Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus nigera

Mojca Saudohar1, Mojca Bencina1, Peter J.I van de Vondervoortb,2, Henk Pannemanc,2, Matic Legisa1, Jaap Visserd,2 and George J. G. Ruijtere,2

National Institute of Chemistry, Department for Biotechnology and Industrial Mycology, Hajdrihova 19, SI-1000 Ljubljana, Slovenia1
Wageningen University, Section Molecular Genetics of Industrial Micro-organisms, Dreijenlaan 2, 6703HA Wageningen, The Netherlands2

Author for correspondence: George Ruijter. Tel: +31 71 5262967. Fax: +31 71 5266876. e-mail: g.j.g.ruijter{at}lumc.nl


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the {Delta}pkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.

Keywords: cAMP-dependent protein kinase, regulatory subunit, signal transduction, morphology

Abbreviations: PKA, cAMP-dependent protein kinase; PKA-R, regulatory subunit of PKA; PKA-C, catalytic subunit of PKA

a The EMBL accession number for the sequence reported in this paper is AJ296317.

b Present address: Wageningen University, Laboratory of Phytopathology, PO Box 8025, 6700 EE Wageningen, The Netherlands.

c Present address: Dr Van Haeringen Laboratorium, Agro Business Park 100, 6708 PW Wageningen, The Netherlands.

d Present address: FGT Consultancy, PO Box 396, 6700AJ Wageningen, The Netherlands.

e Present address: Department of Pediatrics, Leiden University Medical Centre, Building 1 P3-P, PO Box 9600, 2300RC Leiden, The Netherlands.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cyclic AMP is a ubiquitous secondary messenger that controls the activity of the cAMP-dependent protein kinase (PKA). The system is widespread in eukaryotes (Gancedo et al., 1985 ) and is involved in, for example, regulating metabolic and developmental processes (Daniel et al., 1998 ).

cAMP signalling controls a number of developmental events such as growth polarity in the filamentous fungus Neurospora crassa (Bruno et al., 1996 ), cell development of Blastocladiella emersonii (de Oliveira et al., 1997 ) and sexual development of Schizosaccharomyces pombe (Maeda et al., 1994 ). The influence of cAMP via PKA on dimorphic transition was demonstrated for several dimorphic fungi, e.g. Candida albicans (Niimi, 1996 ) and Mucor rouxii (Orlowski, 1991 ). For the plant-pathogenic fungi Ustilago maydis (Gold et al., 1994 ) and Magnaporthe grisea (Mitchell & Dean, 1995 ) and for the human pathogen Cryptococcus neoformans (Kronstad et al., 1998 ), cAMP signalling is directly connected to fungal virulence (reviewed by Borges-Walmsley & Walmsley, 2000 ). PKA is also involved in metabolic regulation, activating glycolysis (Goncalves et al., 1997 ) and diauxic transition in Saccharomyces cerevisiae (Boy-Marcotte et al., 1998 ). The inactivation of Sac. cerevisiae fructose-1,6-bisphosphatase (Jiang et al., 1998 ), the stability of Sac. cerevisiae neutral trehalase (Zahringer et al., 1998 ) and the activation of 6-phosphofructo-1-kinase from Mytilus galloprovincialis (Fernandez et al., 1998 ) are results of PKA phosphorylation. In Aspergillus nidulans conidia PKA is proposed to be involved in mobilization of trehalose by phosphorylation of trehalase (d’Enfert et al., 1999 ).

