Disruption of gene YlODC reveals absolute requirement of polyamines for mycelial development in Yarrowia lipolytica

FEMS Yeast Research 1 (2001) 195^204

www.fems-microbiology.org

Disruption of gene YlODC reveals absolute requirement of polyamines for mycelial development in Yarrowia lipolytica Juan Francisco Jime¨nez-Bremont a , Jose¨ Ruiz-Herrera a

b

a;

*, Angel Dominguez

b

Departamento de Ingenier|¨a Gene¨tica, Unidad Irapuato, Centro de Investigacio¨n y de Estudios Avanzados del Instituto Polite¨cnico Nacional, Apartado Postal 629, 36500 Irapuato, Gto., Mexico Departamento de Microbiolog|¨a y Gene¨tica, Instituto de Microbiolog|¨a Bioqu|¨mica, CSIC/Universidad de Salamanca, 37007 Salamanca, Spain Received 12 March 2001; received in revised form 27 June 2001; accepted 14 July 2001 First published online 1 August 2001

Abstract Polyamines are required for cellular growth and differentiation. In mammals and fungi they are synthesized via a pathway involving ornithine decarboxylase (ODC), which transforms ornithine into putrescine. We have cloned and disrupted the gene coding for ODC in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus. Substrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence. A single transcript 1.7 kb in size was identified by Northern hybridization, and confirmed by rapid amplification of cDNA ends (RACE). Null mutants lacked ODC activity and behaved as polyamine auxotrophs. When low levels of polyamines were supplied to the null mutant, only yeast-like, but not mycelial growth was sustained. This phenomenon was confirmed by introduction of the YlODC gene under the control of an inducible promoter into the null mutant. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Polyamine; Ornithine decarboxylase ; Fungal dimorphism; Yarrowia lipolytica

1. Introduction Yarrowia lipolytica is a dimorphic organism that grows as a mixture of yeast-like and short mycelial cells in solid and liquid media. Study of the environmental factors involved in the regulation of the dimorphic transition indicates that use of N-acetylglucosamine (GlcNAc) induced mycelial growth [1]. Y. lipolytica appears to be a useful model for the study of dimorphism in fungi, since it may be subjected to genetic manipulation [2^4] and transformation [5,6]. Polyamines are essential polycationic micromolecules that play important roles in growth and di¡erentiation in a number of organisms [7^9]. Among the many roles played by polyamines, it has been described that they protect DNA from enzymatic degradation, X-ray irradiation,

* Corresponding author. Tel.: +52 (462) 39 60; Fax: +52 (462) 45 849. E-mail address : [email protected] (J. Ruiz-Herrera).

mechanical shearing and oxidative damage [10]. They also stabilize RNA, prevent ribosome dissociation, stimulate DNA and RNA synthesis, and improve the ¢delity of translation in vitro [10]. In mammals and fungi polyamines are synthesized by a pathway initiated by ornithine decarboxylase (ODC) with formation of putrescine from ornithine. Transient increases in the levels of ODC and polyamines have been shown to take place during the yeast-to-hypha transition in di¡erent dimorphic fungi [11^13], including Y. lipolytica [14]. A role of polyamines in dimorphic transition is also sustained by the observation that ODC inhibitors such as 1,4-diaminobutanone block the yeast-tohypha transition in di¡erent fungi, Y. lipolytica included [10,14,15]. Recently, we have demonstrated that dimorphic fungal mutants a¡ected in ODC displayed a dependence on the concentration of polyamines to engage in their dimorphic transition. Both Ustilago maydis [16] and Candida albicans [17] required higher concentrations of putrescine to grow in mycelial form than to satisfy their auxotrophic requirements. In this study we have proceeded to obtain ODC null mutants of Y. lipolytica by reverse genetics, in order to further investigate this phenomenon.

1567-1356 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 1 3 5 6 ( 0 1 ) 0 0 0 3 3 - 2

FEMSYR 1425 26-11-01

196

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

2. Materials and methods

manufacturer (Amersham Pharmacia Biotech., Piscataway, NJ, USA).

