the Sou− phenotype (seeabove), was transformed with a genomic library(Janbon et al., 1998); the transformants were han-dled as described by Janbon et al. (1998) and ulti-mately screened for those which did not grow orgrew poorly on an L-sorbose plate. The plasmidswere subsequently evicted from the Sou− cells on a5-FOA plate and the cells returning to the originalSou+ phenotype were determined. Finally, DNAwas extracted from each Sou− transformant thatwas grown in liquid SD medium, and transformedinto S. cerevisiae strain B-6929 to resolve polymer-ized plasmids (Goshorn et al., 1992; Janbon et al.,1998). Due to recombinogenic processes within thevector that was passed through S. cerevisiae, a C.albicans genomic fragment was frequently lost;therefore, DNA was extracted from 20 S. cerevisiaecolonies and further analysed. Cells were grown inliquid SD medium containing histidine and trypto-phan but lacking uracil. E. coli was transformedwith DNA from S. cerevisiae for amplification.Plasmids purified from the E. coli transformantswere used to transform C. albicans Sor17 to con-firm the initial trait.

Probes

The use of the SOU1 gene as a probe was describedpreviously by Janbon et al. (1998). The BMH1probe was created from a 1.7 kb ClaI/PstI frag-ment derived from the plasmid pCA79 (Figure 1A).

DNA sequence analysis

DNA sequences were determined by the CoreNucleic Acid Laboratory, University of RochesterMedical Center.

Gene disruption, integration and polymerasechain reaction analysis

The BMH1 gene was disrupted using the URA3blaster method (Fonzi and Irwin, 1993). Regionscontaining the disrupted BMH1 gene in heterozy-gous mutants were analysed by polymerase chainreaction (PCR) using primers P1 (5′-CAATTGC-AAGTATGTGG-3′), P2 (5′-GCGCGTGGCGATG-CACATGGTCAG-3′), P3 (5′-GCTGTGCTACTG-GTGAGG-3′) and P4 (5′-CACCAATGCTCTCCT-TCG-3′) (Figure 1). PCR reactions were carried out

in a 50 µl volume with 30 cycles of amplifica-tion (2 min at 95 ◦C; 1 min at 52 ◦C; and 5 minat 72 ◦C) with a 2 min jump-start at 95 ◦C and afinal 10 min at 72 ◦C. Typical reaction mixturescontained 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2,50 mM KCl, 0.2 mM dNTPs, 10 pmol primers,100 ng genomic DNA, and 5 units Pfu Turbo

DNA polymerase (Stratagene, La Jolla, CA).

Northern blot analysis

Cells were removed from the −70 ◦C stock, inoc-ulated in liquid SD, and grown to the densityof approximately 107 cells/ml. Total RNA wasextracted according to Russo et al. (1991), sep-arated, blotted and hybridized with 32P-labelledBMH1, SOU1 or ACT1 probes, as described byDas et al. (2000). The hybridization signals ofBMH1 and SOU1 transcripts were quantitatedusing Phosphorimager SI (Molecular Dynamics)and normalized against the ACT1 signal.

Southern blot analysis

The BMH1 probe was labelled with digoxigenin-dUTP (DIG DNA Labelling and Detection kit;Boehringer-Mannheim, Indianapolis, IN), accord-ing to the manufacturer’s instructions, and hybri-dized to blots of chromosomes separated by pulsed-field gel electrophoresis (PFGE)

Studies of adaptive mutagenesis

The formation of Sou+ mutants from Sou− strainson L-sorbose medium was analysed as previouslydescribed (Janbon et al., 1999) with minor mod-ifications. Instead of regular agar, LE agarose(Boehringer Mannheim, Indianapolis, IN) was used(Mack et al., 1994), which made the S. cerevisiaescavenger strain unnecessary. Also, a cell masswas used instead of independent subclones pre-pared for fluctuation tests. Approximately 1 × 105

colony forming units (cfu) were spread on duplicateL-sorbose plates, incubated and monitored dailyat approximately the same time for Sou+ colonyappearance. A magnifying glass was used to recordmicroscopic colonies.

In order to analyse cell viability, the same cellsuspension was appropriately diluted and plated induplicate on a number of L-sorbose plates. Everyother day for 8 days, the entire agar disc of a



duplicate plate was transferred to the surface ofan YPD plate, allowing growth of viable cells.As the proportion of cells still viable on 5-FOAplates decreased over time, agar discs with higheramounts of cells were transferred.

The lag time between two events, mutationand appearance of the corresponding microscopiccolony on a plate, can be roughly deduced byre-plating Sou+ mutants and recording the firstday that colonies appeared on the plate, whichis called a ‘reconstruction’ experiment. In orderto perform reconstruction experiments, 14 Sou+colonies, seven each from days 4 and 12, wereplated on L-sorbose medium at approximately500 cfu/plate and monitored daily.

In order to calculate adjusted rates of mutagen-esis on a L-sorbose plate, we considered the dif-ference in time between the presumed mutationalevent and the appearance of a corresponding micro-scopic colony on a plate, as determined by thereconstruction experiment. This approach was firstsuggested by Janbon et al. (1999). Adjusted ratesof formation of Sou+ mutations (mutations/viablecell/day) were calculated using the deduced day ofmutation appearance.

Preparation of a population of cells thatcorresponds to an original population

Cells were removed from −70 ◦C stocks andstreaked for independent colonies on YPD platessupplemented with uridine. After incubation manyyoung dispersed colonies were collected and mixedtogether to prepare a population of cells thatapproximately corresponds to the original popula-tion.

Phenotypic assay on L-sorbose medium

For a spot dilution assay, a strain was removedfrom −70 ◦C stock, streaked for independentcolonies on SD medium and incubated until theyoung colonies appeared. In order to apply cellsto an L-sorbose plate, the following two methodswere used:

1. A total of 16 colonies were individually pre-pared as cell suspensions in distilled water andspotted using a replicator on L-sorbose and SDplates. A total of 16 spots of the control strainwere similarly prepared and placed on the sameplates. This approach allowed more accurate

comparison of control and experimental strainscarrying unstable plasmids.

