INFECI1ON AND IMMUNITY, Nov. 1994, P. 5027-50310019-9567/94/$04.00+0Copyright X 1994, American Society for Microbiology

Molecular and Functional Analysis of the LYSJ Geneof Candida albicans

RICHARD GARRAD,t T. M. SCHMIDT,4 AND J. K. BHATTACHARJEE*

Department of Microbiology, Miami University, Oxford, Ohio 45056

Received 15 April 1994/Returned for modification 14 June 1994/Accepted 26 August 1994

The LYSI gene of Candida albicans has been localized to a 1.8-kb DNA fragment present on the plasmidYpBRG2. YpBRG2 has been shown to complement the saccharopine dehydrogenase mutant Stx4-4A ofSaccharomyces cerevisiae. Transformants of S. cerevisiae Stx4-4A exhibited significant saccharopine dehydro-genase activity, and cells that had lost YpBRG2 after nonselective growth had no enzyme activity. The DNAsequence of the LYSI gene has been determined. The LYS] DNA contains typical yeast upstream regulatorysequences, including the GCN4 motif and candidate sequences responsible for transcription terminationwithin the 3' noncoding region. The fragment contained an open reading frame of 1,146 nucleotides coding fora putative protein of382 amino acids. The open reading frame has 60%o identity at the nucleotide level and 71%similarity at the amino acid level to the LYSS gene of Yarrowia lipolytica, which is believed to code forsaccharopine dehydrogenase. A peptide of 11 amino acids has been found, which is present in S. cerevisiae, Y.

lipolytica, and C. albicans. This peptide can be expanded to 16 amino acids when the sequences from Y. lipolyticaand C. albicans are compared. A motif responsible for the binding of the adenosine residue ofNADH has beendescribed previously and is very similar to this peptide, which may be the site of NADH binding in thesaccharopine dehydrogenase of C. albicans.

The genus Candida is composed of approximately 200diverse yeast species whose common link is the lack of a sexualcycle (24). A minority of the genus is pathogenic, and 80% ofthe clinical isolates are either C. albicans or C. tropicalis (18).C. albicans is the major opportunistic fungal pathogen ofhumans (27), capable of establishing infection whenever thehost immune system or normal flora is perturbed. The inci-dence of C. albicans infections is rising rapidly with theincrease in immunosuppressive disease and therapy (3). Inrecent years, C. albicans has been established as the modelsystem for pathogenic fungi (21). Basic information about thegenome of C. albicans is lacking (21). The sequences ofapproximately 40 genes are available in computerized data-bases, only two others of which are involved in amino acidbiosynthesis (29, 31a). The relatively small database of geneticinformation available for C. albicans places limitations uponconclusions that can be determined from these sequences,although patterns have arisen.The a-aminoadipate pathway for the biosynthesis of lysine

has been demonstrated in phycomycetes, euglenids, yeasts, andother higher fungi (2, 22, 36). This unique pathway is presentin C. albicans and other pathogenic fungi (14). Lysine issynthesized by the diaminopimelic acid pathway in bacteriaand plants and is an essential amino acid for humans andanimals. The a-aminoadipate pathway consists of eight en-zyme-catalyzed steps, and there appear to be seven freeintermediates in Saccharomyces cerevisiae (2). The final revers-ible step of the a-aminoadipate pathway is catalyzed bysaccharopine dehydrogenase (EC 1.5.1.7) encoded by the

* Corresponding author. Mailing address: Department of Microbi-ology, Miami University, Oxford, OH 45056. Phone: (513) 529-5422.Fax: (513) 529-2431.

t Present address: Department of Biochemistry, University of Mis-souri-Columbia, Columbia, MO 65212.

t Present address: Department of Microbiology, Michigan StateUniversity, East Lansing, MI 48824.

LYSI gene of S. cerevisiae and the LYS5 gene of Yarrowialipolytica.

Diagnosis of fungal infections is time-consuming and oftenrequires complete cultural identification protocols (26). Theuniqueness of the a-aminoadipate pathway may offer oppor-tunities to develop molecular probes for the detection offungal pathogens and the ability to inhibit fungal growth byblocking the synthesis of lysine with substrate analogs. Al-though several lysine genes have been cloned, only two othergenes have been sequenced (25, 38). We report here thefunctional characterization and DNA sequence analysis of theLYSI gene of C. albicans.

