Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain1
Author for correspondence: Tahía Benítez. Tel: +34 95 4557109. Fax: +34 95 4557104. e-mail: tahia{at}us.es
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ABSTRACT |
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Keywords: fungal proteases, nitrogen carbon and pH regulation, protein overproduction, proteolysis
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AJ276388.
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INTRODUCTION |
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The amount of proteases can be diminished by modifying the cultivation procedure (Delgado-Jarana et al., 2000 ) or by isolating protease-deficient strains (Berka et al., 1990
; Moralejo et al., 2000
; Archer, 2000
; van den Hombergh et al., 1997a
, b
). Deletion of an aspartic proteinase gene decreased the degradation of the secreted calf chymosin by Aspergillus niger (Dunn-Coleman et al., 1991
) and of thaumatin by Aspergillus awamori (Moralejo et al., 2000
), while deletion of a pepstatin A-sensitive aspartic protease gene of Trichoderma reesei resulted in 94% reduction in the total acidic protease activity and increased cellobiase production (Mäntylä et al., 1998
).
Occurrence of extracellular proteases in strains of T. reesei (Mäntylä et al., 1998 ) and of Trichoderma harzianum (Geremía et al., 1993
; Delgado-Jarana et al., 2000
) has been reported and several acidic, neutral and basic extracellular proteases have been detected (Delgado-Jarana et al., 2000
). Strains of T. harzianum are commonly used in biocontrol against fungal plant pathogens (Harman & Björkman, 1998
). PRB1, a basic proteinase involved in mycoparasitism has been isolated and the gene cloned (Geremía et al., 1993
). Overexpression of the prb1 gene led to T. harzianum strains with improved antagonistic abilities against Rhizoctonia solani (Flores et al., 1997
). There is no information available about the characteristics of Trichoderma neutral proteases or their genes. Some acidic proteases from T. reesei have been totally or partially purified (Pitts, 1992
; Haab et al., 1990
; Dunne, 1982
). One of the aspartate proteases from T. reesei was stable at pH 3·5 and was insensitive to pepstatin A (Haab et al., 1990
), which inhibits most acidic proteases. A second acidic protease, present when T. harzianum was grown on cellulose, possessed similar biochemical characteristics but was pepstatin-sensitive (Dunne, 1982
). However, sequences of these acidic proteases have not been reported so far. In addition, there are almost no available data on acidic proteases from T. harzianum. Two acidic proteases were detected by IEF and zymograms of proteins, obtained after growing T. harzianum CECT 2413 in acidic buffered media or in media supplemented with peptidic nitrogen sources (Delgado-Jarana et al., 2000
). One of these proteases, which was pepstatin-sensitive, was probably responsible, together with acidic pH, for the degradation of an overexpressed ß-1,6-glucanase encoded by bgn16.2 (Delgado-Jarana et al., 2000
).
This study describes the isolation and characterization of a gene from T. harzianum CECT 2413, papA, that encodes an aspartyl protease. Protease regulation and the effect on the production of ß-1,6-glucanase is also discussed.
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METHODS |
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RNA procedures and Northern analysis.
Mycelia were lysed in a mini-beadbeater (Biospec Products) with 2·3 mm diameter steel beads. RNA was isolated as described by Delgado-Jarana et al. (2000) . Ten microgrammes of total RNA from each sample were separated in 1·2% agarose/formaldehyde gels, blotted onto nylon membranes and hybridized as described by Sambrook et al. (1989)
. Blots were probed with the complete papA gene. Probes were labelled using [ß-32P]dCTP with the Amersham oligolabelling method (Amersham Pharmacia Biotech). Loading control of Northern blots was checked using radish 18S rDNA.
Isolation of papA.
papA was isolated with primers (Fig. 1) designed by alignment of several fungal acid proteases, carried out using a CLUSTAL W algorithm. Oligonucleotide sequences were 5'-CGACACCGGCTCCGA-3' (direct) and 5'-GGGCAACGTCACCAAAGA-3' (reverse). PCR conditions were 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 1 min at 55 °C and 1 min at 72 °C, then 7 min at 72 °C, using 5 mM MgCl2. A 900 bp fragment was isolated and used as a probe to screen a genomic library. A 5 kbp SalIPstI fragment, which contained an ORF of 1212 bp and 1 kb of a putative promoter region (Fig. 1
) was isolated.
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Transformation procedure.
Protoplast preparation, transformation and transformant stabilization were carried out according to Herrera-Estrella et al. (1990) . T. harzianum CECT 2413 wild-type and B4, a transformant strain derived from it which overproduces ß-1,6-glucanase (Delgado-Jarana et al., 2000
) were cotransformed with plasmids pLMRS3::papA and pHAT
(Herrera-Estrella et al., 1990
): the latter carries the hygromicin resistance gene (hph) as a selection marker. Cotransformation was conducted with a 1:10 (hph: papA) plasmid ratio.