2.1. Strains, plasmids and culture media

2.3. RNA preparations and Northern analyses

The strains of Y. lipolytica, used in this work, were PO1a (MatA, leu2-270, ura3-302), FJOD (MatA, leu2270, ura3-302, vodc) carrying a disruption of the YlODC gene, FJCO derived from FJOD (MatA, leu2-270, vodc/ pFJ4 (YlODC‡ )) carrying a plasmid with the YlODC ORF under the control of the metallothionein (METI-II) promoter, and FJODC (MatA, leu2-270, vodc, YlODC), an integrative transformant obtained from FJOD by reintroduction of the wild-type ODC gene. All strains were grown in YEPD medium containing 1% yeast extract, 1% peptone and 1% glucose, or in MM medium (0.67% yeast nitrogen base, 1% glucose), supplemented with leucine and/or uracil, each at 50 Wg ml31 , and variable concentrations of putrescine as required. Plasmids used in the study were the following: pFJ1, a pBKS+ plasmid containing the YlODC gene in a 3.5 kb KpnI-KpnI genomic fragment ; pFJ2, obtained from the former by insertion of the YlURA3 gene ; pFJ3 obtained from the former by partial deletion of YlODC ; pFJ4 derived from pINA444, containing the YlODC ORF under the control of the MTPI-II promoter (see Section 3); and PFJ5, a pET-28b plasmid (Novagen, Madison, WI, USA) containing the YlODC ORF driven by the T7lac promoter (see Section 3). Media and procedures used for yeast transformation have been described by Xuan et al. [18,19]. Dimorphic transition was carried out as previously described [1,14]. Growth was measured as optical density at 600 nm, and the proportion of yeast and mycelial cells in the cultures was assessed by phase contrast microscopic observations, counting a minimum of 200 cells. Reported data are representative of at least three di¡erent experiments. The Escherichia coli strain used for transformation and ampli¢cation of recombinant DNA was DH5K [20] grown on LB broth [21]. The polyamine auxotrophic strain of E. coli EWH 319 [22] was used to determine the functionality of the YlODC gene in the bacterium. Other bacterial strains were also grown in M9 minimal salts medium [21] supplemented with kanamycin (50 Wg ml31 ) for plasmid selection.

The RACE procedure was conducted to identify the tsp and poly(A) addition sites of the YlODC gene [27]. To this aim a commercial kit (Gibco Life Technologies, Rockville, MD, USA) was employed. RNA was obtained as described above, and subjected to RT-PCR. The ampli¢ed products were subcloned into p-GEM and sequenced as above. The sequences of the gene-speci¢c antisense primer (910^935 bp) used for 5P-RACE, and the gene-speci¢c sense primer (1517^1535 bp) used for 3P-RACE were, respectively, as follows: 5P-AAGACTCCTTCTTCGTGGCTGACCT-3P (GSP1), and 5P-TCGGACGATTCCTTTGAGTCCTCT-3P (GSP2). Additional nested oligonucleotides necessary for the method were designed according to the sequence previously obtained.

2.2. DNA manipulations

2.6. Other methods

Total DNA from Y. lipolytica was obtained as described by Raeder and Broda [23] for ¢lamentous fungi. Restriction enzyme digestions and DNA ligations were performed according to the recommendations of the manufacturers (Promega, Madison, WI, USA; Fermentas, Hanover, MD, USA). Isolation of plasmid DNA from E. coli was performed using standard procedures [21]. DNA fragments to be used as probes were labeled by random priming with 32 P, and used according to the instructions of the

In order to isolate the YlODC gene, a minigenomic library of Y. lipolytica KpnI fragments of approx. 3.5 kb was prepared in pBKS+. Selection of the restriction enzyme was based on the fact that genomic DNA digested with KpnI displayed a single hybridization band of this size when challenged with the previously described Y. lipolytica ODC gene fragment obtained by PCR [28]. This same fragment was used as a probe to screen the minigenomic library.

RNA was prepared from exponentially grown cells in YEPD, by the method of Percival-Smith and Segall [24], using a kit from BIO 101 (Vista, CA, USA). Prehybridization and hybridization were performed according to standard procedures [21]. 2.4. Sequence analysis The 3.5 kb DNA restriction fragment harboring the YlODC gene was subcloned into pBluescript plasmids (KS+, Stratagene, La Jolla, CA, USA). DNA sequencing was conducted with double-stranded templates on an ABI PRISM 377 DNA automated sequencer (Perkin Elmer). Nucleotide and amino acid sequences were analyzed using the DNASTAR, DNAStrider 1.1 programs and PSORT (version 6.3 WWW). The amino acid sequence of Ylodcp was compared with the Swiss-Prot data bank using the FASTA program [25]. Alignments of amino acid sequences were done with CLUSTAL programs [26]. The sequence data reported here have been assigned EMBL accession No. AJ237707. 2.5. Identi¢cation of the transcription start point (tsp) and polyadenylation site

FEMSYR 1425 26-11-01

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

197

Fig. 1. Nucleotide (nt) sequence of the YlODC gene. The SpeI and NheI restriction sites (waved underlined, bold) of the genomic sequence, the genespeci¢c primers (GSP1 and GSP2) used for RACE (lowercase, bold and underlined), as well as the putative CAAT boxes (bold and encased), and tsp (double underlined A) are indicated. The asterisk locates the putative stop codon, and dotted underline marks the transcription termination motif. Double underlining indicates the consensus polyadenylation signal sequence. The site for addition of a poly(A) tail is indicated by an A inside a box.

