FULL LENGTH RESEARCH PAPER

Cloning and sequence analysis of ornithine decarboxylase genefragments from the Ascomycota

JUAN FRANCISCO JIMENEZ-BREMONT1, MARGARITA RODRIGUEZ-KESSLER1,†,

RAUL RODRIGUEZ-GUERRA2,‡, CARLOS CORTES-PENAGOS3,{, JUAN CARLOS

TORRES-GUZMAN4,§, & JUNE SIMPSON WILLIAMSON5,k

1Division de Biologıa Molecular, Instituto Potosino de Investigacion Cientıfica y Tecnologica, Camino a la Presa de San Jose

2055, Apartado Postal 3-74 Tangamanga, 78210 San Luıs Potosı, San Luıs Potosı, Mexico, 2Instituto Nacional de

Investigaciones Forestales, Agrıcolas y Pecuarias, Campo Experimental Bajıo, Km 6.5 Carr. Celaya-San Miguel de Allende,

Apartado Postal 112, Celaya, Guanajuato, Mexico, 3Escuela de Quımico-Farmacobiologıa, Universidad Michoacana de San

Nicolas de Hidalgo, Tzintzuntzan 173, Apartado Postal, 58420 Morelia, Michoacan, Mexico, 4Instituto de Investigacion en

Biologıa Experimental Facultad de Quımica, Universidad de Guanajuato, Noria Alta s/n, Apartado Postal 187, 36000

Guanajuato, Guanajuato, Mexico, and 5Departamento de Ingenierıa Genetica, CINVESTAV, Unidad Irapuato, Apartado

Postal 629, 36500 Irapuato, Guanajuato, Mexico

(Received 23 January 2006)

AbstractOrnithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the initial step in the biosynthesis of polyamines, the conversion ofornithine to putrescine. Based on the most conserved regions of fungal ODCs, we designed and synthesized oligonucleotidesto amplify homologous fragments of three important plant pathogenic Pyrenomycete fungi (Ascomycota), Magnaporthe grisea,Colletotrichum lindemuthianum and Fusarium solani, and one insect pathogenic fungus Metarhizium anisopliae. Cloning andsequencing of the amplified fragments revealed homologies of between 37 to 88% with other fungal ODCs. The predictedpeptide sequences were compared by Clustal analysis and conserved sequences corresponding to the substrate and cofactorbinding sites were identified. Comparative analyses of the ODC fragments isolated in this study, revealed high homologybetween them (68.3–81.1%) and also with other Pyrenomycetes such as Neurospora crassa (order Sordariales; 68.6–72.9%)and Fusarium graminearum (order Hypocreales; 70.8–88.1%). Data obtained in this work revealed that these fungi constitutea compact group separated from other eukaryotic ODCs.

Keywords: Colletotrichum lindemuthianum, Fusarium solani, Magnaporthe grisea, Metarhizium anisopliae, ornithinedecarboxylase

Database accession number: AY602214, AY325884, AY327897, DQ291140

Introduction

Ornithine decarboxylase (ODC; EC. 4.1.1.17) cata-

lyzes the conversion of ornithine to putrescine and is

the first and rate-limiting step in polyamine bio-

synthesis in most organisms. In plants and some

bacteria a second mechanism for the synthesis of

polyamines exists. This pathway involves the action of

arginine decarboxylase (ADC) to produce agmatine

(Tabor and Tabor 1984). Polyamines are polycations

found to be essential for all organisms (Tabor and

ISSN 1042-5179 print/ISSN 1029-2365 online q 2006 Informa UK Ltd.

DOI: 10.1080/10425170600807009

Correspondence: J. F. Jimenez Bremont, Division de Biologıa Molecular, Instituto Potosino de Investigacion Cientıfica y Tecnologica, 78210San Luis Potosı, Mexico. Fax: 52 444 8342010. E-mail: jbremont@ipicyt.edu.mx

†Tel: 52 444 8342000. Fax: 52 444 8342010. E-mail: mrodriguez@ipicyt.edu.mx.‡Tel: 52 461 6115323. Fax: 52 461 6115323. Ext. 123. E-mail: raulrdzg@yahoo.com.mx.{Tel: 52 443 3142809. Fax: 52 443 3142809. E-mail: neocccs@yahoo.com.§Tel: 52 473 7320006. Fax: 52 473 7320006. E-mail: torguz@quijote.ugto.mx.kTel: 52 462 6239667. Fax: 52 462 6239600. E-mail: jsimpson@ira.cinvestav.mx.

DNA Sequence, June 2006; 17(3): 231–236

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Tabor 1984), playing key roles in a variety of processes

such as nucleic acid packaging, DNA replication,

transcription, translation, membrane stabilization, the

functioning of certain ion channels and resistance to

oxidative stress (Cohen 1998; McCann et al. 1987;

Pegg and McCann 1982; Tabor and Tabor 1985;

Williams 1997). They may also have additional roles

in protection of DNA from enzymatic degradation,

X-ray irradiation, mechanical shearing and oxidative

damage (McCann et al. 1987).