In most cases the inactive form of PKA is a tetrameric protein composed of two regulatory and two catalytic subunits. Upon binding of cAMP, inactive PKA dissociates into two active catalytic subunits and a dimer of regulatory subunits. Two types of PKA regulatory subunits (PKA-R), type I and type II, have been isolated and four different genes encoding PKA-R (I{alpha}, Iß, II{alpha}, IIß) have been identified in higher eukaryotes (Taylor et al., 1992 ). Fungi usually posses a single gene for PKA-R, which encodes a protein similar to either the mammalian type I or type II regulatory subunits. In N. crassa, Sch. pombe, Mag. grisea, Sac. cerevisiae, U. maydis and B. emersonii type II regulatory subunits have been described (Bruno et al., 1996 ; de Voti et al., 1991 ; Adachi & Hamer, 1998 ; Kunisawa et al., 1987 ; Gold et al., 1994 ; Marques & Gomes, 1992 ). PKA from Dictyostelium discoideum (Mutzel et al., 1987 ) and Paramecium tetraurelia (Carlson & Nelson, 1996 ) have a different structure. The regulatory subunit of these enzymes does not contain dimerization domains – they are heterodimeric proteins composed of only one catalytic and one regulatory subunit.

In the filamentous fungus Aspergillus niger, a transient increase in cAMP levels during the early stage of growth in a medium with high initial sucrose concentration has been observed (Legia et al., 1981 ; Gradinik-Grapulin & Legia, 1997 ). Simultaneously with the cAMP peak, a change in morphology from bulbous cells to filamentous hyphae took place (Legia & Gradinik-Grapulin, 1995 ; Gradinik-Grapulin & Legia, 1997 ). Growth tests indicated that even moderate overproduction of PKA-C affected growth and sporulation characteristics of A. niger transformants (Benina et al., 1997 ). During germination of wild-type A. niger the level of expression of pkaC and the specific PKA activity steadily decreased (Benina & Legia, 2000 ). These data suggest that PKA is involved in morphogenesis. To analyse the A. niger PKA enzyme in more detail and study its role in the regulation of morphological and cellular development, we cloned A. niger pkaR and constructed A. niger strains with disrupted or overexpressed pkaC and/or pkaR.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and plasmids.
A. niger strains used in this study are listed in Table 1. A. niger N400 (CBS 120.49) was used for preparation of a genomic library. The strains used for transformations were A. niger NW219 and NW245. A. niger 13mcC with two additional copies of pkaC (Benina et al., 1997 ) and A. niger NW219::pyrA were used as reference strains in some analyses. A. niger NW274 was used for mitotic recombination experiments.


View this table:
[in this window]
[in a new window]
 
Table 1. A. niger strains used in this study

 
Escherichia coli LE392 [{Delta}(lacIZY)6 galK2 galT22 metB1 trpR55 {lambda}-] (Promega), was used for phage amplification and purification, E. coli DH5{alpha} [F-/endA1 hsdR17 ( ) supE44 thi-1 recA1 gyrA (Nalr) relA1 {Delta}(lacIZIA-argF) U169 deoR ({phi}80 {delta}lac {Delta}(lacZ) M15)] (New England Biolabs) and E. coli XL-1 Blue MRF' [{Delta}(mcrA)183 {Delta}(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac{F- proAB laclqZ {Delta}M15::Tn10 (Tetr)}] (Stratagene) were used for plasmid DNA propagation and for in vivo excision, respectively.

Plasmid vectors pBluescript KS (Stratagene) and pEMBL19 (Roche) were used for subcloning. Plasmid pGW635, containing A. niger pyrA (Goosen et al., 1987 ), and plasmid pIM650, containing A. nidulans argB (Johnstone et al., 1985 ), were used for construction of pkaR and pkaC disruption plasmids. Phage ExAssist (Stratagene) was used as a helper phage for phagemid excision.

The following gene fragments were used to generate probes for screening A. niger genomic and cDNA libraries: a 1·51 kbp BglII/BamHI fragment from Mag. grisea pkaR (Adachi & Hamer, 1998 ) and a 1·45 kbp HindIII fragment from A. nidulans pkaR [accession no. AF043231 (gi: 3170247)].