2. Independent colonies were prepared to imitatea reconstitution of an original population (seeabove). Cells were re-suspended in distilledwater, the concentration of the cell suspen-sion determined using a haemocytometer andserial 1/10 dilutions prepared, starting with aconcentration of approximately 9 × 107 cfu/ml.Approximately 5 µl portions of the suspensionswere spotted, such that the number of cellsper spot diminished from left to right, andwith the initial spot containing approximately4.5 × 105 cfu.

Test for hypha and germ tube formation

For hypha formation, cells were pre-grown asindependent colonies on YPD plates supplementedwith uridine, prepared to imitate a reconstitution ofthe initial population (see above), and subsequentlysuspended in distilled water. The suspension wasdiluted and approximately 10 cfu/plate were platedon Spider medium (Liu et al., 1994), and Spidermedium supplemented with uridine. To assay germtube formation, cells were prepared as above anddiluted in either medium 199 or 20% bovineserum in a flat-bottomed microtitre dish. The cellsforming germ tubes within a microscopic field werecounted in suspensions containing 1 × 104 or 1 ×103 cells/ml after incubation for 2 h or overnight.

Pulsed-field gel electrophoresis

Different running conditions for either orthogo-nal field alternating gel electrophoresis (OFAGE)or contour-clamped homogenous electrophoreticfield (CHEF) systems were used to separate pre-cisely different portions of a chromosomal pattern(Janbon et al., 1998; Perepnikhatka et al., 1999;Rustchenko-Bulgac and Howard, 1993). The entireelectrophoretic karyotype could thus be recon-structed from at least three separations that coveredthe total range of C. albicans chromosome lengths.

Results

Cloning of the BMH1 gene

The BMH1 gene was isolated from a C. albicanstotal genomic library (see Materials and methods)



and prepared in the replicative plasmid pRC2312,which is maintained at two to three copies/cell(Cannon et al., 1992). Nine plasmids that inhibitedthe Sou+ phenotype on an L-sorbose plate wereidentified from approximately 6200 transformants.Of the nine plasmids, one, pCA79, completelyinhibited the Sou+ phenotype and was furtherinvestigated. Restriction analyses of the pCA79revealed a 5.9 kbp genomic fragment (Figure 1A).Further analyses of this genomic fragment wascarried out with a specially designed system, whichincluded Ura− Sou− recipient strain CAF4-2, andreplicative plasmids pCA88 and pCA89 that carriedSOU1 in different orientation (see Materials andmethods). Either the entire 5.9 kbp fragment or itsportions were inserted into pCA88 and pCA89 in adifferent orientation, transformed in CAF4-2 cellsand cell phenotypes were tested on an L-sorboseplate. This approach had two advantages. First,functional analysis could be carried out within thegenetic background of a parental strain, which,unlike Sou+ mutants monosomic for chromosome5 or an exceptional Sou+ mutant, did not containmultiple genes with altered expression, as well ashaving the same ratio of SOU1 and a putativeregulator. Second, the approach allowed us to studythe interaction of the two genes independent ofthe variability related to the copy number of theunstable plasmid.

The growth of the control strains CA102 andCA103 overexpressing SOU1 on either SD or L-sorbose media was independent of the orienta-tion of the gene. On SD medium, the growthwas represented by dense papillae, often resem-bling confluent growth, due to plasmid instabil-ity. A similar type of growth was observed forthe control strains on L-sorbose medium, althoughthe amount of growth compared with that on glu-cose plates at the same days was always less (datanot presented). When strain CA184 overexpress-ing the original 5.9 kbp genomic fragment, togetherwith SOU1, was tested on L-sorbose medium, nei-ther conditional confluent growth or any kind ofpapilliated growth was observed after 3 weeks ofincubation (data not presented). Similarly, strainsCA106 and CA107 overexpressing 3.0 kbp por-tion of 5.9 kbp fragment that resulted from EcoRIdigest (Figure 1A), in different orientation, showedno growth or papillae on L-sorbose plates (sum-marized in Table 1). However, when the 5.9 kbp

fragment was cut in two with HindIII, both frag-ments lost the ability to inhibit the Sou+ phenotype(data not presented). In addition, strains carryingthe 5.9 kbp and 3.0 kbp fragments showed substan-tially more variability in growth among their sub-clones when plated on control SD medium, rangingfrom confluent growth in some spots to some papil-lae in others, than did the control strains. This isprobably due to the presence of the negative regula-tor, and not due to the size of the plasmid, becausethe fragments that resulted from HindIII digestionand could not inhibit the Sou+ phenotype had nosuch variability. It is unknown why the presence ofthe negative regulator causes higher plasmid insta-bility.

Sequencing the region on both sides of theHindIII site revealed a 795 bp open reading frame(ORF), which is indicated with a black arrow inFigure 1A. This 795 bp ORF potentially encodes a29 kDa protein. The deduced protein sequence of265 amino acids was 78% and 60–64% identical,respectively, to the S. cerevisiae Bmh1p and to allseven human isoforms of the 14–3–3 protein. Byanalogy with the 14–3–3 protein in S. cerevisiae,we denoted the gene BMH1. The gene sequencewas identical to the C. albicans sequence depositedby D. Cognetti and J. Sturtevant (BMH1 ; Acces-sion No. AF038154). In contrast to other organ-isms, only one 14–3–3 isoform was found in C.albicans, as determined by Southern blot analysis,which was performed under both high- and low-stringency conditions (data not presented). Also,Cognetti et al. (2002) did not find homologues ofBMH1 in C. albicans, using Southern and Westernblot analyses. Furthermore, no similar sequencesindicative of paralogous genes were uncoveredby searching the C. albicans genomic databaseof the Standford Candida Project (http://www-sequence.stanford.edu/group/candida/index.html).

Chromosomal assignment of the BMH1 gene

The BMH1 gene was assigned to chromosome 1by Southern blot analysis (data not presented).

Disruption and reintegration of BMH1 gene

Plasmid pCA119, containing the BMH1 URA3blaster (see Materials and methods), was cut withSml I to remove the vector, and the appropriate



fragment was transformed in the strain CAF4-2.The resulting Ura+ BMH1/bmh1 mutants, CA88and CA91, that were derived from two indepen-dent randomly chosen transformants, as well astheir respective Ura− derivatives, CA94 and CA98(Table 1), selected on 5-FOA medium due to evic-tion of URA3, were confirmed by PCR (Figure 1B).In order to disrupt the second copy of BMH1, CA94and CA98 were transformed with the Sml I digestof plasmid pCA119, and a total of 68 transformantswere screened by PCR. Except for three transfor-mants in which the URA3 blaster was insertedelsewhere in the genome, all remaining transfor-mants had the blaster inserted into the first dis-rupted allele, leaving the second allele intact. Lackof recovery of a double disruptant is consistent withrecent report of Cognetti et al. (2002) that BMH1is an essential gene.