The so-called pop-out technique (see [29] for details) was used for partial replacement of the YlODC wildtype gene. Basically, the method involved integration by homologous recombination of a plasmid (pFJ3) containing a fragment of the YlODC gene, and the URA3 gene as a selectable marker. This created a duplication containing the wild-type copy and the mutant copy of YlODC £anking the plasmid sequences. The second step was the excision of the plasmid carrying the URA3 and wild-type genes, and selection for ura3 mutants by use of 5-£uoroorotic acid resistance. Analysis of the functionality of YlODC gene in E. coli

was performed as follows : the full ORF was PCR-ampli¢ed from a plasmid (pFJ1), and subcloned in phase into pET28b (Novagen, Madison, WI, USA) which contains the strong bacteriophage T7lac promoter for expression in bacteria, and the gene conferring kanamycin resistance. The resulting plasmid (pFJ5) was used for transformation of the polyamine auxotrophic strain of E. coli EWH 319 [22]. ODC activity in intact cells was measured as described previously and expressed as nmoles of CO2 released (mg protein)31 (min)31 [14]. Reported data are representative of at least three di¡erent experiments.

FEMSYR 1425 26-11-01

198

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

Fig. 2. Multiple amino acid alignment as derived by maximal homology of ODCs from Y. lipolytica (Y.l.), N. crassa (N.c.), S. cerevisiae (S.c.), C. albicans (C.a.) and U. maydis (U.m.). Identical residues (asterisks) in the ¢ve polypeptides and conserved amino acid substitutions (dots) are indicated. The putative PEST regions are in italics and underlined. The double-underlined and bold sequence corresponds to the cofactor (pyridoxal phosphate)-binding site. The signature sequence of the conserved decarboxylase family 2 appears in bold and underlined. The encased motif within the second PEST region corresponds to the conserved ODC catalytic sequence.

3. Results 3.1. Isolation of the YlODC gene, and characteristics of the coded enzyme Several positive clones were detected in the minigenomic library described above when screened with the PCR product isolated previously (see Section 2). One of these clones (E. coli 3-32) was selected for further analysis. The plasmid carried by this strain (pFJ1) was propagated, and its KpnI fragment (see Section 2) was sequenced. The sequence revealed the presence of a single ORF of 1350 bp. The deduced coded protein displayed high homology with ODCs from other sources (see below). It contained 449 amino acids with a molecular mass of 49 183.9 Da, and an isoelectric point of 4.74. No consensus splicing sequences were detected. The 5P-RACE method was used to resolve the location of the site of transcription initiation (transcription start point, tsp). According to the results

obtained, transcription initiation takes place at a single site, the A residue located at 3218 bases upstream from the predicted start of translation. Two possible CAAT boxes were located at positions 3340 and 3508 bp from the start point of translation, but no TATA box was identi¢ed. The 3P-RACE method identi¢ed a site for addition of a poly(A) tail, which corresponded to the A residue +75 bases downstream from the predicted termination site of translation. A consensus polyadenylation signal sequence (1402^1408 bp; AATAAA) was identi¢ed 18 bp upstream from the poly(A) addition site (Fig. 1). In agreement with other Y. lipolytica genes [30], YlODC displayed the common transcriptional termination motif TAA...TAGT/ TATGT...TTT. Alignment of the deduced amino acid sequence with those of other cloned ODCs showed a high degree of homology: 50.9% with Neurospora crassa, 46% with Saccharomyces cerevisiae, 45.2% with Coccidioides immitis, 38.7% with C. albicans, and 37.3% with Ustilago maydis

FEMSYR 1425 26-11-01

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

199

zyme in the cytoplasm (66%), nuclei (16%), and the rest in other organelle(s). 3.2. Transcription of the YlODC gene Northern blotting using a 740 bp StuI fragment as a probe revealed a single transcript of about 1.7 kb (Fig. 3, lane 2). RNA obtained from a null mutant (see below) was used as a negative control (Fig. 3, lane 1). No RNA signal was observed in such disruptant. These results indicate that YlODC is transcribed into a single and speci¢c mRNA. 3.3. Disruption of the YlODC gene

Fig. 3. Northern blot analysis of the YlODC transcript. Total RNA obtained from Y. lipolytica P01a cells in the exponential phase of growth was denatured with formaldehyde and subjected to agarose gel electrophoresis, transferred to a nylon membrane and probed with a labeled 809 bp StuI DNA fragment from YlODC (see Fig. 4A). Lane 1, vodc strain FJOD ; lane 2, P01a strain. The position and size of RNA markers (Gibco) are indicated at the left.

enzymes. Higher homology was observed in the central part, compared to the amino- and carboxy-termini of the proteins (Fig. 2). A consensus site, [FY]-[PA]-x-K-[SACV][NHCLFW]-x(4)-[LIVMF]-[LIVMTA]-x(2)-[LIVMA]-x(3)-[GTE], for cofactor (pyridoxal phosphate) attachment [31] was located between amino acid residues 101 and 119. Another consensus site containing a stretch of three consecutive glycine residues ([GSA]-x(2,6)-[LIVMSCP]x(2)-[LIVMF]-[DNS]-[LIVMCA]-G-G-G-[LIVMFY]-[GSTPCEQ]), and proposed to be part of the substrate-binding region [32], was located between amino acid residues 257 and 274. A cysteine residue, which is considered to be within the catalytic site, in the consensus sequence VWGPTCDGID [31], was located at position 387. Two putative PEST regions, characteristic of proteins with a high turnover rate [33], but with low PEST scores (especially one of them) of +1.68 and 36.47, were identi¢ed at residues 21^49 and 375^395 of the protein, respectively, using the algorithm described by Rechsteiner and Rogers [34]. Further examination of the polypeptide sequence was carried out using the PSORT program (see Section 2) in order to determine the possible location of ODC in the cell. The results indicated a probable location of the en-