MATERIALS AND METHODS

Strains, plasmids, and media. S. cerevisiae Stx4-4A (a lyslade2 arg4 gal2) and RC-1 (a LYS) were obtained from theYeast Genetics Stock Center. Escherichia coli DH5a andplasmid pBluescriptSK were purchased from Stratagene, Inc.(La Jolla, Calif.). Plasmid vector YpB1041 and the clonedLYS1 gene from C. albicans in plasmid YpB1078 were ob-tained from S. Scherer (32). Recombinant plasmid YpBRG2was constructed by ligation of a 1.8-kb EcoRI-EcoRV fragmentfrom YpB1078 into pBluescriptSK The fragment was thenremoved from pBluescriptSK by using BamHI and Sall andligated into similarly digested YpB1041 (Fig. 1). Yeast strainswere maintained on yeast extract-peptone-dextrose medium.Minimal medium consisted of dextrose (10 g), yeast nitrogenbase without amino acids (Difco [6.7 g]), sterile distilled waterto 1 liter, and agar (20 g). Appropriate supplements wereadded to minimal medium at a concentration of 5 mg/100 ml(wtlvol) for the growth of auxotrophs. E. coli strains weremaintained on Luria-Bertani medium. Ampicillin was added toLuria-Bertani medium at a concentration of 100 jxg/ml when-ever a plasmid bearing an ampicillin resistance marker waspresent in the strain of E. coli to be cultured on the medium.

Preparation of cell extracts and saccharopine dehydroge-nase assay. Cells were grown in minimal medium (wild type

5027

Vol. 62, No. 11

on February 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

5028 GARRAD ET AL.

BamHl 9.90

Sall 9.70 | EcoRI 0.55

Rsal8.29/ ~~~cUA < Xbal 1.40

YpB11041 Pstl 2.10EcoRI 7.59-

9.90 KbX -Hpal 2.41

Rsal 6.98_ cARS 2 micron

EcoRI 6.72 ORI

Ndel 6.38al 3.39_dH18d inll 3.99

Scal 4.61 EcoRI 4.09/Pvul 4.72

Pstl 4.87

aSau3A 14 79

EcoRV 14.481 0.RIO55\ ~~~~~Xbal1.40

EcoRI 12.78t21cURA3 Pstl 2.10

Hindlil 12.09 //9 Hpal 2.41aal 11.68 cLYSIXhol 11.4S= r YpB1078 2 micror' Xbal 3.39

Xbal I11.08 14.79 Kb a Hindlil 3.99Bgill 108

AMP-R EcoRI 4.09

Sau3A 9.98 ORI Pval 4.61Sail 9.71 vl47

cARS Pstl 4.84

Rsal 8.29 Ndel 6.38I \Rsal 6.68Rsal 6.9

s6

EcoRB 7.59EcoR1 6.72

bBamH I. 50 EcoRI 0.S55

Hindlil 10.70 _> ~~~~~~Xbal 1.40

Sall 9.70-_ cLYS1 cURA Y

Pstl 2.10

YpBRG2 \_ Hpal 2.41

ll1.50Kb9

Rsal 8.29 < ARS 2 micron}\ ^ ~~~~~~~~Xbal3.39

EcoRI 7.59 \0i Hindlll 3.99

Rsal 6.98EcR\40J / _ \\\ ~~~~EcoRI 4.09

EcoRI6.72 l{|Rsal 6.68 ||\ Scal 4.61

Ndel 6.38 | Pvul 4.72

Pstl 4.87

C

FIG. 1. Plasmids used in this study. YpB1041 and YpB1078 (a andb) were supplied by Scherer et al. (15, 32). The LYS1 gene wassubcloned into YpB1041 with pBluescriptSK (Stratagene, Inc.) as anintermediary. The resulting subclone is YpBRG2 (c).

and transformants) or minimal medium plus lysine (mutants)at 30"C with constant shaking. The cells were harvested duringthe late logarithmic phase. Crude enzyme preparations wereobtained by disruption of the cells in a Braun homogenizerflask (34). The crude extract was dialyzed and used as theenzyme source for the saccharopine dehydrogenase assays.The amount of protein in the dialyzed extracts was determined

by the method of Bradford (4) with the Bio-Rad protein assaykit (Bio-Rad Chemical Division, Richmond, Calif.).The activity of saccharopine dehydrogenase was measured in

the reverse direction of the reaction by monitoring the oxida-tion of NADH to NAD+ spectrophotometrically at 340 nmafter conversion of lysine and oa-ketoglutarate to saccharopine(19). The reaction mixture consisted of 50 mM lysine, 20 mMox-ketoglutarate (neutralized with 2.0 N NaOH), 100 ,uMNADH, 250 mM potassium phosphate buffer (pH 7.5), 0.1 to1.0 mg of dialyzed protein extract, and sterile distilled water toa final volume of 2.0 ml. The mixture without lysine served asthe control. The enzyme was assayed within a linear range atthree concentrations of protein, and the values were averagedand determined to be within 1 standard error of the mean.Transformation of S. cerevisiae Stx4-4A. The transformation

method was similar to those of Rodriguez and Tait (30) andSherman et al. (33). S. cerevisiae cells transformed with thecloned C. albicans LYS1 gene were selected on minimalmedium devoid of lysine. Plates were incubated at 30°C for 5 to7 days.Curing of autonomously replicating plasmids. A colony

produced from a putatively plasmid-transformed yeast cell waspicked off a selective transformation plate and streaked forisolation on selective medium. This plate was incubated, afterwhich an isolated colony was inoculated into both nonselectiveand selective broth media. The cultures were grown to maxi-mum A550, and then 0.1 ml was streaked from the broth tononselective media. Multiple colonies were streaked from thisnonselective medium to selective and nonselective media.