It has been observed that during fungal differen-

tiation, the activity of the key enzyme for polyamine

synthesis, ornithine decarboxylase (ODC), as well

as polyamine levels are increased several-fold (Calvo-

Mendez et al. 1987; Ruiz-Herrera 1994; Jimenez-

Bremont et al. 2001). Most of the economically

important plant pathogenic fungi (Fusarium

oxysporum, Rhizoctonia solani, Verticillium dahliae and

Phytophthora infestans) are prevented from growing by

low concentrations of difluoromethylornithine

(DFMO), a specific and highly potent ODC inhibitor

(Walters 1995; West and Walters 1989).

We have isolated and cloned conserved fragments

of genes encoding ODCs from three important

phytopathogenic Pyrenomycetes (Ascomycota),

Magnaporthe grisea (order Diaporthales), Colleto-

trichum lindemuthianum (order Phyllachorales),

Fusarium solani (order Hypocreales) and one

insect pathogenic fungus, Metarhizium anisopliae

(teleomorph unknown but anamorph related to

Ascomycota; Alexopoulus et al. 1996). In this study

we describe the isolation, sequence analysis and

homologies of these fragments with the aim of further

expanding the molecular information concerning

Ascomycota ODCs.

Materials and methods

Fungal strains

Stains of Magnaporthe grisea “4091”, Colletotrichum

lindemuthianum “1088” (Rodrıguez-Guerra et al.

2005), Fusarium solani “San Luis” and Metarhizium

anisopliae “Caro19” were used in this study. Strains

were maintained at 2708C in 50% (v/v) glycerol. They

were transferred to YEPD liquid media (1% yeast

extract, 1% peptone and 1% glucose), incubated at

288C for 2–3 d in an orbital shaker (150 rpm) and used

to obtain genomic DNA as described by Raeder and

Broda (1985) for filamentous fungi.

PCR amplification and cloning of genomic fragments

from ornithine decarboxylase genes

The oligonucleotides for PCR were designed on the

basis of conserved regions of different fungal ODCs:

Neurospora crassa (GenBank P27121), Coccidioides

immitis (GenBank AAF35284), Paracoccidioides

brasiliensis (GenBank AAF34583), Phaeosphaeria

nodorum (GenBank CAB56523) and Tapesia yallundae

(GenBank AAK38838). Three oligonucleotides were

synthesized: two sense oligonucleotides, 5 Ma

50-gccagcacctccgctggaag-30 coding for residues

95–101 from N. crassa (RQHLRWK) and 5-IMa

50-ggcttcgactgtgcctcc-30 coding for residues 131–136

(GFDCAS); and one anti-sense oligonucleotide, 3 Ma

50-atctggggtcctacttgcgacggcatcgaccg-30 coding for

residues 417–426 (IWGPTCDGID). Reaction mix-

tures (50ml) for each PCR amplification contained:

200 ng of genomic DNA, 0.2 pmoles of each

oligonucleotide, 10 mM dNTPs, 10 mM Tris–HCl

pH 8.3, 3.0 mM MgCl2, 2.5 U Taq DNA polymerase

(Invitrogen, San Diego, CA, USA). The “touch

down” protocol employed was as follows: 5 min at

948C, and 5 cycles each with annealing temperatures

of 35, 40, 45, 50 and 558C for 1 and 2 min at 728C.

Nested amplifications using the 5-IMa oligonucleo-

tide were performed under the following conditions:

1 min at 948C, 1 min at 45–558C, and 2 min at 728C.

After 30 cycles, extensions were continued at 728C for

10 min. Samples were analysed by agarose gel

electrophoresis. Fragments of the expected size were

cloned in TOPO-pCR II (TA Cloning Kit

Invitrogen, San Diego, CA, USA) and sequenced on

both strands. Escherichia coli DH5a and Top10 were

used for transformation, plasmid amplification and

preparation.

BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)

searches of all the obtained sequences revealed high

homology to published ornithine decarboxylase

sequences. Analysis of protein domains and functional

sites were carried out using the InterProScan (http://

www.ebi.ac.uk/InterProScan/) program. Comparisons

and protein sequence alignments were carried out

using the Clustal (Higgins and Sharp 1988) and the

ClustalW (http://www.ebi.ac.uk/clustalw/) programs.

For the construction of dendrograms we used

MEGA version 3.1 (Kumar et al. 2004). PEST motifs

were identified using the PESTfind algorithm

(https://emb1.bcc.univie.ac.at/). The nucleotide

sequences reported in this paper were deposited in

the GenBank nucleotide sequence database under

the Accession numbers AY602214, AY325884,

AY327897 and DQ291140, respectively.