The Y. lipolytica ODC gene was deleted using the popout technique as described in Section 2. The URA3 gene obtained by digestion of pINA444 [35] with SalI was ligated to the only SalI site of the polylinker in pFJ1 plasmid to obtain pFJ2. Most of the ORF (1223 bp, 90.5%) was deleted in this plasmid by digestion with StuI and NheI (Fig. 4A). The resulting pFJ3 plasmid was linearized by digestion with SnaBI, and used to transform the P01a strain of Y. lipolytica. Ura‡ transformants were selected, and checked by Southern hybridization, utilizing the whole ODC gene KpnI-StuI fragment as a probe. Southern analysis revealed that two of the mutants contained both the wild-type 3.5 kb KpnI fragment, plus a deleted 2.1 kb fragment of the YlODC gene (Fig. 4B, lane 2). After counterselection on 5-£uoroorotic acid, loss of the wild allele was con¢rmed by hybridization with the same probe (Fig. 4B, lanes 3^5). 3.4. Determination of the phenotype of the odc null mutant The odc null mutants obtained above behaved as polyamine auxotrophs. Further studies were carried out with one of them (FJOD). When it was grown in complete medium, and afterwards transferred to polyamine-free synthetic medium, the mutant grew at a reduced rate compared to the wild-type after two cycles of growth, but Table 1 Determination of the auxotrophic nature of odc strain FJOD of Y. lipolytica Strain and conditionsa

P01a (parental) FJOD plus 5 mM putrescine FJOD

Growthb at incubation cycle No. in MM 1

2

3

2.1 1.4 1.3

2.0 1.4 0.8

2.0 1.3 0.1

a Cells were grown in complete medium. After 24 h growth was measured, cells were washed and reinoculated into minimal medium (MM). After further periods of 24 h, the process was repeated to complete three growth cycles. b As OD at 600 nm. Inoculum, OD = 0.05.

FEMSYR 1425 26-11-01

200

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

Fig. 4. Replacement of the YlODC gene. Gene disruption was carried out by the pop-out technique. A: Scheme of the strategy followed; see text for details. B: Southern blot analysis of parental and transformants probed with a KpnI-StuI fragment from the YlODC gene. Genomic DNA samples were digested with KpnI. Lane 1, parental strain; lane 2, transformant strain where the URA3-YlODC fragment was integrated; lanes 3, 4, 5, odc transformants obtained by pop-out of the wild-type gene.

required three transfers to putrescine-free medium in order to stop growing (Table 1). Addition of 5 mM putrescine restored the growth rate to a value similar to that of the parental strain. ODC activity from the parental and mu-

tant strains was measured in cells grown for 5 h in polyamine-free minimal medium. ODC speci¢c activity in P01a was approx. 1.2 nmoles (mg protein)31 (min)31 . On the other hand, speci¢c activity of the mutant was about 0.04,

FEMSYR 1425 26-11-01

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

201

ORF under the control of the bacteriophage T7lac promoter (pFJ5). Transformants were recovered on kanamycin-containing LB agar. Transfer to minimal medium revealed that transformants were prototrophic, and that they, in contrast with the parental strain, displayed ODC activity (not shown). 3.5. Polyamine requirement for growth and mycelial development

Fig. 5. Comparative growth rate and dimorphism of parental strain P01a and odc strains of Y. lipolytica. Both strains were incubated in minimal medium (MM) at 28³C for two successive periods of 24 h to reduce the polyamine pool. Then both strains were inoculated into MM (0.15 OD at 600 nm) and incubated under conditions that promote hyphal growth. At intervals OD600 was measured. 8, P01a; F, FJOD in the presence of 1 mM putrescine ; R, 0.1 mM putrescine; a, 0.01 mM putrescine; *, 0.001 mM putrescine; b, no putrescine. Inset: percentage of mycelial cells after 12 h of growth.

a value probably representing the background. To con¢rm that phenotype of the FJOD strain was solely due to disruption of the YlODC gene, it was transformed with the plasmid containing the YlODC and YlURA3 genes (pFJ2, see above) opened at the NarI site. Transformants were recovered in uracil-less medium. Several Ura‡ transformants grown in this medium were transferred to a medium without putrescine, and prototrophic strains recovered. These strains were found to have recovered ODC activity, and the wild-type gene as determined by PCR (not shown). Functionality of the gene in bacteria was evidenced by transformation of a polyamine auxotrophic strain of E. coli EWH 319) with the plasmid carrying the YlODC Table 2 ODC activity in cells of strain FJCO grown in the presence of variable amounts of CuSO4 a CuSO4 (mM)

ODC activityb

0 0.2 0.4 0.8 1.2 2.0

0.7 1.6 2.48 7.75 7.92 8.32

a

Cells were grown at 28³C for 18 h in MM with the required additions. At this time, di¡erent amounts of sterile solution of CuSO4 were added, and 3 h later, the cells were recovered, washed, and ODC activity was measured. b Nanomoles of CO2 released per mg protein in 1 min.