Nucleotide sequencing. DNA sequencing was performed bythe dideoxy chain termination method (31). Both strands of theinsert were sequenced. In order to facilitate sequencing,exonuclease III-digested clones were made of the 1.8-kbfragment within pBluescriptSK with the Erase-a-Base system(Promega). The DNA sequence and protein data were ana-lyzed by using various programs available with the GeneticsComputer Group software developed at the University ofWisconsin (6).

RESULTS

Complementation of S. cerevisiae Stx4-4A and functionalcharacterization of the LYS] gene of C. albicans. The ability ofthe cloned LYS1 gene present in YpBRG2 to complement thesaccharopine dehydrogenase mutant Stx4-4A of S. cerevisiaewas demonstrated by colony counts from two transformationexperiments. The number of colonies obtained in the presenceof YpBRG2 DNA was consistently severalfold higher thantransformations with vector alone or without DNA (results notpresented).

Plasmid loss experiments were performed to determine lossof lysine prototrophy (LYS1J ) from transformed cells as aresult of growth on nonselective medium. S. cerevisiae Stx4-4Acells transformed with the C. albicans LYS1 gene (fromYpBRG2) showed a 60% loss of C. albicans LYSI plasmidcontaining DNA as exhibited by an inability to grow onselective medium (data not shown).

Wild-type S. cerevisiae, a lysl mutant of S. cerevisiae Stx4-4A,transformants of this mutant, and plasmid-cured strains wereassayed for saccharopine dehydrogenase activity (Table 1).Plasmid-cured strains and the lysl mutant grown in lysine-supplemented minimal medium exhibited little or no activity.Wild-type and Lysl+ transformed strains grown in minimalmedium exhibited significant saccharopine dehydrogenase ac-tivity.

Sequence analysis of the LYSI gene of C. albicans. The DNA

INFECT. IMMUN.

on February 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ANALYSIS OF THE LYS1 GENE OF CANDIDA ALBICANS 5029

TABLE 1. Saccharopine dehydrogenase activity of the wild type,lysl mutants, and Lysl + transformed strains of S. cerevisiae

Strain Lysine Mean saccharopinegenotype dehydrogenase activity ± SDaWild type LYS1 24.6 ± 0.40STX4-4A lysi 4.00 ± 0.50STX4-4A-8(YpB 1078)b Lysl + 13.8 ± 2.50STX4-4A-8(YpB 1078)C lysi 3.00 ± 0.50STX4-4A-3(YpB RG2)b Lysi + 44.7 + 11.1STX4-4A-3(YpB RG2)C lysi 4.50 ± 0.44STX4-4A-4(YpB RG2)b Lysl + 34.7 ± 11.3STX4-4A-4(YpB RG2)C lysi 7.00 ± 0.47STX4-4A-5(YpB RG2)b Lysi + 37.2 ± 9.60STX4-4A-5(YpB RG2)C lysi 6.50 ± 0.44

a Saccharopine dehydrogenase specific activity is expressed as micromoles ofNADH converted to NAD+ at 340 nmol/min/mg of protein.

b Mutant strain transformed with the stated plasmid.c Transformed strain that lost the plasmid after growth on nonselective

medium.

BaoHI EcoRI

/- /

101

CTCTGTCTCTCTCTCTTTCTCAcTTAGAGAATAIAIA ACCACATTACAATTCATrTATTCTACATTGAACAATTTGAATG AAACATM201 - - - - - - - - - -

GAGACAGAGAGAGAAAGAGTGAATCTcTTATATTTGGTGTAATGTTAAGTAAATAAGATGTAAcTTG1GTTAAAACTTACrnTJJ GTAAAA

M S K S P V I L H L R A EATACCTI ATTACTTMACTTCMCTAATAATCAACTATACTAGCTAACTCATATACTAATTATGTCTAAATCACCAGTArTTCTCAMAAGAGCAGAA

301 - --- -- - ---- --- -- -

401

T K P L F A R A A L T P S T T K 0 L IDOA G F F I Y V F S S 0 S TACTAAACCATTAGAAGCTAGAGCTGCTTTAACTCCTTCTACTACTAAACAATTACTCGATGCTGGAMGAAATTTATGTTGAAGAATCTTCTCAATCTA