The integrative vector pCA116, carrying BMH1,was linearized with SphI to target integrationto the LEU2 locus, and both Ura− heterozy-gous BMH1/bmh1 mutants, CA94 and CA98,were transformed, thus generating two reconsti-tuted Ura+ BMH1/bmh1BMH1 strains, CA111 andCA115, respectively, having a second ectopic copyof BMH1.

To obtain Ura+ derivatives from Ura− heterozy-gous BMH1 /bmh1 ‘pop-outs’, CA94 and CA98,the empty vector pRC3915 was targeted to theLEU2 locus, as described above, thus generat-ing two independent transformants, CA110 andCA114. Similarly, to obtain BMH1/BMH1 controlstrains that are Ura+, the empty vector pRC3915was integrated into the LEU2 locus of the Ura−original strain CAF4-2, thus generating two inde-pendent transformants, CA61 and CA62.

Sou phenotypes and electrophoretickaryotypes of sequential derivatives of strainCAF4-2

According to proposed hypothesis, the copy num-ber of chromosome 5 controls the copy numberof the major negative regulatory gene, CSU, thatis carried on this chromosome, and CSU thencontrols the Sou phenotype (Janbon et al., 1998).Conversely, the disruption of one copy of majornegative regulator CSU might imitate the loss ofone homologue of chromosome 5, resulting in cellgrowth on L-sorbose medium. Accordingly, thedisruption of one copy of any other secondary

negative regulator might also affect, although toa lesser degree, Sou phenotype. All the above-mentioned sequential derivatives of strain CAF4-2having either a single copy of BMH1 or a sec-ond copy ectopically integrated in the genome wereanalysed for their electrophoretic karyotypes (datanot presented), and growth on L-sorbose mediumwith spot dilution assay (Figure 2; see Materi-als and methods), as summarized in Table 2. Asexpected, and as presented in Figures 2B and 2Cfor the strain CA61, the Ura+ BMH1/BMH1 con-trol strains CA61 and CA62 were Sou−, but accu-mulated Sou+ mutants (see Introduction). Ura+ sin-gle deletants BMH1/bmh1, CA88 and CA91, whichwere not exposed to 5-FOA medium, had a normalchromosomal pattern similar to that of the origi-nal strain CAF4-2, and were also Sou−; however,contrary to the controls, they gave rise to a sub-stantially higher number of Sou+ mutants. TheirUra− ‘pop-outs’, CA94 and CA98, acquired mono-somy of chromosome 5 and Sou+ phenotype (datanot presented). Similar chromosome alteration wasfound by us in a systematic study of electro-karyotypes of mutants selected on 5-FOA medium,and will be discussed elsewhere (M. Welling-ton and E. Rustchenko, unpublished data). Ura+mutants BMH1/bmh1BMH1, CA111 and CA115,which derived from the above-mentioned mutantswith monosomic chromosome 5 due to reintroduc-tion of a second copy of BMH1, possessed theduplicated remaining homologue of chromosome5, and recovered the Sou− phenotype. We attributechromosome duplication to spontaneously occur-ring non-disjunction, which is common in lowerfungi. Cultivation of a mutant monosomic for chro-mosome 5 in liquid rich medium was shown tofavour proliferation of cells that reconstituted dis-omy of this chromosome because of the cell higherrates of growth, presumably due to gene balance(Janbon et al., 1998). We assume that repetitivegrowth, before and after the BMH1 reintroduction,of our monosomic mutants in liquid YPD mediumresulted in enrichment of the cultures with normaldiploid cells, which was then followed by the selec-tion of faster growing diploid transformants on SDmedium.

Although mutants reconstituted for the secondcopy of BMH1 and chromosome 5 restored Sou−phenotypes, the number of Sou+ mutants that theyproduced on an L-sorbose plate was intermediatebetween control and single deletant strains. The



Figure 2. Comparison of Sou+ colonies produced from cell suspensions spotted on the same L-sorbose plate by Ura+ Sou−strains having either one or two copies of the BMH1 gene. (A) Control growth on glucose medium (SD) after 2 days ofincubation of the following strains: 6, BMH1/BMH1 control strain CA61; 7 and 9, BMH1/bmh1 mutants CA88 and CA91,respectively; 8 and 10, BMH1/bmh1 BMH1 reconstituted mutants CA111 and CA115, respectively. Suspensions of eachstrain were prepared as serial 1/10 dilutions, with a starting concentration of approximately 9 × 107 cfu/ml. Approximately5 µl portions of the suspensions were spotted with a replicator, such that the number of cells per spot diminished fromleft to right, and with the initial spot containing approximately 4.5 × 105 cfu. (B) and (C) Growth of strains listed in (A) onL-sorbose medium after 6 and 8 days of incubation, respectively. Only two spots containing 4.5 × 105 and 4.5 × 104 cfuare shown in each. The genotypes are abbreviated as follows: BMH1/BMH1, +/+; BMH1/bmh1, +/−; and BMH1/bmh1BMH1, +/−+

lack of complete recovery, which was reproducedin several experiments, indicated changes in thegenetic constitution as a result of the manipula-tions. As we recently discovered, the exposure to5-FOA medium induces high genetic instabilityin addition to various chromosome alterations (M.Wellington and E, Rustchenko, data not presented).In this respect, we tested whether the transfor-mation procedure itself might interfere with Souphenotype. The original strain CAF4-2 was sub-jected to a mock transformation, and subsequentlytested for the growth on an L-sorbose plate usingspot assay. No difference in comparison with theuntreated strain CAF4-2 was observed.

Recently, exogenously supplied uridine wasreported to restore the ability to form hyphae inHWP1 homozygous null mutants (L. L. Sharkey,W.-L. Liao and W. A. Fonzi, personal communica-tion), which prompted us to investigate a possibledependence of the Sou phenotype on the copy num-ber and genomic position of gene URA3. Controlstrains, as well as single deletants and reconstitutedmutants, were spotted on L-sorbose plates contain-ing uridine. The production of Sou+ mutants byeach strain appeared to be identical with or withouturidine (data not presented), thus indicating that

Sou phenotype is not dependent on the level ofexpression of genomic copies of URA3.

