Cloning, sequence analysis and expression of the gene encoding imidazole glycerol phosphate dehydratase in Cryptococcus neoformans

Gene, 145(1994)135-138 0 1994 Elsevier Science B.V. All rights reserved.

135

0378-l 119/94/$07.00

GENE 07989

Cloning, sequence analysis and expression of the gene encoding imidazole glycerol phosphate dehydratase in Cryptococcus neoformans (HZS3; hisB; hid; histidine biosynthesis; fungi)

Aulma R. Parked, Tracey D.E. Mooreb, Jeffrey C. Edmanb, John M. Schwab” and V. Jo Davissona aDepartment of Medicinal Chemistry and Pharmacognosy, Purdue University. West Lafayette, IN 47906, USA; and “Hormone Research Institute and Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA. Tel. (l-415) Received by G.P. Livi: 10 January

1994; Revised/Accepted:

3 March

1994: Received at publishers:

476-7986

31 March

1994

SUMMARY

A cDNA from Cryptococcus neoformans, encoding imidazole glycerol phosphate dehydratase (IGPD), was isolated by complementation of a his3 mutant strain of Saccharomyces cerevisiae. The C. neoformans HIS3 cDNA encodes an approx. 22-kDa protein with a high degree of amino-acid sequence similarity to IGPDs from ten other microorganisms, as well as Arabidopsis thaliana. Most striking are two conserved HHXXE regions and several conserved His, Asp and Glu residues. The cDNA was engineered for expression in Escherichia coli and an approx. 26-kDa protein was identified by SDS-PAGE. DNA and N-terminal sequence analyses confirmed that this protein was C. neoformans IGPD. Furthermore, IGPD assays of crude extracts from IGPD-producing E. coli cells demonstrated that the C. neoformans protein was catalytically active.

INTRODUCTION

The nt sequences of genes controlling histidine biosynthesis from various microorganisms have been reported Correspondence Chemistry Lafayette, 494-6790;

to: Dr.

V.J.

Davisson,

Department

of Medicinal

and Pharmacognosy, 1333 RHPH, Purdue University, West IN 47097-1333, USA. Tel. (l-317) 494-5238; Fax (1-317) e-mail: [email protected]

Abbreviations: A, absorbance (1 cm); aa, amino acid(s); ACLCB. AIDS Center for Laboratory and Computational Biochemistry (Purdue University); Ap, ampicillin; bp, base pair(s); C., Cryptococcus: Cm, chloramphenicol; Cn, C. neoformans; dATP, deoxyadenosine 5’-triphosphate;

dNTP,

deoxyribonucleoside

triphosphate;

HIS3, gene

encoding IGPD; HPP, histidinol phosphate phosphatase; IAP. imidazole acetol phosphate; IGP, imidazole glycerol phosphate; IGPD, IGP dehydratase; IPTG, isopropyl-B-o-thiogalactopyranoside: kb, kilobase(s) or 1000 bp; LB, Luria-Bertani (medium); NCBI, National Center for Biotechnology Information; nt, nucleotide(s); oligo. oligodeoxyribonucleotide; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; TAE, Tris-acetate-(ethylenedinitrilo)tetraacetic acid; Tc, tetracycline; 2 x YT, 2 x yeast-tryptone (medium); X, any aa; [I, denotes plasmid-carrier state. SSDl 0378-l

119(94)00200-C

(Struhl, 1985; Chiariotti et al., 1986; Carlomagno et al., 1988; Fani et al., 1989; Limauro et al., 1990; Derlorme et al., 1992; Goldman et al., 1992; Wei et al., 1993). Since histidine is an essential aa, enzymes of the histidine biosynthetic pathway are attactive targets for the design of antifungal, antibacterial and herbicidal agents. Knowledge of the mechanisms of the histidine biosynthetic enzymes is critical for such an approach. In order to understand factors involved in the virulence of Cryptococcus neoformans (Cn), selectable markers that allow investigators to carry out molecular studies on Cn are necessary (Edman and Kwon-Chung, 1990). The Cn HIS3 gene may prove useful in this respect, as it has been in Saccharomyces cerevisiae (Garfinkel et al. 1988; Sikorski and Hieter, 1989; Christianson et al., 1992). Here, the cloning and sequence analysis of a HIS3 cDNA encoding IGPD from Cn are described, and we demonstrate that E. coli is a suitable host for the expression of Cn HZS3. Although sequences of eleven IGPD encoding genes are known, published sequence alignments have been limited to a few microorganisms (Fani