F DIT K F Y F A V G A wKFS K T A P K F R I F I K K

CTM0 ATATTAAAGAATATGAAGCTGTTGGTGcTAAAATAGTACCTGAAGGTTCATGGAAAACTGCTCCTAAAGA+AGAATTAT- MGa -MAAAAGAGAAAACTATAAMCTTATACTTC GACAAC CACGATTTTW|ATCATGGACTTCCAAGTACCTa TGACGAGGATTcTCTCTTAATAAA^AACcAAATmTTC

sequence of the C. albicans LYS1 gene is shown in Fig. 2(GenBank accession no. U13233). A single open reading frameof 1,146 bp is present. The deduced amino acid sequence ofsaccharopine dehydrogenase stretches for 382 residues and isincluded beneath the nucleotide sequence. The open readingframe codes for a protein with a molecular weight of approx-imately 42,450. The nucleotide sequence is 60% similar to thatof the LYS5 gene of Y lipolytica (38) according to thealignment method of Needleman and Wunsch (27). The5'-flanking region of the C. albicans LYS1 gene contains asingle GCN4 binding site at positions -315 to -306, a TATAbox at positions -128 to -123, and a poly(dA-dT) tract atpositions -80 to -68. Position -3 relative to the ATGinitiator codon is a purine (A) that has been identified asimportant for recognition by eukaryotic ribosomes (20). The 3'noncoding region of the C. albicans LYS1 gene contains a setof sequences of the TAG.....TAGT... TT[ motif (positionsare marked with an asterisk in Fig. 2) that may play a role intermination of transcription. This region also contains tworegions (1675 to 1682 and 1735 to 1742) that are almostidentical to the yeast transcription termination signal, TTIlTTATA (16).The calculated molecular weights of saccharopine dehydro-

genase from S. cerevisiae and Y lipolytica are 39,000 and40,566, respectively. Figure 3 shows the optimal alignment ofthe Y. lipolytica LYS5 and C. albicans LYSI predicted peptides.The peptides are 78.9% similar and 70.4% identical. Twohighly conserved areas are underlined in Fig. 3; the stretch ofamino acids between residues 210 and 225 (C. albicans se-quence) is present and is identical in S. cerevisiae, Y lipolytica,and C. albicans. The codon bias index (1) for the C. albicansLYS1 predicted amino acid sequence was found to be 0.71.This is very similar to the 0.7 value found in the Y lipolyticaLYS5 gene (38) and corresponds to those of moderatelyexpressed yeast genes (1).

ATTACCTGAAAATGAAACmCCCATTAATTCATGAACATATTCAAMGCTCATTGTTATAAAGATCAAGCTGGTTGGCAAGATGTTrT7AAAAAGATTC601-+-+-+-+-+-+-+-+-+-+

TAATGGACTMfI,ACTTGAAAGGGTAATTAAGTAMTGTATAAGTTAAACGAG;TAACAATAMCTAGTrCGACCAACCGrrCTACAAAAmTTMCTAAG

P 0 C N G T L F F L F N D 0 G R R V A A F G F Y A G F A G A ACCACAAGGTAATGGTATATTATATGAMAGAATrMX, AGAAAATGATCAAGG;TAGGAGAGTTGCTGCCMGGAT 1l ATGCTGGATrGCTGG(GGCTG

701 - +a - a - a--a--+--a--a+-a+GGTGTTCCATTACCATATAATATACTAAATCTTAAAAATCTTMACTAGTTCCATCCTCTCAACGACGGAAACCTAAATACGACCTAAACGACCCCGAC

TIG V L D S F K 0 L 1NG T K G T K G E G F G G E L P G V T P YCCATTGGGITATTAGATTGGAGTTTAAACATTGAATGGTAATACTAAAGGTACTAAAGGTGMGGTGAAGGTGGTGAATTACCTGGGGTGACTCCATA

801 -- ---- --- - ----+----- -- --+GGTAACCCCATAATCTAACCTCAAAATTGTTAACTTACCATTATIATTTCCATGATTCACTTCCACTTCCACCACTTAATGGACCCCACTGAGITAT

HindIII

P N E N F L I K D V K I E L E K A L T K N G G 0 Y P K C L V I G ATCCTAATGAAAATGAATrAATTrAAGATGTTAAAATTGAATTAGAAAAAGCTAACTAAAAATGGGGGTCAATATCCTAAATGTCTTGTTATTGGTGCC

90A1-+++A-T_C+A__T+C+T+ACaaA-Ar~~~~~~ K 1 6 1aP a LrrU N I A K n D M A F A K G GTTGGGTAGATG;TGGATCTGGTGCCATTGAMAmAAAAAAATTGGTATCC CTGATGATAATATTGCTAAATGGGATATGGCTG;AAACTGCTAAAGGTG+0-- - - - - --