Isolation of extracellular proteins.
For protein detection and measurement of enzymic activity, culture supernatants were concentrated by ammonium sulphate precipitation and dialysed against 50 mM potassium acetate buffer, pH 5·5, at 4 °C. Protein concentration was determined by the Lowry method, using BSA as the standard.
Enzyme activity.
ß-1,6-Glucanase activity was determined by measuring the amount of reducing sugars released from pustulan (Calbiochem) (De la Cruz et al., 1995 ). The reducing sugar content was determined by the procedure of Somogyi (1952)
and Nelson (1957)
. An enzymic unit was defined as the amount of enzyme that catalyses the release of reducing sugar groups equivalent to 1 µmol glucose min-1 under the above-described assay conditions. Protease activity was measured by the azocasein procedure of Holwerda & Rogers (1992)
. An enzymic unit was defined as the amount of enzyme that hydrolyses 1 µg azocasein (Sigma A-2765) min-1 under the specific assay conditions.
IEF and zymograms.
IEF was performed with 10 µg extracellular proteins in a Multiphor II unit (Amersham Pharmacia). Ampholyte polyacrylamide gels with a pH gradient between 3·5 and 9·5 were used. Zymograms were carried out by overlaying the IEF gel with a 1·5 mm thick 2% agarose gel containing 1% BSA in 50 mM acetate buffer, pH 5·5. Both gels were incubated between two glass plates for 24 h at 37 °C. Proteolytic activities were detected as haloes of BSA degradation.
Western blot analysis.
Western blotting was performed using discontinuous SDS12% PAGE. Prestained molecular mass protein standards (Bio-Rad) were included for molecular mass determination. Transfer onto Immobilon-P membranes (Millipore) was performed using a Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) following the manufacturers instructions. Membranes were blocked for 2 h with a solution of 5% skimmed milk in 10 mM Tris/HCl pH 8, 1 mM EDTA, 150 mM NaCl and 0·1% Triton X-100. Filters were then incubated overnight at room temperature with rabbit polyclonal antibody against BGN16.2 (Delgado-Jarana et al., 2000 ), diluted appropriately in blocking solution, and then washed with blocking buffer. Alkaline phosphatase-conjugated anti-rabbit IgG antibody (Sigma) was used as the secondary antibody.
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RESULTS |
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Sequence analysis of papA
The nucleotide sequence of the 3 kb cloned fragment revealed that it contains about 1 kb which corresponds to the promoter region, and the entire nucleotide sequence of the papA gene (GenBank accession no. AJ276388) (Fig. 1
). The 5' non-coding sequence of papA was examined for the presence of motifs involved in the regulation of papA expression. The sequence, applying the MatInspector algorithm, contains multiple copies of the 5'-GATA-3' motif, which is the recognition sequence of the nitrogen regulatory protein AreA from Aspergillus (Ravagnani et al., 1997
) and several copies of the 5'-CCCCT-3' motif, which corresponds to the recognition sequence of the stress proteins, STRE, involved in gene regulation under stress conditions in Saccharomyces cerevisiae (Martínez-Pastor et al., 1996
). These motifs have also been found in the basic proteinase (prb1) and endochitinase (ech42) gene promoter sequences of T. harzianum (Cortés et al., 1998
). Only one copy of the motif 5'-GCCARG-3', the recognition site for the PacC protein mediating pH regulation in Aspergillus (Tilburn et al., 1995
), was detected. Putative binding sites for regulatory proteins involved in mycoparasitism (MYC) described in the prb1 and ech42 promoter sequences of T. harzianum (Cortés et al., 1998
), identical to MYC1 (5'-GCTTCA-3') and nearly identical to MYC2 (5'-TTGGCAA-3'), MYC3 (5'-GGGCAC-3') and MYC4 (5'-GGCAWTCGGCAT-3'), and with the same relative position (5'-MYC1 MYC2 MYC3 MYC43') were also detected, although their role in vivo has still to be demonstrated. In this promoter region there are also TATAA and CAAT motifs, which are related with the initiation of transcription (Tilburn et al., 1995
). The sequence prior to the translation initiation triplet is highly conserved, since the TCAAA pentamer correlates with the proposed (T/C)CAA(A/C) consensus sequence previous to the AUG codon (Goldman et al., 1998
). No polyadenylation signals were detected in the gene sequence.