136 et al., 1989; Goldman

et al.. 1992). We now report

comprehensive

sequence

striking

of aa identity.

regions

alignment,

revealing

several

GAATTCC TrmrMTATrrc~CCATcACACCATCCCGCCCw+C

-46

a

-1

. ~TCTGAACGCATTGCTWTG~AAA~ACCACGAGCGAGACGCATATCTC~CACT

60

MSERIASVERTTSETH

I

S

C

T

20

ATCGACCTCGACCACATCCCAGGTGTCACCGAGCAGAAC

120

IDLDHIPGVTEQKINVSTGI

40

GGGTTCCT’XACCATATGTAGCGCTCGCAAAGCACGGCGGCATCTCTCTCCAACTG

EXPERIMENTAL

180

GFLDHMFTALAKHGGMSLQL

AND DISCUSSION

60

CAGTGCAAGGGCGACCTTCACATTGACGACGACCACCACACGGCGGAAGACTYXGCTTTGGCT

240

QCKGDLHIDDHHTAEDCALA

(a) Determination of the Cn HIS3 gene sequence A Cn HIS3 cDNA clone was isolated by complementation in an S. cerevisiae his3 mutant library yeast/E.

(Edman

and Kwon-Chung,

coli shuttle

TRPl marker;

strain.

1990) cloned into the

vector pYcDE-SfiNot

J.C.E., unpublished)

the S. cerevisiae strain

A Cn cDNA (contains

was transformed

IH1837(MATa

ura3-52 his3); a gift from I. Herskowitz,

the into

ste14-2 adel trpl UCSF.

Pooled

yeast transformants were plated onto synthetic dextrose medium containing adenine and uracil but lacking histidine, and plasmid DNA was isolated from a histidine prototrophic transformant and introduced into E. coli. A llOO-bp NotI-EcoRI cDNA insert was subcloned into pBluescript II KS+ to create pBluescript KS-HIS3. Both strands of the Cn HIS3 cDNA were sequenced by the method of Sanger (1977). A 609-bp ORF was identified as encoding an IGPD (Fig. 1). The coding region predicts a translated product of 21976 Da. (b) The aa sequence alignments of IGPD proteins The predicted aa sequence of Cn IGPD was aligned with all available IGPD sequences (Fig. 2). Cn IGPD has 63% identity with IGPD from S. cerevisiae and shares a high degree of identity with all other known IGPDs. Most striking are two conserved HHXXE regions, seven conserved His, four conserved Glu and five conserved Asp residues. Currently, no experimental evidence exists to assign functional roles for these highly conserved residues. However, since all IGPDs examined thus far require a metal cofactor (usually Mnzf), it is postulated that several of these highly conserved aa interact with the metal cofactor or the substrate. In E. coli and S. typhimurium, the IGPD protein is bifunctional, since it also exhibits histidinol phosphate phosphatase (HPP) activity (Martin et al.. 1971). The sequence alignment in Fig. 2 predicts that, like other fungal IGPDs, Cn IGPD does not have HPP activity (Broach, 1981; Chiariotti et al., 1986). Enzyme activity assays from our laboratory confirm that the protein has a single catalytic function (data not shown). (c) Production of Cn IGPD in E. coli Our initial attempt to develop regulated expression of the Cn HIS3 cDNA in E. coli used the tat promoter vector pJF119EH (Ftirste et al., 1986). This construct, pHZS3-tat, conferred the his+ phenotype to transformed

80

CTTGGCGAAGCGTTTAAAAAGGCGCTTGGAGAGAGGAAGGGAATCAAGCGATA’I’XATAT LGEAFKKALG

E

RKG

I

K

300

R

YGYlOO

S

S

GCTTA’,YXTCCCCTKA’AGTCGCTIWXAGGGCTGTGAT.X3ACATTTCTTCCCGGCCG AY

A

P

L

D

E

S

L

S

RA”