Results and discussion

Fragments encoding partial ornithine decarboxylase

genes from Fusarium solani (GenBank DQ291140),

Metarhizium anisopliae (GenBank AY325884),

Magnaporthe grisea (GenBank AY327897) and

Colletotrichum lindemuthianum (GenBank AY602214)

were isolated using oligonucleotides representing

conserved amino acid motifs in the different ODCs.

In the case of F. solani, one round of PCR

with external oligonucleotides (5 and 3 Ma) yielded

J. F. Jimenez-Bremont et al.232

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a single amplification product of 986 bp. In the case of

C. lindemuthianum, M. anisopliae and M. grisea, two

different amplification products were obtained using

oligonucleotides 5 and 3 Ma. However, when the

amplified products were subjected to a further round

of PCR (nested PCR) using the oligonucleotide

5-IMa, a single band was obtained in each case, with a

length of 834, 830 and 858 bp, respectively. The

corresponding amino acid sequences of the amplified

fragments were predicted and compared with selected

fungal ODC sequences reported in GenBank:

Neurospora crassa (M68970), Paracoccidioides brasilien-

sis (AF212867), Phaeosphaeria nodorum (AJ249387),

Tapesia yallundae (AF333773), Yarrowia lipolytica

(AJ237707; Jimenez-Bremont et al. 2001), Ustilago

maydis (X88796), Candida albicans (U85005) and

F. graminearum (EAA75548), as shown in Figure 1.

The central region of the protein in comparison with

the amino- and carboxy-termini presents the highest

homology (Figure 1). Characteristic domains present

in ODC proteins were observed in all the analysed

fragments:

1. A consensus site for pyridoxal-phosphate cofactor

binding was localized between residues 15–35 in

F. solani (YAvKCHpderLLqlLaalG) (Figure 1,

doubly underlined). In the mouse ODC, a

conserved lysine residue located in the sequence

PFYAVKC is responsible for direct attachment of

the pyridoxal-phosphate group (Poulin et al. 1992).

This lysine residue is conserved in all the ODC

sequences compared (Figure 1), but is missing in

the shortest DNA fragments amplified here.

2. The conserved sequence of family 2 decarboxy-

lases, containing a stretch of three consecutive

glycine residues (AaaygysLktLDVGGGFC) and

proposed to be part of the substrate binding region

(Moore and Boyle 1990), was located between

amino acid residues 172–190 of F. solani (Figure 1,

dotted line).

3. A cysteine residue, which is considered to be within

the catalytic site located at the consensus sequence

(IWGPTCDG(I)D) (Poulin et al. 1992), is located

between amino acids 305–314 in F. solani. Three

changes were detected in this motif, valine instead

of isoleucine in ODCs from U. maydis and Y.

lipolytica, serine instead of glycine in U. maydis, and

leucine instead of isoleucine in C. albicans (Figure 1,

shadowed).

4. Antizyme is a spermidine-induced protein that

binds and stimulates ornithine decarboxylase

degradation (Li and Coffino 1992). Originally

described in higher eukaryotes, antizyme has also

been described to be active in fungi (Chattopad-

hyay et al. 2001). A comparison of the amino acid

sequences of mouse and fungal ODCs in the

antizyme-binding region reveals that 10 out of

24 residues are identical to those in F. solani,

and correspond to amino acids 68–91 (Figure 1,

wavy line).

5. Two putative PEST regions (sequences rich in

proline (P), glutamic acid (E), serine (S) and

threonine (T) residues) characteristic of proteins

with a high rate of turnover (Rechsteiner and

Rogers 1996) with PEST scores of 20.05 and

þ0.93, were identified at residues 68–95 and 405–

427 of N. crassa (Williams et al. 1992), respectively,

using the algorithm described by Rechsteiner and

Rogers (1996) (Figure 1, underlined). These PEST

sequences were also identified at residues 56–77

(Score 23.69) and 360–391 (Score 21.23) of

F. graminearum and were absent in the F. solani

sequence. A remarkable feature is that the second

putative PEST box harbors the active site of the

enzyme. Both PEST motifs had a low score in

agreement with results obtained for the Yarrowia

lipolytica ODC (Jimenez-Bremont et al. 2001).

The partial sequences of the ODC genes isolated

from C. lindemuthianum, F. solani, M. grisea and

M. anisopliae present a high degree of homology

between them and with other ODCs from fungi

belonging to the Ascomycota at both the nucleotide

and amino acid sequence level as shown in Table I.