The papA gene seems to consist of an intronless ORF of 1212 bases, which encodes a polypeptide of 404 aa. However the existence of introns of small size that do not alter the reading frame cannot be ruled out. By comparison with the N-terminal region of secreted acid proteases of other filamentous fungi, analysed with the Signal IP v1.1 algorithm, it may be suggested that the PAPA protein is synthesized as zymogen, following the general rules for fungal acid proteases. This region contains an N-terminal preproregion of 52 aa (Fig. 1), formed by a leader peptide of 20 aa, with a potential proteolytic site between aa 20 (A) and 21 (L) identified with the Signal IPV1.1 algorithm. The leader peptide is followed by an N-subterminal peptide of 32 aa that could be cleaved to activate the enzyme. This peptide ends with the amino acids KR (at positions 51 and 52). A potential protease KEX2 site follows KR (52) in other proteins (Bussey, 1988
). This proteolysis would produce a mature active protein. The mature form of the enzyme would be a 352 residue protein with a calculated molecular mass of 36·7 kDa and a calculated pI of 4·35. There are two DTG motifs for aspartyl proteases. The papA sequence also reveals the existence of two lobes, each of which has its own active DTG site (Rawlings & Barret, 1995
). No potential post-translational modification signals were detected in the deduced primary sequence.
PAPA and homologous proteins found in other organisms
The mature PAPA protein exhibits 4356% identity with aspartyl proteases of Colletotrichum gloeosporioides (54%), Endothia parasitica (56%), Podospora anserina (48%), Penicillium roqueforti, Penicillium janthinellum (4347%), and Aspergillus oryzae, A. niger and A. awamori (4446%). Alignment of the deduced PAPA amino acid sequence and those aspartyl protease sequences of different filamentous fungal species with which the PAPA sequence displays the highest similarity are shown in Fig. 2. The sequences have been aligned to achieve maximal homology, which corresponds to the amino-terminal region, by using the CLUSTAL W program.
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Patterns of papA mRNA accumulation and of protease and ß-1,6-glucanase activities
The strains were cultivated in MM with 2% glucose as the carbon source and either 0·5% ammonium or 1% yeast extract as the nitrogen source, as described in Methods. The mRNA level of papA was similar in the transformants 13OE1, which overexpressed only papA, and B4OE4, which overexpressed both papA and bgn16.2 (Fig. 4c). Data on protease activity, measured in the supernatants of the cultures of the four strains, indicated that in MM with ammonium, papA single transformant 13OE1 or the double transformant B4OE4 had similar values, and were also similar to those of the wild-type and strain B4. Protease values increased about 15-fold in supernatants from papA transformants and 34-fold in supernatants from the wild-type and B4 strain, when they were cultivated in medium with yeast extract compared to medium with ammonium (Table 2
). Extracellular proteins were obtained from the supernatants of MM with either ammonium or yeast extract by ammonium precipitation; the proteins were separated by SDS12% PAGE and were stained with Coomassie Brilliant Blue R-250. PAPA and/or BGN16.2 proteins were observed only in supernatants from cultures with yeast extract (Fig. 5a
). A zymogram of extracellular proteins from MM with yeast extract is presented in Fig. 6
. There is a single protein with protease activity which was overexpressed in the papA transformant 13OE1, which possesses a pI of 4·5. Protease inhibition was obtained when pepstatin, an aspartyl protease inhibitor, was used; no inhibition was observed when PMSF was used instead.
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DISCUSSION |
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In a different strain of T. harzianum, a basic protease of 31 kDa, PBR1 (Geremía et al., 1993 ), probably equivalent to the basic proteinase detected in T. harzianum CECT 2413 (Delgado-Jarana et al., 2000
), was identified as a subtilisin which was induced by fungal cell walls and played a fundamental role in mycoparasitism against phytopathogenic fungi (Flores et al., 1997
) and biocontrol of root-knot nematodes (Sharon et al., 2001
). The role of other fungal basic proteases such as those of Metarhizium anisopliae, Bauveria bassiana (St Leger et al., 1997
) or Sclerotinia sclerotiorum (Poussereau et al., 2001
) has also been regarded as highly significant during antagonistic interactions with other organisms, and their regulation seems to depend on cell wall components of the antagonized hosts.
With regard to acidic proteases, several pepsin-like aspartyl proteases have been described in T. reesei (Dunne, 1982 ; Haab et al., 1990
) and T. harzianum (Delgado-Jarana et al., 2000
) with pIs that varied from 4·3 to 4·9. None of them have been reported to be completely purified, with the exception of a trypsin-like protease (Suárez et al., 2000
) and the aspartyl protease called trichodermapepsin, which has been purified and cloned (Pitts, 1992
; Mäntylä et al., 1994
). Some of these acidic proteases are pepstatin-inhibitable, but others are insensitive to this inhibitor (Haab et al., 1990
) and they have been detected in casein- or cellulose-supplemented media. The pepsin-like aspartyl protease gene from T. harzianum CECT 2413, papA, isolated and characterized in this study, possesses characteristics that are similar to those of the trichodermapepsin of T. reesei (Mäntylä et al., 1994
) (Fig. 1
). The PAPA protein, which has been only partly purified, showed inhibition by pepstatin when it was overproduced (Fig. 6
).