As previously observed with U. maydis and C. albicans [16,17], requirement of polyamines for dimorphism was more stringent than its requirement for growth. When incubated in liquid synthetic medium under conditions that induce mycelial growth, requirements of the odc mutant FJOD were satis¢ed by polyamine concentrations as low as 1 WM. At this concentration, growth was reduced by only 25% compared to that obtained in the presence of 1 mM putrescine. On the other hand, dimorphic transition was highly dependent on putrescine concentration. Mycelial cells were absent in the medium supplemented with 1 WM putrescine, and its population increased as polyamine supply was raised (Figs. 5 and 6). Similar results were obtained on solid media. Colonies of the odc strain FJOD grown on agar plates of minimal medium containing 1 WM putrescine appeared smooth, and made of yeastlike cells, whereas those grown in the presence of 5 mM polyamine looked as rough as the parental strain, and were made of a mixture of mycelial and yeast-like cells (Fig. 6). Regulation of intracellular polyamine levels was brought about by use of an inducible promoter. For these experiments we used the inducible promoter of the Y. lipolytica MTPI-II genes coding for metallothioneins in the 5P-3P sense [36]. A 1-kb fragment was excised from a plasmid containing the full promoter (pSGS70) with EcoRI and BamHI, and subcloned into pINA444 to produce plasmid pINA444Cu. The YlODC containing its own ATG and terminator was PCR ampli¢ed and cloned into the BamHI site of this plasmid to produce pFJ4. Construction was con¢rmed by restriction analysis and Table 3 Growth and dimorphic transition of strains P01a and FJCO in the presence of variable amounts of CuSO4 a CuSO4 (mM) 0 0.2 0.4 0.8 1.2 2.0

Strain P01a (parental)

Strain FJCO

Growth

Mycelium (%)

Growth

3.9 3.7 3.6 3.4 3.1 2.9

69 66 70 68 66 67

1.6 1.3 1.8 2.1 2.1 2.2

Mycelium (%) 0 0 0 32 38 52

a Cells were grown at 28³C for 18 h in MM with the required additions. At this time, di¡erent amounts of a sterile solution of CuSO4 were added, and incubation was continued for 15 h. Growth as OD600 and morphology were measured.

FEMSYR 1425 26-11-01

202

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

Fig. 6. Morphology of P01a and vodc strains grown in liquid and solid media. Both strains were grown in minimal medium containing di¡erent polyamine concentrations at 28³C under conditions that promote hyphal growth. 1: Morphology of inoculum in liquid medium. A: P01a strain; B: FJOD strain in medium with 1 WM putrescine; C: FJOD strain in medium with 5 mM putrescine. 2: Cell morphology scored after 12 h of incubation in liquid medium. A,B,C: As in 1. Magni¢cation bar, 10 Wm. 3: Colonial morphology in solid medium after 3 days of incubation. A,B,C: As in 1. Colony diameter: A,C, 3 mm ; B, 2 mm.

sequencing. Transformation of null odc mutants with this plasmid led to recovery of putrescine prototrophy. Cells of one of these transformants (Y. lipolytica FJCO) grown in a medium containing increasing amounts of copper sulfate displayed an ODC activity proportional to the salt content up to 2 mM (Table 2). Growth of the strain and dimorphic transition were also dependent on the concentration of copper added to the medium, except that at the lowest copper concentration used, a decrease in growth was observed (Table 3). As deduced from data for the parental strain presented in the same table, this decrease was due to the toxicity of the metallic ion. Such a toxic e¡ect was compensated at higher copper concentrations by an increased level in YlODC transcription. Copper had no inhibitory e¡ect on mycelial growth of the fungus (Table 3). 4. Discussion It is interesting to note that the exact mode of action of polyamines, especially in di¡erentiation processes, remains largely unknown. It has been observed that during fungal

di¡erentiation, activity of the key enzyme for polyamine synthesis, ODC, as well as polyamine levels are increased severalfold [11^13]. Studies of the e¡ect of ODC inhibitors on several fungi have revealed that they blocked di¡erentiation processes when present at levels that had minimal e¡ect on growth [13^15,37,38]. Similar results have been reported for Y. lipolytica [14]. Criticism that may be raised to the use of inhibitors was eliminated by the observation that dimorphism in vodc mutants of U. maydis and C. albicans was much more sensitive than cellular growth to the addition of low polyamine concentrations [16,17]. In the present study we have analyzed this phenomenon in Y. lipolytica in order to further analyze the problem. Isolation and characterization of the YlODC gene revealed high homology of the coded protein with ODCs from other fungi, and some relevant features. The gene lacks a TATAA box, a feature that coincides with the U. maydis gene [16]. Absence of this box is not uncommon in fungi [39,40], nevertheless two CAAT boxes at 3508 and 3340 bp from the ATG were identi¢ed. The initial point of transcription was located at 3218 bp within a CA dinucleotide, a feature agreeing with other genes of