AACCCATCTACACCTAGACCACGGTAACTAAATAAATTTTTTTAACCATAGG;GAcTACTATTATAACGATTACCCTATACCGACTTT,GACGATTCCAC

N R K L T T T V D V S A D T T N P H P T P V Y F T A T V F N PAAATAGAAAATTGAcTACTATFGTTGATGmCTGCTGATACTACTAATCCTCATAATCCAATCCCAGTATATGAAATTGcTACAGTMTTCAATGAACCA

1201 --+--+++-+-++--a-- a

HindIII

ACCGTTAAGTAAACTTGATAAAGGTCCTAAATTATCAGTATGTCAATTGATCAMACCTTCMTArTACCTAGAGAAGCTTCAGAA wTTTrT|GCTA1301-+-+-+-+-+-+-+-+-+-+

TGGCAAM ICAAMGAACTAMCCAGGAPrAATAGTCATACAAGTTAACTAGTAAATGGAAAAATAATGGATCTCTF CGAAGTCTTAAAAAACGAT

D L M4 P S L L E L P N R D T S P V W V R A K 0 L F D K H V A R L D

1501 ------a-a---a+--a-----+--a------ a-a-----------ATrTCTCATCATCATCCAAATGTTCAGTTCATTTACACAAATTAMATAAAATAAMAGAAAATAAAATAAAATAAAGTAAAGTAAAGAATTAATCAT

TCTGTGTATATTGGGATCTATrAGTAAAATAGTAGCACTATTATTAT'TCTAATGTTACACTAACTT| Cr CrmmAATATTATTCTTTMlGAm1160 - -- ---- - ----- --- ---- --

AGACACATATAACCCTAGATAATCATTTTATCATCGTGATAATAATAAGATTACAATGTGATTGAAAAGAAAAGAATTATAATAAGAAAAAACTAAA

EcoRV HindIII SlIl/ / /

DISCUSSION

The C. albicans LYS1 gene for the synthesis of saccharopinedehydrogenase has been cloned into both pBluescript andYpB1041 vectors. Previously it was reported that the C.albicans LYS1 gene lay on the 1.8-kb HindIlI fragment fromthe original plasmid, YpB1078 (14); however, this does notappear to be the case. The data presented here unequivocallyidentify the C. albicans LYS1 gene. The gene is located within

TCrACC101 ------ 106

AGCTGG

FIG. 2. The nucleotide and predicted amino acid sequence of theC. albicans LYS1 gene. The putative GCN4 box, TATA box, andpoly(dA-dT) regions are shown underlined upstream of the openreading frame. Stop codons are indicated with asterisks.

P F 0 F I V D L D T F T N C T Y L 1, K P T P P F T N K F N N FGTCCATTCCAAGAAArrMGATCTG(;ATATMCAT'TAATTGTAMAMATCTAAACCAATCCCACCAMAT'TAATAAAGAAATMGAATAATGA

- - - - . . - . I - - - I I . . . . - . - I I - . . . . . I .AAGATrrAATGCCATCATTATrGGMTrACCAAATAGAGATAMCTCCAGTATGGGTTAGAGCTAAACAATTAMGATAAACAC(;lTGCCAGACTrGA

VOL. 62, 1994

- ---------I---------I--------- --------- ----------I----------I----------I---------I--------------------TrCTAAArrACGGTAGTAATAACCTrAATGGMATCTCTATGAAGAGGTCATACCCAATCTCGAMGTTAATAAACTAMr.TGCAACGGTCTGAACT

on February 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

5030 GARRAD ET AL.

1 MSKSPVIu0RAETIpLEA9AALTPSTn9lLDA;FEIYVEESSQSTFDIKE 54

1 ITrAPVKuIEKPLEHRsALTPTrrrIAa-EVFVEKSPLRIFDDQE 52

55 YEAVGAKIVPEGS rAPIERHEPLFLPuEENLIHEHIQFXCYKD 103

53 FVDVGATLVEEGSWVSAPEDRIIGLKEIPE.ESFPLSHEHIQFAHCYKD 101

104 QAD3VLKPFPQGNGILYDI AIGhGA GJLD 153

102 E GVET 151

154 WSFQLNGE PGGEEGGELUPGVTYPNuELNKIDVKELaKALTKtNG 2031.1.1 ::::1111.:1111.11::1 :1. 1:.G 1

152 WAE ...THPDS2LGSYNTVDIKK.DLAVEK.G 191

204

1921111111111111111 :1:111::1I :111.11 11111111

SKLP LPEIG4EARKVGIPG'ErIEUEI 241

254 VDIDIFINCiIYIspIPPFINEIINRTIVDVSDTNPHNPIP 303

242 ADADIF]3IYLSPIPPFINYDUNKTRKLSV IHNPVP 291

304 VYEIAIVNPVETKVKEV,-ELM2DAKE6FIDIWFS 353

292 VYTIAlaTVPEITALVPEKL,-CAJLLE_EaWSEALLPS 341

354 LIETNPDrSPWMRAN92LDKHVAR 379

342 ULQPQUTAP2VWRAKALFDKHVIR 367

FIG. 3. Alignment of the predicted amino acid sequences of the C.albicans LYSI gene (upper sequence) and the Y lipolytica LYS5 gene