I

D

360

I

R

P120

E

“140

D

S160

TACT’ITATXGCCACCTM3CC“TCACTCGGGAAAAGGTTGGAGATTTATCGACffiAAATG Y

F

M

C

H

L

P

F

T

R

E

KVG

D

L

420 ST

GTGTCTCACCT’KTTCAGTCGTITGCCTTTGCCGCCGCCGGTGT~CCCWCACA~ACWG “S

H

L

L

QS

FAFAAGVT

L

480 H

I

ATCCGAGGAGAAAACAACCACCACATCGCCGAGTC’FGCCTTCAAGGCGCTCGCTCKGCT I

RG

E

NNH

H

I

AE

S

AFKA

L

540 ALA

180

ATCCGAATGGCGATtAGCAGAACCGGCGGCGATGACGTTCCTAGTACCAAGGGTGTCCTT IRMA

I

S

RTGGDD”

P

S

600

TKGVL

200

GC’ITTATAATAATAGTAATAATAATRATAATAACTATTCATATCATACATTCGGTCTTTGGTG AL’****.* f 210

660

GTA’KX’CTGCTCCAAATGGGCATGTATCGCGCG

720

TFTKATTATTCAGAAAA

Fig 1. Sequences

738

of Cn HIS3

cDNA

and

IGPD

(* indicates

stop

codon). The start codon is underlined and aa are aligned with the third nt of each codon. Sequencing was performed by the method of Sanger

( 1977) using the Sequenase kit, version 2.0 (US Biochemical,

Cleveland,

OH, USA). Single-stranded plasmid templates were isolated from transformed E. coli XL-l Blue which had been infected with bacteriophage VCSM13

(Stratagene,

La Jolla, CA, USA). T7 and T3 oligodeoxyribo-

nucleotide (oligo) primers (Stratagene) and four internal primers ( 19-mers, Center for Macromolecular Structure, Purdue University) were used to sequence

the sense and anti-sense

strands.

All sequencing

reactions included [c(-‘*S]dATP (5 pCi) and pyrophosphatase (0.004 units) (US Biochemical) to eliminate variations in band intensity. Reactions were analyzed on 8% polyacrylamide-6.8 M urea gels, which were dried and exposed to Kodak X-OMAT AR film for 12-24 h. The GenBank

accession

No. is UO4329.

E. coli strain FB 251 (hisB). However, attempts to induce high level production of Cn IGPD using the pHZS3-tat construct in E. coli strain FBl (A hisGDCBHAFI/E) were unsuccessful (data not shown). On the other hand, expression of Cn HIS3 cDNA cloned into the T7 RNA polymerase vector PET-lla (Novagen, Madison, WI, USA) afforded useful levels of protein. E. coli BL21 (DE-3)[pLysS] (Novagen) harboring the plasmid construct pHZS3-T7 were grown to an A,,, nm of 1.2, and IPTG was added to induce expression of HZS3. Production of Cn IGPD was confirmed by SDS-PAGE (Fig. 3), which revealed the presence of a major protein that migrates with an apparent molecular mass of 26 kDa. The soluble characteristics of this protein were established by analysis of IGPD activity in cell-free extracts of the induced cultures. IGPD specific activity was 19 units/mg in these cells which is 20-fold over the detectable background activity in the noninduced cell cultures. No HPP activity (Martin et al., 1971) was detected, consistent with the monofunctionality of HIS3, deduced from