Notable difference in growth between twooriginal mutants with deleted copy of BMH1

It is important to note that, despite deriving fromtwo transformants on the same selective SD plate,single deletants CA88 and CA91 were different.CA91 was slow-growing on either control glucoseor test sorbose media (Figure 2). Furthermore,compensatory mutation(s) for growth occurred insome colonies of CA91 growing faster on sorbosemedium.

The mutants lacking one copy of the BMH1gene have a higher survival on L-sorbosemedium and an increased ability to producemonosomic Sou+ mutants

An important question to address was whetherBMH1 controls rates of Sou+ mutant formation bymeans of the rate of loss of chromosome 5. In orderto determine the rates of mutagenesis, we stud-ied adaptive mutagenesis in a representative pair ofthe normally growing sequential Ura+ derivatives



CA88, BMH1/bmh1, and CA111, BMH1/bmh1BMH1. As presented in Materials and methods,our experiments included the following: daily mon-itoring of the appearance of Sou+ mutants; sur-vival of the original Sou− cells on toxic L-sorbose medium (Brockman and De Serres, 1963);reconstruction experiment; and calculation of theadjusted rates of mutagenesis. We found that in alltested strains the Sou+ colonies appeared continu-ously over a period of 2 weeks (Figure 3A), despitethe progressive death of Sou− cells (Figure 3B).The average frequencies of Sou+ mutants in thecontrol strain CA61, the single deletant CA88,and the reconstituted CA111 were 3 × 10−4, 4 ×10−3 and 2 × 10−4, respectively. Reconstructionexperiments were performed with 14 representa-tive Sou+ mutants of CA88, BMH1/bmh1, derivedon days 4 and 12 after CA88 cells were incu-bated on an L-sorbose plate. All the mutants grewup on L-sorbose medium within 3–4 days (datanot presented), most probably indicating that theday 12 mutants appeared after the contact withL-sorbose medium, and not because they wereslow growing pre-existing mutants. All of theseresults were consistent with previously investigatedmutant appearences on a sorbose plate of C. albi-cans laboratory strain 3153A (Janbon et al., 1999),CAF4-2 and others (E. Rustchenko, unpublisheddata). The deduced lag in time of approximately4 days, which presumably separated two events(formation of a Sou+ mutation and formation ofa microscopic Sou+ colony), was used to calcu-late adjusted rates of mutagenesis. As presentedin Figure 3C, the rates of mutagenesis increasedapproximately two orders of magnitude within thefirst 4–5 days, consistent with the results of Janbonet al. (1999). Together with reconstruction experi-ment data, this result firmly established that themajority of mutations occurred after contact withL-sorbose medium due to adaptive mutagenesis (seeIntroduction). Most importantly, there was no dif-ference in rates of mutagenesis among the strainsstudied, i.e. the BMH1 copy number does not con-trol frequency of chromosome 5 monosomy duringadaptive mutagenesis. Thus, the simple explanationof BMH1/bmh1 deletants producing the increasednumber of Sou+ colonies (Figure 3A) is that thesemutants survived better on L-sorbose medium.

These results established that BMH1 controls theSou phenotype, but does not control adaptive muta-genesis by means of chromosome 5 monosomy.

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Figure 3. The results of plating Sou− cells having either oneor two copies of BMH1 on L-sorbose medium. The followingstrains are used: ž, the control CA61 (BMH1/BMH1); °, thesingle deletant CA88 (BMH1/bmh1); and �, reconstitutedmutant CA111 having two copies of BMH1 (BMH1/bmh1BMH1). (A) Accumulation of Sou+ colonies. (B) Survival ofSou− cells. (C) Adjusted rates of Sou+ mutants. Both thededuced times of formation of the mutations (top number)and the time of the appearance of the correspondingcolonies (bottom number, in parentheses) are presented.See Figure 2 for other designations

Sou+ mutants derived on L-sorbose plate fromeither single deletants or strains reconstitutedfor BMH1 contained monosomy ofchromosome 5

Although we established that BMH1 does notcontrols the rates of mutagenesis on an L-sorboseplate, it was unclear whether adaptation of single



deletants occurs by the usual means of forma-tion of monosomy of chromosome 5, similar toSou+ mutants that are derived from regular strains(reviewed in Rustchenko, 2003). A total of fiveSou+ mutants were randomly chosen from days 4,9 and 12 after plating Ura+ Sou− cells of CA88and CA91, BMH1/bmh1 single deletants, on L-sorbose medium, as illustrated in Figure 3A. Also,a single day 4 Sou+ mutant from the Ura+ Sou−strain CA111, BMH1/bmh1 BMH1, was randomlychosen. Intact chromosomes of the Sou+ mutantswere prepared and separated in PFGE, as describedin Materials and methods. All electrophoretic kary-otypes of the Sou+ mutants showed that one copyof chromosome 5 was lost (data not presented).When strains CA88, CA91 and CA111 were grownon YPD plates and their electro-karyotypes wereprepared, these strains remained disomic at chro-mosome 5 and had no changes in any of the otherchromosomes (data not presented).

The inverse dependence between the copynumber of the BMH1 gene and the amount ofthe SOU1 transcript

As exemplified in Figure 4, we have used North-ern blot analyses to study the effects of alterationof copy number of BMH1 on SOU1 mRNA. Twoseries of sequentially derived mutants, each start-ing with pre-5-FOA single deletant BMH1 /bmh1,either CA88 or CA91, were prepared, as presentedin Figure 5, Table 1 and Results. Within eachseries, a reconstituted mutant was compared witheither pre- or post-5-FOA single deletant as well aswith a control strain. Overexpression strains werealso prepared and analysed. For each experiment,every strain was grown de novo (see Materialsand methods). Because of instability, independentlygrown cultures of the same strain can result indifferent populations with different characteristics.For this reason, the measurements from each blotwere calculated as percentages of the control level,and presented as individual graphs in Figure 5.Under these circumstances, averaging and statis-tical analysis of sets of experiments would be mis-leading. The results, by and large, indicated that thelevel of the SOU1 transcript was inversely depen-dent on the level of the BMH1 transcript, althoughthe precise relative values of the BMH1 and SOU1mRNAs often varied in sequential strains from amutagenized original deletant CA91 (see below).