(lower sequence). Identical amino acids are shown by an adjoiningline, very similar amino acids are shown by two dots, and similar aminoacids are shown by one dot. The stretches of highly conserved residues(16 or more amino acids) between the two proteins are underlined; theregion between residues 210 and 225 (C. albicans sequence) is presentand identical in S. cerevisiae, C. albicans, and Y lipolytica.

a 1.8-kb EcoRV-EcoRI fragment and demonstrates full func-tional activity when transformed into a S. cerevisiae saccharo-pine dehydrogenase mutant.The C. albicans LYSI gene has been mapped to chromo-

some 4 by Magee et al. (23). The C. albicans LYS1 gene hasnow been sequenced. The gene has a typical TATA box and a

poly(dA-dT) region upstream of the open reading frame forLYS1 at positions -128 to -123 and -79 to -67, respectively.Only one GCN4 box is present in the 5' upstream region atposition -315. Usually more than one GCN4 box is present ingenes regulated by this system (17). However, two genes fromS. cerevisiae, ILV2 and ARG3, have only one GCN4 box andare under general control (5, 7). The GCN4 box of the C.albicans LYS1 is a perfect match for the consensus TGACTC.Analysis of a series of mutations of the S. cerevisiae HIS3upstream region has led to an expanded sequence, RRTGACTCATTT, found to be ideal for GCN4 derepression (17).The RR in this sequence designates purines; however, the 3'ATTll have been found to be the important nucleotidesoutside of the core TGACTC for the optimum binding ofGCN4 protein. The GCN4 binding site in the C. albicans LYSIgene has ATIT 3' to the core, which may negate the need formore than one of these sites upstream of the open readingframe of the gene.The open reading frame of C. albicans stretches for 1,146

nucleotides and has the codon bias index of a moderatelyexpressed gene. This is expected for an amino acid biosyntheticgene. The predicted amino acid sequence for saccharopinedehydrogenase from C. albicans has strong accord with thosefrom S. cerevisiae and Y lipolytica. Although the protein fromS. cerevisiae has never been sequenced, a great deal is knownabout the enzyme from the work of Fujioka et al. (8-13). Apeptide of 11 amino acids isolated from the S. cerevisiaeprotein, which contained an essential cysteine residue capableof carboxymethylation by iodoacetate, is present and identicalin predicted amino acid sequences from both C. albicans and Y

lipolytica (between residues 210 and 225 of C. albicans). Thispeptide can actually be expanded to 16 residues if the Ylipolytica and C. albicans sequences are compared. The con-servation of this amino acid sequence and the data reported byFujioka et al. (8-13) indicate that this peptide is of significancein the function of saccharopine dehydrogenase.

Further data suggest that the conserved 16-amino-acid pep-tide between residues 210 and 225 in C. albicans is involved inNADH binding. Work by Taylor and Thornton (35) outlined aprocedure for the recognition of the Pap super secondarystructure within proteins with 75% accuracy. Wierenga et al.(37) determined 11 critical amino acids within a stretch ofbetween 28 and 32 residues that were essential in forming thestructure they termed the "ADP binding-fold" or "finger-print." Residues 224 to 194 (31 amino acids) of C. albicanshave 9 of 11 amino acids matching the fingerprint in the correctpositions. The differences between the peptides are conserva-tive changes. The hypercritical residues of this fingerprint areglycines at positions 6, 8, and 11 and an acidic amino acid(glutamate or aspartate) at the last position of the peptide.These residues are present and identical in the predictedamino acid sequence of the C. albicans LYS1 gene.The only stumbling block to the formation of the ADP-

binding fold in the C. albicans LYS1 gene is the fact that thepeptide in the Candida protein runs from the carboxyl termi-nus to the amino terminus, not vice versa. However, the natureof this motif is such that the peptide may be produced beforethe relatively loose associations of this structure, hydrophobicinteractions along the core, assemble the motif. Structuralevidence shows that the residues between 224 and 194 of the C.albicans LYS1 gene form the 1cox motif, including the residuesaround the second loop, two glycines and a proline, allowingfor turns within the structure, and the predicted secondarystructure (data not shown), which, although it doesn't unequiv-ocally describe the acsj motif, certainly does not rule out thepossibility of its formation.