As expected, ODCs from F. graminearum and F. solani

share the greatest homology (84.1 or 88.1% nucleo-

tide and amino acid, respectively) since they belong to

the same genus. Furthermore, the M. anisopliae ODC

also shows a high homology (77 or 81%) with both

Fusarium species. Additionally, M. grisea and C.

lindemuthianum present homologies of approximately

70% between them and with the other genus analysed.

Arginine decarboxylase (ADC), a key enzyme

involved in putrescine biosynthesis in plants and

bacteria, has been used as molecular marker for

phylogenetic studies. This gene is encoded by a single

or low-copy nuclear gene, and possesses highly

conserved regions that provide several target sites for

PCR priming for amplification and sequence analysis,

making the alignment of sequences across a wide

range of taxonomic levels possible. Additionally, the

variable regions are potentially informative for the taxa

surveyed (Galloway et al. 1998). In evolutionary

terms, ODC is related to the 50-pyridoxal-phosphate-

dependent enzymes and possesses many of the

characteristics described for ADC. The degree of

homology between the isolated ODC fragments

(Table I) correlates with the taxonomical classification

of the fungi from which they were obtained, opening

the possibility to use ODC sequences as molecular

markers for phylogenic analysis. A phylogenetic tree of

ODCs belonging to 26 organisms representing fungi,

plants, mammals and bacteria was constructed using

MEGA 3.1 version (Kumar et al. 2004) (Figure 2).

As shown in the Figure, the isolated ODC frag-

ments constitute a compact group separated from

Ornithine decarboxylase gene fragments from the Ascomycota 233

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other fungal ODCs and the other eukaryotic and

prokaryotic organisms.

An intron of 47 bp in the coding sequence, with the

characteristic intron-exon boundaries of the Asco-

mycota (Kupfer et al. 2004), was identified in the F.

solani ODC partial genomic fragment isolated in this

study. Other Ascomycota ODC open reading frames

(ORFs) have also been shown to be interrupted by

Figure 1. Multiple amino acid alignment as derived by maximal homology of ODCs from genomic DNA fragments isolated in this study and

with the corresponding sequences of genomic DNA encoding other fungal ODCs: Candida albicans (Ca), Colletotrichum lindemuthianum (Cl),

Fusarium graminearum (Fg), Fusarium solani (Fs), Magnaporthe grisea (Mg), Metarhizium anisopliae (Ma), Neurospora crassa (Nc),

Paracoccidioides brasiliensis (Pb), Phaeosphaeria nodorum (Pn), Tapesia yallundae (Ty), Ustilago maydis (Um), Yarrowia lipolytica (Yl). An asterisk

(*) indicates identical amino acids conserved in the twelve polypeptides, a dot (.) indicates conserved substitutions and a colon (:) indicates

semi-conserved substitutions. The putative PEST regions are underlined. The doubly-underlined sequence corresponds to the cofactor

(pyridoxal-phosphate) binding site; and the location of the intron is also indicated in this region. The signature sequence of the conserved

decarboxylase family 2 is indicated by a dotted line. The shadowed motif corresponds to the conserved ODC catalytic sequence. The sequence

indicated by the wavy line corresponds to the region of the antizyme-binding site.

J. F. Jimenez-Bremont et al.234

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a small intron as described for Coccidioides immitis,

62 bp (Guevara-Olvera et al. 2000); F. graminearum,

60 bp (GenBank EAA75548); N. crassa, 70 bp

(Williams et al. 1992); P. brasiliensis, 72 bp (Nino-

Vega et al. 2004); P. nodorum, 51 bp (Bailey et al.

2000) and T. yallundae, 70 bp (Mueller et al. 2001).

Interestingly, all of these introns split the ORF

between the tyrosine (Y) and alanine (A) residues of

the consensus site for pyridoxal-phosphate cofactor

binding (PFY-intron-AVKC) (Figure 1). These

amino acids are located at positions 17 and 18 of

the F. solani ODC protein sequence, respectively. The

function of the intron is unknown, but it may be

implicated in regulation. It is important to note that

the presence of intron sequences in the coding region

of Odcs is not a rule. Until now no introns have been

reported in the ODCs belonging to yeasts and plants

(Jimenez-Bremont et al. 2004; Fonzi and Sypherd

1987); while in mammals the ODC coding region is

split by multiple introns (Katz and Kahara 1988).

Whether the shorter fungal ODCs isolated in this

study also contain intron sequences remains to be

determined.

Isolation, characterization and physiological rel-

evance of ornithine decarboxylase has been extensively

studied in other fungal models. Data obtained in this

work opens the possibility to further understand ODC

regulation and also polyamine metabolism in filamen-

tous Ascomycetes.

Acknowledgements

This work was partly supported by the Fondo

Mixto de Fomento a la Investigacion Cientificia y

Technologica CONACYT – Gobierno del Estado de

Guanajuato (Project FOMIX GTO-04-CO2-97).

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– 72.9 Mg

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