Figure 4. Northern blot analysing the levels of SOU1 andBMH1 transcripts in different Ura+ strains overexpressingBMH1 or/and SOU1, as well as in Ura+ Sou− strains havingeither one or two copies of the BMH1 gene. Signalswere also obtained for ACT1 transcript, as indicated. 1,CA182, control having plasmid with no insert; 2, CA181,overexpression of BMH1; 3, CA103, overexpressionof SOU1; 4 and 5, CA106 and CA107, respectively,overexpression of BMH1 and SOU1 on the same plasmid;6–10, control strain and strains with altered number ofgenomic copies of BMH1, as presented in Figure 2. Forexplanation of the strains, see Table 1

Alteration of BMH1 genomic copy number

In this study, we used the following Ura+ deriva-tives of Ura− strain CAF4-2 (BMH1/BMH1 ): thecontrol strain CA61 having URA3 incorporatedin LEU2 ; the original pre-5-FOA single dele-tants BMH1/bmh1 CA88 and CA91 having URA3in BMH1 ; the corresponding post-5-FOA singledeletants CA110 and CA114 having URA3 inLEU2 ; and the mutants BMH1/bmh1BMH1 CA111and CA115 having URA3 and BMH1 in LEU2(Table 2). Although these strains had one copy ofthe URA3 gene incorporated at either the BMH1 orthe LEU2 locus, our results indicated that neitherURA3 copy number nor its position interfered withthe Sou phenotype (see Sou phenotypes and elec-trophoretic karyotypes of sequential derivatives ofstrain CAF4-2).

The level of the BMH1 expression in thesingle deletants diminished, as expected, in allbut one graph, Figure 5B2. The SOU1 mRNAsincreased in all graphs. This dependence wasmore prominent in both pre-5-FOA CA88 and itspost-5-FOA derivative CA110, whose growth on



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Figure 5. Graphical presentation of the SOU1 and BMH1 transcript levels, as determined with five Northern blots, A–E,in which the original single deletants CA88 and CA91 and their derivatives are shown separately. Experimental values ofeach strain, as estimated by Phosphorimager, were normalized to the level of corresponding ACT1 mRNAs. Normalizedlevels of the SOU1 and BMH1 mRNAs in the control strains CA61 and CA182 were defined to be 100%, although theywere not the same. The percentage of the transcripts in the overexpression strain CA181 were calculated against theCA182, whereas the strains with altered genomic copies of BMH1 were calculated against CA61. Both CA61 and CA182are designated as ‘controls’ in the figure text. Graphs of the following Ura+ Sou− strains are presented: �, controls CA182(BMH1/BMH1 p[URA3 LEU2]N) and CA61 (BMH1/BMH1); ž, pre- and post-5-FOA single deletants (BMH1/bmh1) CA88and CA110, respectively; °, pre- and post-5-FOA single deletants (BMH1/bmh1) CA91 and CA114, respectively; � and �,reconstituted mutants (BMH1/bmh BMH1) CA111 and CA115, respectively; and ♦, CA181 (BMH1/BMH1 p[BMH1]N). Thedashed lines designate either an unexpected lack of diminution of BMH1 (B2) or curves, which ignore an outlying point(A2, B1 and E2). See Figure 2 for other designations



different media appeared to be normal (see Figure 2for CA88). This series of sequential derivativesdoes not seem to be heavily mutagenized bygenetic manipulations (see below). The levels ofBMH1 and SOU1 expression ranged between38–73% and 155–199% in CA88, respectively(Figures 5A1–5C1), and 60–74% and 133–182%in CA110, respectively (Figures 5D1 and 5E1).

The amount of SOU1 mRNA in slow-growing,mixed populations of pre-5-FOA single dele-tant CA91 (Figure 2) and its post-5-FOA deriva-tive CA114 (data not presented) also increased,ranges 186–194% for CA91 (Figures 5A2–5C2)and 127–219% for CA114 (Figures 5D2 and 5E2).However, contrary to expectation, BMH1 mRNAlevel was not always notably diminished, e.g. 84%(Figure 5A2), or practically not diminished, e.g.99% (Figure 5B2). It appeared that post-5-FOACA114, which is separated from pre-5-FOA origi-nal transformant CA91 by several rounds of growthin liquid YPD medium, showed more dimin-ished levels of BMH1 mRNAs, 60% and 74%(Figures 5D2 and 5E2). Obviously, the inconsis-tency with BMH1 expression in pre-5-FOA singledeletant CA91 cannot be attributed to toxic 5-FOAexposure, which was shown by us to induce a largespectrum of chromosome alterations, as well ashomologous recombination and high instability (M.Wellington and E. Rustchenko, unpublished data).In this work we also report the loss of one homo-logue of chromosome 5 in CA94 and CA98 dueto exposure to 5-FOA medium (see Sou pheno-types and electrophoretic karyotypes of sequentialderivatives of strain CAF4-2). However, other pro-cedures could be also mutagenic, e.g. although thetransformation procedure did not appear to interferewith Sou phenotype (see Sou phenotypes and elec-trophoretic karyotypes of sequential derivatives ofstrain CAF4-2), it could nevertheless interfere byproducing mutations that affected BMH1 expres-sion in CA91. Another possibility is the mutagenicconsequences of homologous integration in thegenome (M. A. Kabir and E. Rustchenko, unpub-lished data). We believe that the mutagenic pro-cedure led to diminished growth of the originalsingle deletant CA91, which was followed bycompensatory mutations for growth in some cells(Figure 2), thus creating a heterogeneous and prob-ably an unstable population (see also Notable dif-ference in growth between two original mutantswith deleted copy of BMH1 ). Apparently, the

relationship between BMH1 and SOU1 mRNAscan occasionally be distorted in such population.It is important to note that independently preparedbatches of the same heterogeneous population dif-fer to different extents. This may explain why anunexpected lack of diminution was obtained insome, but not all, repeats.