Interestingly enough, Wierenga et al. (37) point out thatproteins using NADPH rather than NADH as a coenzymewould be unlikely to have an acidic amino acid at the terminusof the motif. This would create an unfavorable interactionbetween this residue and the 2'-phosphate of NADPH. Theseproteins replace the terminal acidic amino acid with a hydro-phobic residue. The predicted amino acid sequence of the Ylipolytica LYS5 gene forms a peptide very similar to that justdescribed for C. albicans. However, the peptide is one aminoacid shorter in Y lipolytica; this makes the terminal amino acida leucine, a hydrophobic residue. It would be of great interestto know if the Y lipolytica saccharopine dehydrogenase re-quires NADPH rather than NADH as a coenzyme.Taken together, these data suggest that the amino acid

stretch between 194 and 224 in the C. albicans LYSI predictedpeptide is responsible for binding of the coenzyme NADH.The terminal glutamate may form hydrogen bonding betweenits acidic side chain and the 2'-OH of the adenosine of NADH.

It appears the saccharopine dehydrogenases of C. albicans,Y lipolytica, and S. cerevisiae are similar and are obviouslyhomologs. As noted, the nucleotide similarities between C.albicans and Y lipolytica for saccharopine dehydrogenase are60%; of interest would be a determination of the nucleotidesimilarity of the sequence of the S. cerevisiae gene whenavailable.The analyses of the polypeptides from the C. albicans LYSI

gene and the Y lipolytica LYS5 gene have indicated conservedareas and one area between residues 157 and 165 of the C.albicans sequence that is completely missing from the Ylipolytica sequence. Perhaps these regions could be the bases of

INFECT. IMMUN.

on February 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ANALYSIS OF THE LYS1 GENE OF CANDIDA ALBICANS 5031

two oligonucleotide probes. A probe made from a conservedregion might have general use in the detection of fungalspecies, and a probe from a unique region might be useful forthe diagnosis of C. albicans. Since the a-aminoadipate pathwayis unique in fungi, the divergence of nucleotide sequences fromlysine genes may have a useful role in determining evolutionarypatterns within this group of organisms.

ACKNOWLEDGMENTS

We thank P. Magee and S. Scherer for the generous gift of plasmidsYpB1078 and YpB1113 containing C. albicans genes and vector YpB1041.This research was supported by a Shoupp award from Miami

University, a grant from Eli Lilly and Co. to J.KB., and a Sigma Xigrant to R.G.

ADDENDUM IN PROOF

After submission of the manuscript, a search of the data-bases indicates the addition of the LYS1 gene sequence forsaccharopine dehydrogenase from S. cerevisiae (GenBank ac-cession no. X77632). The S. cerevisiae and C. albicans openreading frames have 69.1% similarity at the nucleotide level.Both conserved peptides present in the C. albicans and Ylipolytica putative amino acid sequences are also present in theS. cerevisiae LYS1 sequence with a single amino acid difference.

REFERENCES1. Bennetzen, J. L, and B. D. Hall. 1982. Codon selection in yeast. J.

Biol. Chem. 257:3026-3031.2. Bhattacharjee, J. K 1985. The a-aminoadipate pathway for the

biosynthesis of lysine in lower eukaryotes. Crit. Rev. Microbiol.12:131-151.

3. Bodey, G. P., and V. Fainstein (ed.). 1985. Systemic candidiasis, p.135. Raven Press, New York.

4. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

5. Crabeel, M., R. Huygen, K Verschueren, F. Messenguy, K Tinel,R. Cunin, and N. Glansdorff. 1985. General amino acid controland specific arginine repression in Saccharomyces cerevisiae: phys-ical study of the bifunctional regulatory region of theARG3 gene.Mol. Cell. Biol. 5:3139-3148.

6. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensiveset of sequence analysis programs for the VAX. Nucleic AcidsRes. 12:387-395.

7. Falco, S. C., K S. Dumas, and K J. Livak. 1985. Nucleotidesequence of the yeast ILV2 gene which encodes acetolactatesynthase. Nucleic Acids Res. 13:4011-4027.

8. Fujioka, M. 1975. Saccharopine dehydrogenase substrate inhibi-tion studies. J. Biol. Chem. 250:8986-8989.

9. Fujioka, M. 1984. Chemical mechanism of saccharopine dehydro-genase (NAD, L-lysine forming) as deduced from initial rate pHstudies. Arch. Biochem. Biophys. 230:553-559.

10. Fujioka, M., and Y. Nakatani. 1970. A kinetic study of saccharo-pine dehydrogenase reaction. Eur. J. Biochem. 16:180-186.

11. Fujioka, M., and Y. Nakatani. 1972. Saccharopine dehydrogenase:interaction with substrate analogues. Eur. J. Biochem. 25:301-307.

12. Fujioka, M., Y. Takata, H. Ogawa, and M. Okamoto. 1980. Theinactivation of saccharopine dehydrogenase (L-lysine forming) bydiethyl pyrocarbonate. J. Biol. Chem. 255:937-942.

13. Fujioka, M., and Y. Tanaka. 1981. Role of arginine residue insaccharopine dehydrogenase (L-lysine forming) from baker'syeast. Biochemistry 20:468-472.