m Ll SC SC ??I Ab

Sk An AC Pn EC .?a

1 85 * . ........ f .............. ..&SERIASVERTTSIiTSiISCTIDL D ................. ..HIFGVTEQKIL;VSTOIGBL~*~~=G~SLPLPCKOD...LHIDDIiHTAlDCALALGEA F ............... ..MTRISHITRNTKBTQIELSINLDG T ...................... ..GQADISTDIGPLDlBIILTLLTFBSDFDLKIIGHOD~~G~P~IIDVAI~GKC I ............... ..MSRVGRVER~~~LVEIDLDG T ...................... ..GKTDIATOVCIY~DQLGRBGLFDLTVKTDOD...LHIDSHHTIPDTALALGAA F ................~EQKALVKRITNPTKIQIAISLKGGPLAIEHSIFPEK~~VAEQA~SQVI~HTOIG~L~I~~KESGWSLIVECIO D ...LHIDDRHTTIDCGIALGQA F ............MASPLPVRAAALSRDmJITSIQIALSIDGOELPQDADPRCKQD...LHIDD~AIDCCIAVGTT F ..........~DQSLANGVGGASIERNTTBTAIRVAVNLDO T ...................... ..GVYDVKTOVGPLDBYLEQLSRHSLMDLSVAAEOD...VHIDA~PWSGIAIGQA V ....~TEPAQKKQKQTVPERKAFISRI~~IQIAISLNGGYIQIKDSILPAKKDDDVASQATQSQVIDIHTOVGBLDBIIIHALAKIISGWSLIVECICI D ...LHIDDIU#'ITIDCGIALGQA F ....~QISRYPQLNLSSVPRIASVHRIlG~NVQ~~ T ...................... ..GICXAATOIPPLDHYLDQLSEGLFDV~TOD...VHIDD~IIDIALAIGT RL LUQSISCSASSSSS~GRIG.~R~~~~K~~~~ ...................... ..VADSSSOIPPLDIMHQISSRGLIDLDVQAKODWE...IDDHaPNIDVGITLG QAL VFAVFTAS~P~GKSGVR~ISR~~S~~KLSL~T ...................... ..GKSKVSSQIGPLMMTALAK%SRFDLELDCKOD...TWIDD~I~TLGEA F YDR~L~PMIG~QLT~RDRY~R~~~~Q~DRE ...................... ..GGSKINTOVGFF~DQIATHGGF~EI~GD...LYIDD~LD~~ ERL YHR~~IGEQLTKRDRY~~~IDVS~~RE ...................... ..GNSKIWTOVGPF~DQIATHF~EITVKCID...LYIDD~BDTGLALR EAL

01 Ll SC SC 78 A!3

Sk At An FYI EC .%I

Fig 2. Amino-acid sequence alignment of IGPDs available through the literature and through sequence databases. Residues conserved across all IGPD sequences are indicated in bold. For E. coti (EC), S, r~~~~~~r~~~(Sa) and P. ~jc~f~~~ue(Pn), only the IGPD domains are included; the A. thaiiuna (At) sequence starts at aa 56. Start codons for the other IGPDs are underlined. Numbering is based upon Cn IGPD. The percent identity (first value) and similarity (second value) between C. negjiirmans (Cnn)IGPD and IGPDs from other organisms are as follows: S. cerevisiae: (SC) 63, 75 (Struhi, 1985); S. kluyveri (Sk): 60, 78 (GenBank accession no. 531235); Trichoderma harzianum (7%): 60, 76 (Goldman et al., 1992); ~~y~o~~r~~ru ni~~t~u~ue(Pn): 58, 74 (GenBank accession no. Z11591); Azos~irit~um~~~~j~e~se(A&: 51, 72 (Fani et al., 1989); E. coli (EC): 50, 71 (Chiariotti et al., 1986); Su~monellQ~yp~i~~~~u~(Su): 49,71 (Carlomagno et al., 1988); Arubidopsis r~iu~iunu(At): SO.69 (E. Ward, personal communization); Srrepro~y~es co&color (St): 48, 64 (Limauro et al., 1990); Anaburna sp. (An):46, 65 (Wei et al., 1993); Lactococcus luctis (AI): 43, 65 (Derlorme et al., 1992). Comparison scores were generated using the Genetics Computer Group (Madison, WI, USA) program Bestfit. The Anabaena sp. his5 sequence has been reported, but IGPD activity has not been demonstrated.

12

3

4

5

6

7

8 "a __1_ :;:4 45.0

Fig. 3. Production of Cn IGPD in E. coli. E. coli BL21(DE3)[pLysS] cells were transformed with pHIS3-T7, plated onto LB containing Ap (100 pggiml) and Cm (25 pg/ml) and grown overnight at 37°C. A single colony was inoculated into 4 ml 2 x YT (Ausubel et al., 1989) containing Ap ( 1.50 &ml) and Cm (25 pg/ml) and grown at 37°C for 12 h. 1 ml of this culture was used to inoculate 100 ml of fresh medium. Cells were grown to an A550nm of 1.2, and expression of IGPD was induced by adding IPTG (Gold Biotechnology, St. Louis, MO, USA) to a final concentration of 1 mM. lo-ml aliquots were taken at 0, I,&4 and 6 h. Cells were harvested by centrjfugation and stored at - 80°C. Cell pellets were resuspended in 2 ml of 100 mM triethanolamine (PI-I 8.1). cooled on ice and disrupted by four 15-s sonic&ion periods separated by 10-s rests. Cell debris was removed by centrifugation at 27ooO xg for 15 min. The supernatant phase was analyzed using the Bio-Rad protein assay and by 0.1% SDS-15% PAGE (4 pg protein per lane). Lanes I