When the second copy of BMH1 was reintro-duced in mutants CA111 and CA115, the SOU1and BMH1 transcript levels consistently dimin-ished and increased, respectively, to the approxi-mately normal level in 7/10 cases. In three excep-tions, Figures 5A2, 5B1 and 5E2, both BMH1 andSOU1 mRNA amounts were increased. A sim-ple explanation for these exceptions could be thatconstruction of the reconstituted mutants CA111and CA115 involved passage of CA88 and CA91through mutagenic 5-FOA medium, which inducedinstability. Thus, the abnormal mRNA increase insome, but not all, graphs could be explained byovergrowth of the mutated cells in some cultures.Once again, more inconsistency in form of largervariability occurred in CA115 (Figures 5A2 and5E2), which was derived from a slow-growingmixed population. Presumably, CA115 includedmutations that were induced by BMH1 disruptionand exposure to 5-FOA.

A stronger dependence between BMH1 genomiccopies and SOU1 transcript could not be demon-strated due to essentiality of BMH1, which pre-vents recovery of a double disruption bmh1 /bmh1(see above). However, the less mutagenized orig-inal transformant CA88, as well as its sequentialderivative CA111, showed quite clearly that theamount of SOU1 mRNA is inversely related tothe amount of BMH1 mRNA. Despite some incon-sistencies that were mostly related to the debili-tated original transformant CA91 and its sequentialderivative CA115, the overall data support pro-posed dependence.

Overexpression of BMH1 and concomitantexpression of SOU1 and BMH1 on replicativeplasmids

We further analysed the level of SOU1 transcriptin CAF4-2 cells that were transformed withvarious derivatives of the low copy number vectorpRC2312. The control strain CA182, carryingpRC2312, was used to measure the basal levelof BMH1 transcript, as well as the normal lowlevel of SOU1 transcript, which does not allow



confluent growth on an L-sorbose medium (seeIntroduction). We found relatively small, but con-sistent, increase by approximately 37% in sixmeasurements ranging from 133% to 145%, ofBMH1 mRNA that was either overexpressedalone in strain CA181 on pCA121 (BMH1 /BMH1[BMH1 ]N; Figures 4, 5B1 and 5C1) or overex-pressed in strains CA106 and CA107 together withSOU1, plasmids pCA102 and pC103, respectively(BMH1 /BMH1 [BMH 1 SOU 1]N; data not pre-sented). In strain CA181 overexpressing BMH1alone, this increase of BMH1 mRNA producedthe diminution of mRNA of two regular genomiccopies of SOU1 to approximately 75% of the con-trol level (Figures 4, 5B1 and 5C1). These data areconsistent with the finding that SOU1 expressionis increased in mutants having one copy of BMH1deleted, as presented above.

SOU1 mRNA level increased by 214–277%in overexpression strain CA103, which containedplasmid pCA89 carrying SOU1 gene alone. Bycontrast, the overall SOU1 transcript level dimin-ished by approximately 82% in strains CA106 andCA107 (BMH1 /BMH1 [BMH 1 SOU 1]N) overpro-ducing both genes BMH1 and SOU1, consistentwith the view that overexpression of BMH1 dimin-ishes the level of the SOU1 mRNA. Nevertheless,in these strains we observed variability of 109%,119%, 127% and 295% in four measurements ofSOU1 mRNA. Although we did not investigatethe reason for the variation in this work, C. albi-cans replicative plasmids are notorious for beingunstable, spontaneously integrating in the genome,polymerizing and rearranging (see e.g. descriptionof the variable growth of strains CA106 and CA107on SD medium in the section Cloning of the BMH1gene, or the cloning procedures in Materials andmethods). The culture with a high level of SOU1mRNA could be overgrown, e.g. by cells withrecombined plasmids that lost BMH1.

Test for specificity of BMH1 regulation

We investigated the specificity of BMH1 regulationof the utilization of L-sorbose vs. the utilization ofthe other carbon sources. For this purpose all Ura+mutants BMH1/bmh1 and BMH1/bmh1 BMH1, aswell as control strain CA61, were compared fortheir growth on the synthetic media containingthe following carbon sources: L-sorbose, lactose,galactose, glycerol, D-arabinose and glucose. The

cell suspensions were prepared as 10-fold dilutionsand plated as spots, similarly to the strains inFigures 2A–2C. The spots developed confluentgrowth within 2 days on all carbon sources exceptL-sorbose.

BMH1 gene is not involved in formation ofhyphaeS. cerevisiae BMH1 was shown to be implicated infilamentation (Roberts et al., 1997). Also, Cognettiet al. (2002) reported that C. albicans BMH1 con-trols hyphae formation. We set about to analysethe relationship between C. albicans BMH1 andhyphal growth. The Ura+ control strains CA61 andCA62, BMH1/BMH1, and the pre-5-FOA Ura+ sin-gle deletants CA88 and CA91, BMH1/bmh1, aswell as Ur++ reconstituted mutants CA111 andCA115, BMH1/bmh1 BMH1, were plated on Spidermedium. Because it was recently shown that a defi-ciency in expression of the transformation markerURA3 in genetic background of double deletantsby HWP1 resulted in the loss of filamentation,and was subsequently restored by exogenous uri-dine (L. L. Sharkey, W.-L. Liao and W. A. Fonzi,personal communication), all the above-mentionedstrains were also plated on Spider medium sup-plemented with uridine. After 10 days of incu-bation on Spider medium, control colonies hadwell-developed mycelium, mutants having secondcopy of BMH1 in LEU2 locus had dramaticallyreduced mycelium, and only some, but not allcolonies of single deletants developed dramaticallyreduced mycelium. Colonies on Spider mediumwith uridine were not significantly different fromcolonies on Spider medium without uridine. In2 weeks all strains on Spider medium had propor-tionally more growth of mycelium than before, butthe difference between the degree of the develop-ment of mycelium between mutants with one andwith two copies of BMH1 remained. However, thesame mutants, CA88 (BMH1/bmh1 ) and CA111(BMH1/bmh1 BMH1 ) on Spider medium with uri-dine appeared identical, and had a well-developedmycelium, although this was somewhat shorter thanin the control strains (data not presented). It isclear that neither the copy number and position ofsecond allele of BMH1 nor the position of a singleallele of URA3 gene, which differ in BMH1/bmh1and BMH1/bmh1 BMH1 mutants (Table 2), wereimportant for the hyphal formation if the strainswere grown on a medium containing uridine. In



addition, all tested strains formed a similar pro-portion of germ tubes when exposed to medium199 or 20% bovine serum (data not presented).The loss of hyphae formation reported by Cognettiet al. (2002) was undoubtedly due to the dimin-ished expression of URA3 under the condition ofthe lack of uridine.