FEMSYR 1425 26-11-01

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

Y. lipolytica [41,42]. In S. cerevisiae the corresponding initial transcription site for ODC was located at 3211 bp [43], indicating a similar size of the upstream region with the Y. lipolytica gene. The theoretical length of the mRNA obtained from the addition of 218 bp to the tsp, 1350 bp of the ORF, and 75 bp of the end region, plus the poly(A) (50 bp), was in agreement with the observed size of the transcript in the gel. Speci¢c motifs of the enzyme identi¢ed in other organisms [31,32], such as the active and the cofactor- and substrate-binding sites, were present in the Y. lipolytica ODC. Functionality of the gene was demonstrated by its capacity to revert the phenotype of both Y. lipolytica and E. coli polyamine auxotrophs. It is interesting to note that genes coding for ODC from S. cerevisiae [44], C. immitis [45], and Y. lipolytica all have the capacity to complement the bacterial odc mutant, although the GC content of their coding genes is noticeably dissimilar : 40, 52 and 56%, respectively. Putative PEST regions characteristic of proteins with a high turnover rate [33] were identi¢ed in the Y. lipolytica enzyme. Interestingly, the second putative PEST box is located at the active site of the enzyme. Both PEST motifs had a low score in agreement with other fungal ODCs [16,46,47]. Nevertheless, since an antienzyme mechanism for regulation of ODC levels in Schizosaccharomyces pombe has been identi¢ed [48], it is possible that they may be operative, as it occurs for animal ODCs. Disruption of 90% of the YlODC ORF led to polyamine requirement for growth, as it occurs in other fungal odc mutants: S. cerevisiae [43], N. crassa [49], U. maydis [16] and C. albicans [17]. These data agree with the idea that fungi utilize this single route for polyamine biosynthesis. The Y. lipolytica mutant released only minimal levels of CO2 when ODC activity was measured. Since whole cells were used for these assays, it may be argued that these extremely low levels of carbon dioxide came from an activity other than ODC. The mutant helped to demonstrate that the requirement of di¡erent polyamine concentrations for growth and differentiation in fungi is a general phenomenon. Addition of di¡erent levels of putrescine revealed on one side that the fungus can grow with minimal amounts of the polyamine (1 WM) well beyond the minimal concentrations found in other systems [16,17]. Nevertheless even lower amounts of spermidine (10310 M) were found to allow slow growth of an S. cerevisiae auxotroph [50]. More interesting was the observation that dimorphic transition was obtained only with much higher concentrations of putrescine, as previously reported for U. maydis and C. albicans [16,17]. An e¡ect of permeability problems of extracellular polyamines as a possible explanation for the phenomenon was eliminated when the null mutant was transformed with a plasmid carrying the wild gene under the control of the metallothionein promoter of the fungus. This system also helped to demonstrate a ¢ner control of the phenomenon. Growth, enzymatic activity and dimorphism were all de-

203

pendent on the concentration of copper added to the system. Growth inhibition at the lowest copper concentration is probably due to its toxic e¡ect. The basal levels of growth and ODC activity (but not dimorphism) obtained in the absence of copper are explained by the fact that the promoter has a basal constitutive activity [36]. As tentative hypotheses for the requirement of high polyamine concentrations for mycelial growth, we may suggest that some mRNAs involved in di¡erentiation may be less stable than the rest, requiring higher concentrations of polyamines to be stabilized. Alternatively, requirement of polyamines for adequate binding of transcription factors involved in expression of those genes may be an attractive possibility. And ¢nally, the existence of a polyamine-induced frame shifting during translation, operative in di¡erentiation, could be invoked. The observation of the existence of this mechanism in ODC regulation in S. pombe [48] may be an argument to sustain this last hypothesis. Acknowledgements This work was partially supported by CONACYT, Mexico, DGICYT and the Junta de Castilla y Leo¨n, Spain, and the European Union. Stay of J.F.J.-B. in Salamanca, Spain, was supported by fellowships from CONACYT, Mexico, and ALFA Program Micologia No. 5.0118.9 from the European Union. Thanks are given to Prof. Garry T. Cole, Medical College of Ohio, for his gracious hospitability to J.F.J.-B. during the realization of several experiments. J.F.J.-B. is a predoctoral fellow from CONACYT at Centro de Investigacio¨n y de Estudios Avanzados del IPN (Unidad Irapuato). J.R.-H. is National Investigator, Mexico.