aa sequence alignments (Fig. 2). The partial sequence of purified Cn HIS3 was determined using Edman degradation chemistry. (Laboratory for Macromolecular Structure, Purdue University). The N-terminal Met resi-

and 8, Bio-Rad low molecular mass protein standards; lane 2, PET-lla, no IPTG, 4 h; lane 3, PET-lla, +IPTG, 4 h; lane 4, pHIS3-T7, no IPTG, 2 h; lane 5, pHIS3-T7, + IPTG, 2 h; lane 6, pHIS3-T7, no IPTG, 4 h; lane 7, pHIS3-T7, +IPTG, 4 h. Methods: Construction of expression vector pHIS3-T7: Oligo primers (Center for Macromolecular Structure, Purdue University) that incorporated an overlapping 5’ EcoRI/NdeI site and a 3’ BamHI site were used in a PCR to amplify HIS3. The sense primer nt sequence was S-CGGAATTCATATGTCTGAACGCATTG~TTC, and the antisense primer nt sequence was S’-CGGGATCC~A~ATAAAGCAAGGACACCC (restriction sites underlined; start codon doubly underlined). The amplified PCR product was purified using a Magic PCR prep column (Promega, Madison, WI, USA), digested with BamHI and EcoRI, and cloned into pBluescript I1 SK+ (Stratagene) between the BamHI and EcoRI sites using T4 DNA ligase (Promega). E. coli XL-1 Blue cells were transformed with this reaction mixture, and colonies were selected on LB plates containing Ap (50 pg/ml) and Tc (15 pg/ml). Plasmid DNA was isolated and pBluescript-HIS3 construct was digested to completion with BarnHI, followed by a partial NdeI digest on the linearized template to liberate the HIS3 cDNA. The DNA fragments were separated on a TAE 1.5% agarose gel, and the 616-bp fragment was extracted using the Geneclean kit (BiolOl, La Jolla, CA, USA). The purified DNA was cloned between the BamHI and NdeI sites of PET-lla (Novagen) using T4 DNA ligase (Promegaf, to give pHIS3-T7. DNA sequence analysis of pHtS3-T7 confirmed that the Ndel-BamHI fragment containing the entire HIS3 coding region had been faithfully cloned into PET-1 la.

138 due was not detected, but the next 25 aa corresponded to the predicted aa sequence. The observed mobility of Cn IGPD on SDS-PAGE was 16% greater than anticipated based upon the predicted molecular mass. The basis of this anomalous electrophoretic mobility cannot be explained at this time.

P.: Multifunctional yeast high-copy-number 110 (1992) 119-122.

shuttle

vectors.

Gene

Derlorme, C., Ehrlich, S.D. and Renault, P.: Histidine biosynthetic genes in Lactococcus lactis supsp. lactis. J. Bacterial. 174 (1992) 6571-6579. Edman.

J.C. and Kwon-Chung,

Cryptococcus neoformans marker for transformation.

of the URAS gene from

K.J.: Isolation

var. neoformans and its use as a selective Mol. Cell. Biol. 10 (1990) 4538-4544.

Fani, R.. Bazzicalupo, M., Damiani, G., Bianchi, A., Schipani. C.. Sgaramella, V. and Polsinelli, M.: Cloning of the histidine genes of Azospirillum hrasilense: organization nucleotide 224-229.

ACKNOWLEDGEMENTS

We thank Prof. Carmelo B. Bruni for E. coli strains FB 1 and FB 251 and Prof. Ira Herskowitz for S. cerevisiae strain IH1837(MATa ste14-2 adel trpl ~~3-52 hid). We thank Eric Ward for communicating the A. thaliana sequence prior to publication. Financial support has been provided in the form of a Purdue Research Foundation Fellowship (to A.P.) and award 6056 from the Showalter Trust Fund.

of the ABFH gene cluster and

of the hisB gene. Mol. Gen. Genet. 216

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region

tucP expression

in a multi-host-range

Garfinkel, D.J., Mastrangelo, M.F., Sanders, N.J., Shafer, Strathern, J.N.: Transposon tagging using ty elements Genetics 120 (1988) 955108. Goldman. G.H., Demolder, J., Geremia,

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