Discussion

By discovering that the loss of one homologueof chromosome 5 provides an increase of theSOU1 transcript and the ability to utilize L-sorbose,we initiated a study of negative regulation ofthe Sou phenotype in C. albicans. We proposedthat chromosome 5 carries a negative regulatorCSU of structural gene SOU1 (Introduction). Thispaper describes an additional negative regulatorof SOU1, which was cloned from C. albicansgenomic library, localized to chromosome 1, andis thought to belong to a category of secondaryregulators. The gene encodes a member of the14–3–3 protein family, and is an orthologue ofthe BMH1 gene of S. cerevisiae, but, unlike theS. cerevisiae BMH1, does not have paralogues andalso is essential (Cognetti et al., 2002; see Results).

Because of the essentiality of BMH1, we pre-pared and tested C. albicans strains in which onlyone copy, but not two copies, of BMH1 was dis-rupted and then reconstituted. Precautions weretaken not to assay Ura− ‘pop-out’ strains that camedirectly from a 5-FOA plate. Due to the expo-sure to toxic 5-FOA, these strains acquired at leastmonosomy of chromosome 5 (see Results; Tables 1and 2), subsequently leading to Sou+ phenotype(some other mutagenic consequences of the expo-sure to 5-FOA are also mentioned in Results). Thisartifactual acquisition of the phenotype that couldbe easily confused with the BMH1 role in SOU1regulation can be viewed as the foremost exam-ple of the pitfalls of using gene disruption to studygene function.

In order to conduct functional assays, we there-fore used either two pre-5-FOA single dele-tants having URA3 inserted in BMH1 or twopost-5-FOA single deletants that recovered chro-mosome 5 disomy, presumably due to spontaneousnon-disjunction (see Janbon et al., 1998), in whichURA3 was inserted in LEU2, and which had novisible change in their electrophoretic karyotypes.

In this model system, two strains having one recon-stituted genomic copy of BMH1 in LEU2 locuswere also prepared. It is important to note thatduplication of the remaining homologue, strictlyspeaking, does not return the cell to an origi-nal genomic condition. First, C. albicans is welldocumented to have allelic diversity (reviewed inRustchenko-Bulgac et al., 1990; Johnson, 2003),which means that homologous chromosomes areexpected to have multiple differences. Thus, withduplication of a homologue, many genes changefrom heterozygosity to homozygosity. Second, cur-rent resolution of chromosomal separations maynot allow small deletions to be seen, which meansthat by loss of one and duplication of anotherhomologue some non-essential genes could be lost.As we determined in this work, 5-FOA is notthe only inducer of undesirable mutations. Thegene disruption procedure that preceded expo-sure to 5-FOA can be also mutagenic, as demon-strated with one of two pre-5-FOA single deletantsCA91 (Figures 2, 5A2–5C2; and see Results). Inthis regard, Rieg et al. (1999) reported variationin growth rates in different clones of aaf1 nullmutants, which they attributed to the disruptionprocedure. It remains to be seen whether these dif-ferences were due to mutations induced by 5-FOAor by both 5-FOA and the disruption procedure.Also, Andaluz et al. (2002) reported that both celltransformation with a disruption cassette, as well asthe subsequent exposure of Ura+ transformats to 5-FOA medium caused extensive rearrangements inboth chromosomes R.

In designing a system for the overexpressionof BMH1, we could not use regular monosomicSou+ mutants, e.g. CA183–CA187, or the excep-tional Sor17 mutant (Table 1). The mutants witheither altered chromosome copy number or anyother chromosome alteration have multiple changesof gene expression (Marichal et al., 1997; alsodiscussed in Perepnikhatka et al., 1999), whosecumulative action could affect mRNA level ofthe gene of interest, as determined with North-ern blot analyses. We transformed, instead, a labo-ratory Sou− strain having two weakly expressedgenomic copies of SOU1 with replicative plas-mids carrying either BMH1 alone, or both SOU1and BMH1. The level of BMH1 mRNA was mea-sured reproducibly in the corresponding strains, asexemplified in Figures 5B1 and 5C1, establishinga rather modest increase of approximately 37%.



The small but consistant increase is indicative ofautogenous regulation of BMH1, although furtherstudies are required to establish such a hypothesis.

We suggest that the amount of the SOU1 tran-script is inversely dependent on BMH1 expres-sion (as summarized in Figure 5, and discussedin Results in more detail), despite the limitationsimposed by a number of factors, including the fol-lowing: the essentiality of BMH1 ; the use of Sou−instead of Sou+ strains for BMH1 overexpression;the debilitated condition of one of the originalBMH1/bmh1 mutants due to disruption procedures;mutagenesis due to exposure to 5-FOA; sometimesinconsistent BMH1 and SOU1 mRNA levels inreconstituted mutants; and plasmid instability. It israther clear that the increase of BMH1 transcriptdue to overexpression on a plasmid diminished thelevel of the SOU1 transcript produced by bothtwo normal SOU1 genomic copies (Figures 5B1and 5C1) or combined genomic and plasmid-borncopies (see Results), although presumably plas-mid instability was responsible for a higher thanexpected level of SOU1 transcript in one measure-ment. Conversely, the disruption of one genomiccopy of BMH1 produced the increase of the amountof SOU1 transcript, which, in most cases, wasrestored after the reintroduction of the secondgenomic copy. These measurements were consis-tent with differences in the survival of the samestrains and in the production of Sou+ mutantson sorbose plates (Figures 2, 3). Other workershave also obtained erroneous phenotypes of recon-stituted mutants, e.g. the intermediate phenotypereported by Kohler and Fink (1996). Demonstrat-ing that procedures used to alter gene copy numberin genome are mutagenic provides a rationale forthe inconsistencies of the restored phenotypes.

The integration of a third copy of BMH1 ingenome of L-sorbose utilizing mutants Sor17 andSor19 (see Materials and methods for their kary-otypes) did not diminish the growth of these strainson L-sorbose medium (data not presented). How-ever, an introduction of four or five BMH1 plas-mid copies in the same strains, Sor17 or Sor19,that resulted in total number of approximatelysix or seven copies/cell, inhibited the growth ofthese strains on L-sorbose medium, which was theapproach used to clone BMH1 (see Results).