References [1] Rodriguez, C. and Dominguez, A. (1984) The growth and characteristics of Saccharomycopsis lipolytica : morphology and induction of mycelium formation. Can. J. Microbiol. 30, 605^612. [2] Wickerham, L.J., Kurtzman, C.P. and Herman, A.I. (1970) Sexual reproduction in Candida lipolytica. Science 167, 1141^1145. [3] Gaillardin, C., Charoy, V. and Heslot, H. (1973) A study of copulation, sporulation and meiotic segregation in Candida lipolytica. Arch. Microbiol. 92, 69^83. [4] Ogrydziak, D., Bassel, J., Contopoulou, R. and Mortimer, R.K. (1978) Development of genetic techniques and the genetic map of the yeast Saccharomycopsis lipolytica. Mol. Gen. Genet. 163, 229^ 239. [5] Barth, G. and Gaillardin, C. (1996) Yarrowia lipolytica. In: Nonconventional Yeasts in Biotechnology (Wolf, K., Ed.), pp. 313^388. Springer, Berlin. [6] Davidow, L.S., Apostolakos, D., O'Donnell, M.M., Proctor, A.R., Ogrydziak, D.M., Wing, R.A., Stasko, I. and DeZeeuw, J.R. (1985) Integrative transformation of the yeast Yarrowia lipolytica. Curr. Genet. 10, 39^48.

FEMSYR 1425 26-11-01

204

J.F. Jime¨nez-Bremont et al. / FEMS Yeast Research 1 (2001) 195^204

[7] Heby, O. (1981) Role of polyamines in the control of cell proliferation and di¡erentiation. Di¡erentiation 14, 1^20. [8] Tabor, C.W. and Tabor, H. (1984) Polyamines. Annu. Rev. Biochem. 53, 749^790. [9] Tabor, C.W. and Tabor, H. (1985) Polyamines in microorganisms. Microbiol. Rev. 49, 81^99. [10] McCann, P.P., Pegg, A.E. and Sjoerdsma, A. (Eds) (1987) Inhibition of Polyamine Metabolism: Biological Signi¢cance and Basis for New Therapies, 371 pp. Academic Press, Orlando, FL. [11] Inderlied, C.B., Cihlar, R.L. and Sypherd, P.S. (1980) Regulation of ornithine decarboxylase during morphogenesis of Mucor racemosus. J. Bacteriol. 14, 699^706. [12] Calvo-Mendez, C., Martinez-Pacheco, M. and Ruiz-Herrera, J. (1987) Regulation of ornithine decarboxylase in Mucor bacilliformis and Mucor rouxii. Exp. Mycol. 11, 270^277. [13] Ruiz-Herrera, J. (1994) Polyamines, DNA methylation, and fungal di¡erentiation. Crit. Rev. Microbiol. 20, 143^150. [14] Guevara-Olvera, L., Calvo-Mendez, C. and Ruiz-Herrera, J. (1993) The role of polyamine metabolism in dimorphism of Yarrowia lipolytica. J. Gen. Microbiol. 139, 485^493. [15] Martinez-Pacheco, M., Rodriguez, G., Reyna, G., Calvo-Mendez, C. and Ruiz-Herrera, J. (1989) Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diaminobutanone. Arch. Microbiol. 151, 10^14. [16] Guevara-Olvera, L., Xoconostle-Ca¨zares, B. and Ruiz-Herrera, J. (1997) Cloning and disruption of the ornithine decarboxylase gene of Ustilago maydis. Evidence for a role of polyamines in its dimorphic transition. Microbiology 143, 2237^2245. [17] Herrero, A.B., Lo¨pez, M.C., Garc|¨a, S., Schmidt, A., Spaltmann, F., Ruiz-Herrera, J. and Dominguez, A. (1999) Control of ¢lament formation in Candida albicans by polyamine levels. Infect. Immun. 67, 4870^4878. [18] Xuan, J.W., Fournier, P. and Gaillardin, C. (1988) Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration. Curr. Genet. 14, 15^21. [19] Xuan, J.W., Fournier, P., Declerck, N., Chasles, M. and Gaillardin, C. (1990) Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica. Mol. Cell. Biol. 10, 4795^4805. [20] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557^580. [21] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [22] Hafner, E.W., Tabor, C.W. and Tabor, H. (1979) Mutants of Escherichia coli that do not contain 1,4 diaminobutane (putrescine) or spermidine. J. Biol. Chem. 254, 12419^12426. [23] Raeder, U. and Broda, P. (1985) Rapid preparation of DNA from ¢lamentous fungi. Lett. Appl. Microbiol. 1, 17^20. [24] Percival-Smith, A. and Segall, J. (1984) Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 142^150. [25] Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444^2448. [26] Higgins, D.G. and Sharp, P.M. (1988) Clustal : a package for performing multiple sequence alignments on a microcomputer. Gene 73, 237^244. [27] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1989) Current Protocols in Molecular Biology. Wiley, New York. [28] Torres-Guzman, J.C., Xoconostle-Cazares, B., Guevara-Olvera, L., Ortiz, L., San-Blas, G., Dominguez, A. and Ruiz-Herrera, J. (1996) Comparison of fungal ornithine decarboxylases. Curr. Microbiol. 33, 390^392. [29] Boeke, J.D., Trueheart, J., Natsoulis, G. and Fink, G.R. (1987) 5-Fluororotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154, 164^174.