In this study, glucose represses papA transcription. Substitution of glucose by glycerol in media with either ammonium or casein does not derepress papA. By contrast, other protease genes such as acp1 from S. sclerotiorum are strongly expressed when glycerol considered a neutral carbon source is added to the medium instead of glucose (Poussereau et al., 2001 ). In addition, papA was regulated by at least the nitrogen and carbon sources and by ambient pH (Fig. 4
). The results are consistent with the presence of potential sites for nitrogen (AreA) and pH (PacC) control described in Aspergillus and other filamentous fungi (Espeso et al., 1993
) that are also found in the papA promoter, but the lack of potential CreA sites suggests carbon regulation may take place by a system different from CreA, as has been suggested in other fungi (Espeso et al., 1993
; Ronne, 1995
). Other protease genes are also repressed by glucose and ammonia, such as pepA from A. niger (Berka et al., 1990
), and/or induced by peptidic nitrogen sources (casein), such as the aspA protease gene from Penicillium roqueforti (Gente et al., 1997
). In this study basic and acidic pHs completely repressed papA gene expression, even in the presence of casein (Fig. 4
). A similar phenomenon at alkaline pH has been described for aspA from P. roqueforti (Gente et al., 2001
). At low pH the PAPA protease might be denatured and/or pH cleaved as occurred to the ß-1,6-glucanase (Delagado-Jarana et al., 2000
). At alkaline pH papA product might not be processed into its mature, active form (Gente et al., 2001
). Those peptides which should activate papA are not liberated from casein.
Overexpression of PAPA did not affect cell viability, and in papA transformants with multiple copies of the gene, the papA gene product could be detected in yeast extract (Figs 5 and 6
) but not in MM minimal medium with 2% glucose and 0·5% ammonium. Overexpressed PAPA coexisted with overexpressed ß-1,6-glucanase and both proteins were only present in media with organic nitrogen sources such as yeast extract (Figs 5
and 6
). The absence of PAPA in MM minimal medium with glucose and ammonium probably resulted from proteolysis and denaturation due to a double effect of aspartyl proteases and extreme pHs, as occurred with BGN16.2 (Delgado-Jarana et al., 2000
). PAPA is detected at higher pHs and when alternative protease substrates (i.e. yeast extract) are present in the medium. Peptidic nitrogen sources are frequently added during protein overproduction to avoid proteolysis of the desired protein (Jeenes et al., 1991
). Lack of PAPA when BGN16.2 is proteolysed (Figs 5
and 6
) indicated that this protease is not responsible for BGN16.2 degradation.
Disruption of papA would indicate whether or not PAPA plays a role in ß-1,6-glucanase degradation. However, attempts to disrupt the protease gene in the wild-type and the B4 transformant have been unsuccessful. The hygromycin resistance gene hph that was used as the selection marker was flanked at both ends by 1·4 kb fragments of the papA gene. In all cases the transformants isolated possessed several ectopic copies of the hph gene but maintained unaltered the endogenous papA gene. The reason for the lack of papA gene disruptants could be the small size of the homologous fragments rather than papA being essential. As occurs in other filamentous fungi (Asch & Kinsey, 1990 ) T. harzianum CECT 2413 is probably very reluctant to undergo homologous recombination, which does not take place unless the homologous region possesses a minimal size.
Acidic proteases seem to be responsible for degradation of extracellular proteins such as chitinases and glucanases, cellulases and other homologous or heterologous proteins overproduced for different applied purposes (Margolles-Clark et al., 1996 ; Delgado-Jarana et al., 2000
; Hagspiel et al., 1989
; Nyyssönen et al., 1993
). In this study, overexpression of PAPA did not result in degradation of overproduced ß-1,6-glucanase. Rather, a 30% increase in ß-1,6-glucanase activity was detected in double bgn16.2 papA transformants, compared to single bgn16.2 transformants (Table 2
). This increase could result from a dilution effect due to the BGN16.2 and PAPA proteins being the targets of the same proteases, although the reason is not yet known for sure.
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ACKNOWLEDGEMENTS |
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This work was financially supported by the grants Junta de Andalucía PAI CVI-107 and CICYT IFD97-0668, IFD97-0820 and AGL2000-0524.
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Received 13 November 2001;
revised 4 January 2002;
accepted 10 January 2002.