Contrary to the monosomy of chromosome 5,which was responsible for approximately five-to seven-fold increase of SOU1 transcript in

regular Sou+ mutants (Janbon et al., 1998), theapproximately two-fold increase of the amount ofSOU1 mRNA in single deletants BMH1/bmh1 wasnot sufficient for producing growth on L-sorbosemedium. The increase, however, led to the approx-imately 10-fold increase in Sou− cell survivalon the otherwise toxic L-sorbose, which in turnresulted in approximately 10-fold higher produc-tion of Sou+ mutants monosomic for chromosome5 (Figure 3). Undoubtedly, the increased produc-tion of Sou+ mutants is due to a simple increasein cell number on a plate, because there was nochange in rates of mutant production in singledeletant CA88 vs. control and reconstituted strains,CA61 and CA111, respectively. We conclude thatBMH1 is not involved in mechanism of adaptationto L-sorbose by monosomy of chromosome 5 thatis coupled with adaptive mutagenesis. We speculatethat BMH1 is involved in repression of SOU1 tran-script in order to maintain Sou− phenotype whencarbon sources better than L-sorbose are available.

Because the test for the utilization of four addi-tional carbon sources, glycerol, lactate, galactoseand D-arabinose (see Results), clearly showed thatC. albicans 14–3–3 protein is not a negative reg-ulator of assimilation of any of these nutrients, webelieve that BMH1 participates in the control of theutilization of some, but not all, carbon sources. Incontrast, van Heusen et al. (1992) previously sug-gested that BMH1 controls general carbon utiliza-tion in S. cerevisiae. Heterozygous BMH1 /bmh1strains of S . cerevisiae grew poorly on glucosemedium and even more poorly on acetate andglycerol medium. This does not appear to be acase of control of utilization of specific carbonsources; for example, many temperature-sensitivemutants grow poorly on all media, with the retar-dation of growth exacerbated on media contain-ing non-fermentable substrates such as acetateand glycerol. In plants, the 14–3–3 proteins areinvolved in regulation of enzyme activities for car-bon and nitrogen metabolism, such as glutaminesynthetase, nitrate reductase, trehalose 6-phosphatesynthase and sucrose phosphate synthase (Finnieet al., 1999).

Overall, our results showed that the amount ofthe SOU1 transcript depends upon gene dosageof at least two negative regulators, BMH1, onchromosome 1 and the hypothetical gene CSUon chromosome 5. This complexity is similar tothe regulation of utilization of various carbon



sources in other well-studied microorganisms. Ourinterpretation of this data is that BMH1 contributesto a general cellular homeostasis in which manydifferent genes may function as negative regulatorsof secondary carbon sources, in order to keepthe corresponding genes turned off or at a lowlevel of activity. The CSU, which is controlledby a newly discovered mechanism of chromosomecopy number and which we denote as a ‘major’negative regulator, could serve as an ‘override’,thus allowing ready adaptation to environmentalchanges. It seems that at least because of itsessentiality, BMH1 can not be a major regulator ofthe utilization of certain nutrients in C. albicans.It remains to be seen whether Bmh1p and Csupdirectly interact with each other and how theyaffect the transcription of SOU1. As emphasizedby Finnie et al. (1999), the 14–3–3 protein mightinduce a rapid change between metabolic states inresponse to environmental changes.

14–3–3 proteins, although evolutionarily con-served in eukaryotes, have unusually diverse func-tions (Morrison 1994; Wang and Shakes, 1996),and it is not too surprising that BMH1 regulatesSOU1. These proteins function as adapters, facili-tating the interaction of other proteins (Marais andMarshall, 1995; Morrison 1994; Vincenz and Dixit,1996), and chaperones or stabilizers of the activityor structure of other proteins (Aitken, 1996; Alamet al., 1994; Vincenz and Dixit, 1996). The com-mon property of 14–3–3 proteins, the ability tointeract with other proteins, might define the largevariety of functions. As recently reviewed (Aitken,1996; Finnie et al., 1999; van Hemert et al., 2001;Xing et al., 2000), 14–3–3 proteins participate inmultiple signalling pathways by binding to proteinkinases, such as Raf-1, KSR-1, BSR, U-α, PKC,Ask1, and apoptosis-promoting protein, BAD, aswell as tyrosine and tryptophan hydroxylases thatare important in neurotransmitter synthetic path-ways. Also, 14–3–3 proteins participate in check-point control pathways and mitochondrial import(Alam et al., 1994), associate with Polyomavirusmiddle tumor antigen (Pallas et al., 1994), and bindto receptors and membrane proteins (Aitken, 1996;Finnie et al., 1999). These interactions can resultin either the activation or inactivation of functions.

The lower fungi Schizosaccharomyces pombeand S. cerevisiae each have two isoforms of14–3–3 proteins (van Heusen et al., 1995; Fordet al., 1994; van Hemert et al., 2001). The two

14–3–3 paralogues of Sz. pombe are required forfunctioning of Rad24p and Rad25p, in cell cycleDNA damage checkpoints (Ford et al., 1994). Theoverexpression of BMH1 and BMH2 in S. cere-visiae stimulates cell elongation and filament for-mation (Roberts et al., 1997). A concomitant dis-ruption of both BMH1 and BMH2 blocks the for-mation of either phenotype, although the cells arestill capable of invading agar. Interaction of the14–3–3 protein and Ste20p has been postulated,thus assigning S. cerevisiae 14–3–3 proteins tothe RAS/MAP signalling cascade. According to ourdata (see Results), BMH1 of C. albicans is notimplicated in hyphae formation.

Our findings further substantiate the involvementof the 14–3–3 proteins in a wide range of regula-tory processes in evolutionarily diverse organisms.Furthermore, the involvement of the BMH1 inSOU1 regulation is particularly interesting becauseit is a component of newly discovered regulationrelated to chromosome copy number, which is stillin the initial stage of investigation.

Acknowledgements

We thank Dr G. Janbon for valuable discussions and Dr J.Greenberg for reading and commenting on the manuscript.This work was supported by NIH Grants AI29433 andGM12702.

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role of the 14–3–3 protein in carbon metabolism of the pathogenic yeast candida albicans

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