[30] Perez-Campo, F.M., Nicaud, J.M., Gaillardin, C. and Dominguez, A. (1996) Cloning and sequencing of the LYS1 gene encoding homocitrate synthase in the yeast Yarrowia lipolytica. Yeast 12, 1459^1469. [31] Poulin, R., Lu, L., Ackermann, B., Bey, P. and Pegg, A.E. (1992) Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by di£uoromethylornithine. J. Biol. Chem. 267, 150^ 158. [32] Moore, R.C. and Boyle, S.M. (1990) Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli. J. Bacteriol. 172, 4631^4640. [33] Rogers, S.R., Wells, R. and Rechsteiner, M. (1986) Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 23, 364^368. [34] Rechsteiner, M. and Rogers, S.W. (1996) PEST sequences and regulation by proteolysis. Trends Biochem. Sci. 21, 267^271. [35] Fournier, P., Guyaneux, L., Chasles, M. and Gaillardin, C. (1991) Scarcity of ars sequences isolated in a morphogenesis mutant of the yeast Yarrowia lipolytica. Yeast 7, 25^36. [36] Prado, M. (1994) Ana¨lisis de la Sensibilidad al Cobre de Yarrowia lipolytica. Clonacio¨n y Caracterizacio¨n Estructural de los Genes CFR1, MTPI y MTPII. Doctoral Thesis, University of Salamanca. [37] Stevens, L., Mckinnon, I.M. and Winter, M.D. (1977) The e¡ects of 1,4 diaminobutanone on polyamine biosynthesis in Aspergillus nidulans. FEBS Lett. 75, 180^182. [38] Ruiz-Herrera, J. and Calvo-Mendez, C. (1987) E¡ect of ornithine decarboxylase inhibitors on germination of sporangiospores of Mucorales. Exp. Mycol. 11, 287^296. [39] Oberholzer, U. and Collart, M.A. (1999) In vitro transcription of a TATA-less promoter, negative regulation by the Not 1 protein. J. Biol. Chem. 380, 1365^1370. [40] Sakurai, H., Ohishi, T. and Fukasawa, T. (1996) Core promoter elements are essential as selective determinants for function of the yeast transcription factor GAL11. FEBS Lett. 398, 113^119. [41] Gaillardin, C. and Heslot, H. (1988) Genetic engineering in Yarrowia lipolytica. J. Basic Microbiol. 28, 161^174. [42] Treton, B.Y., Le Dall, M.T. and Gaillardin, C. (1992) Complementation of Saccharomyces cerevisiae acid phosphatase mutation by a genomic sequence from the yeast Yarrowia lipolytica. Curr. Genet. 22, 345^355. [43] Fonzi, W.A. and Sypherd, P.S. (1987) The gene and the primary structure of ornithine decarboxylase from Saccharomyces cerevisiae. J. Biol. Chem. 262, 10127^10133. [44] Fonzi, W.A. and Sypherd, P.S. (1985) Expression of the gene for ornithine decarboxylase of Saccharomyces cerevisiae in Escherichia coli. Mol. Cell. Biol. 5, 161^166. [45] Guevara-Olvera, L., Hung, C.Y., Yu, J.J. and Cole, G.T. (2000) Sequence, expression and functional analysis of the Coccidioides immitis ODC (ornithine decarboxylase) gene. Gene 242, 437^448. [46] Williams, L.J., Barnett, G.R., Ristow, J.L., Pitkin, J., Perriere, M. and Davis, R. (1992) Ornithine decarboxylase gene of Neurospora crassa: isolation, sequence, and polyamine-mediated regulation of its RNAm. Mol. Cell. Biol. 12, 347^359. [47] Lo¨pez, M.C., Garc|¨a, S., Ruiz-Herrera, J. and Dominguez, A. (1997) The ornithine decarboxylase gene from Candida albicans. Sequence analysis and expression during dimorphism. Curr. Genet. 32, 108^ 114. [48] Ivanov, I.P., Matsufuji, S., Murakami, Y., Gesteland, R.F. and Atkins, J.F. (2000) Conservation of polyamine regulation by translational frameshifting from yeast to mammals. EMBO J. 19, 1907^ 1917. [49] Davis, R.H., Hynes, L.V. and Eversole-Cire, P. (1987) Nonsense mutation of the ornithine decarboxylase structural gene of Neurospora crassa. Mol. Cell. Biol. 7, 1122^1128. [50] Cohn, M.S., Tabor, G.W. and Tabor, H. (1980) Regulatory mutations a¡ecting ornithine decarboxylase activity in Saccharomyces cerevisiae. J. Bacteriol. 140, 649^654.

FEMSYR 1425 26-11-01