14. Garrad, R. C., and J. K Bhattacharjee. 1992. Lysine biosynthesisin selected pathogenic fungi: characterization of lysine auxotrophsand the cloned LYSI gene of Candida albicans. J. Bacteriol.174:7379-7384.

15. Goshorn, A. K, S. M. Grindle, and S. Scherer. 1992. Gene

isolation by complementation in Candida albicans and applica-tions to physical and genetic mapping. Infect. Immun. 60:876-884.

16. Henikoff, S., and E. H. Cohen. 1984. Sequences responsible fortranscription termination on a gene segment in Saccharomycescerevisiae. Mol. Cell. Biol. 4:1515-1520.

17. Hinnebusch, A. G. 1988. Mechanisms of gene regulation in thegeneral control of amino acid biosynthesis in Saccharomycescerevisiae. Microbiol. Rev. 52:248-273.

18. Hopfer, R L 1985. Mycology of Candida infections, p. 1-12. InG. P. Bodey and V. Fainstein (ed.), Candidiasis. Raven Press, NewYork.

19. Jones, E. E., and H. Broquist. 1966. Saccharopine, an intermediateof the a-aminoadipate pathway of lysine biosynthesis. J. Biol.Chem. 240:2531-2536.

20. Kozak, M. 1986. Point mutations define a sequence flanking theAUG initiator codon that modulates translation by eukaryoticribosomes. Cell 44:283-292.

21. Kurtz, M. B., R Kelly, and D. R Kirsch (ed.). 1990. Moleculargenetics of Candida albicans, p. 21-73. CRC Press, Inc., BocaRaton, Fla.

22. Lejohn, H. J. 1971. Enzyme regulation, lysine pathways and cellwall structures as indicators of evolution in fungi. Nature (Lon-don) 231:164-168.

23. Magee, B. B., Y. Koltin, J. A. Gorman, and P. T. Magee. 1988.Assignment of cloned genes to the seven electrophoreticallyseparated Candida albicans chromosomes. Mol. Cell. Biol. 8:4721-4726.

24. Meyer, S. A., D. G. Ahearn, and D. Yarrow. 1984. Genus 4, p. 1-12.In N. J. W. Kreger-van Rij (ed.), Candida. Elsevier, Amsterdam.

25. Morris, M. E., and S. Jinks-Robertson. 1991. Nucleotide sequenceof the LYS2 gene of Saccharomyces cerevisiae: homology toBacillus brevis tyrocidine synthetase 1. Gene 98:430-434.

26. Musial, C. E., F. R Cockerill Im, and G. D. Roberts. 1988. Fungalinfections of the immunocompromised host: clinical and labora-tory aspects. Clin. Microbiol. Rev. 1:349-364.

27. Needleman, S. B., and C. D. Wunsch. 1970. A general methodapplicable to the search for similarities in the amino acid sequenceof two proteins. J. Mol. Biol. 48:443-453.

28. Odds, F. C. 1979. Candida and candidosis. Leicester UniversityPress, Leicester, United Kingdom.

29. Pereira, S. A., and G. P. Livi. 1993. Cloning and expression of theARO3 gene encoding DAHP synthase from Candida albicans.Gene 132:159-165.

30. Rodriguez, R L., and R C. Tait. 1983. Recombinant DNAtechniques: an introduction. Benjamin/Cummings, Reading, Mass.

31. Sanger, F., S. Nicklen, and A. R Coulson. 1977. DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

31a.Scherer, S., et al. Unpublished data.32. Scherer, S., and P. T. Magee. 1990. Genetics of Candida albicans.

Microbiol. Rev. 54:226-241.33. Sherman, F., G. R Fink, and J. B. Hicks. 1986. Methods in yeast

cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

34. Sinha, A. K., and J. K. Bhattacharjee. 1970. Control of a lysinebiosynthetic step by two unlinked genes of Saccharomyces cerevi-siae. Biochem. Biophys. Res. Commun. 39:1205-1208.

35. Taylor, W. R, and J. M. Thornton. 1984. Recognition of super-secondary structure in proteins. J. Mol. Biol. 173:487-514.

36. Vogel, H. J. 1960. Two modes of lysine synthesis among lowerfungi: evolutionary significance. Biochim. Biophys. Acta 41:172-174.

37. Wierenga, R K., P. Terpstra, and W. G. J. Hol. 1986. Prediction ofthe occurrence of the ADP-binding Pap fold in proteins, using anamino acid sequence fingerprint. J. Mol. Biol. 187:101-107.

38. Xuan, J.-W., P. Fournier, N. Declerk, M. Chasles, and C. Gaillar-din. 1990. Overlapping reading frames at the LYS5 locus in theyeast Yarrowia lipolytica. Mol. Cell. Biol. 10:4795-4806.

VOL. 62, 1994

on February 11, 2020 by guest

http://iai.asm.org/

Dow

nloaded from