Culture media and growth conditions.
For the preparation of conidiospores, A. niger strains were grown at 30 °C for 3–4 days on complete medium (CM), originally described for A. nidulans (Pontecorvo et al., 1953 ), using 1% (w/v) glucose as a carbon source, with appropriate supplements and solidified with 1·5% (w/v) agar. Since disruption of the pkaR gene in A. niger resulted in an inability to form conidiospores, we could not maintain a {Delta}pkaR mutant in the form of conidiospores. Instead, mycelium of an A. niger {Delta}pkaR mutant was picked from a plate and stored in 30% (v/v) glycerol at -70 °C. For submerged growth, medium was inoculated with 106 conidiospores ml-1. One litre of minimal medium (MM) contained 6 g NaNO3, 1·5 g KH2PO4, 0·5 g KCl, 0·5 g MgSO4.7H2O, pH 6·0, and 0·2 ml trace metal solution (Visniac & Santer, 1957 ). Glucose was used as a carbon source. For the growth of auxotrophic strains, appropriate supplements were added: 10 mg nicotinamide l-1, 200 mg leucine l-1, 1220 mg uridine l-1 and 200 mg arginine l-1. Mycelium was grown at 30 °C in a rotary shaker at 250 r.p.m.

Germination kinetics were analysed by microscopic examination of slides coated with minimal medium containing 2% glucose and 1·5% agar and spot-inoculated with 2x104 conidiospores. Spores were incubated at 30 °C. The percentage of germinated spores was followed in time by examining 50–100 spores at 30 min intervals.

DNA manipulation.
A. niger chromosomal DNA was isolated as described by de Graaff et al. (1988) . Propagation and isolation of plasmid DNA, Southern blot analysis and other DNA manipulations were essentially done as described by Sambrook et al. (1989) . [{alpha}-32P]dATP-labelled probes were synthesized using random hexamer primers (Sambrook et al., 1989 ). Automated sequencing based on the dideoxy chain-termination procedure (Sanger et al., 1977 ) was done with ALFexpress (Amersham Pharmacia Biotech) using the T7 DNA polymerase sequencing kit (Amersham Pharmacia Biotech). DNA sequence was analysed with PC/GENE and WINSTAR computer program packages. Further analysis of the nucleotide sequence was performed using EDITSEQ and MAPDRAW from the PC/GENE software package, MATINSPECTOR v2.2 (Quandt et al., 1995 ) and Prosite and InterPro databases.

The A. niger pkaR gene and the corresponding cDNA clone were isolated from an A. niger N400 genomic library in the {lambda} replacement vector EMBL4 (Promega) and a cDNA library from A. niger N400 in {lambda}ZAP II (Stratagene), respectively. Plasmids containing the cloned cDNAs were obtained from the {lambda}ZAP II phages by in vivo excision, according to the manufacturer’s instructions.

Mitotic recombination was performed as described by Bos et al. (1988) . Heterokaryon formation between NW276 and NW275 was induced by protoplast fusion (Van Diepingen et al., 1998 ). Haploidization was induced with benomyl.

To construct a plasmid for simultaneous overexpression of pkaC and pkaR, pIM492, an 8 kbp EcoRI fragment, containing pkaR, was inserted into pEMBL19. After removal of one EcoRI site, a 5 kbp EcoRI pkaC fragment from pPKAC1 (Benina et al., 1997 ) was inserted into the other EcoRI site giving pIM492 (Fig. 1).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1. Partial restriction maps of pIM490 containing pkaR, pIM492 containing pkaC and pkaR, the pkaR disruption plasmid, pIM493 and the pkaC disruption plasmid, pKOG10. The arrows below the restriction maps indicate the probes used for Southern and Northern analysis. The coding regions and the directions of transcription are presented by filled arrows; open boxes indicate non-coding regions.

 
To construct pIM493, a plasmid to disrupt pkaR, a 1·7 kbp SalI fragment of pkaR from pIM490 was inserted in pBluescript, the plasmid obtained was digested with XhoI/BglII and ligated to the 2·8 kbp BamHI/XhoI fragment of argB from pIM650 (Fig. 1). The 3·3 kbp XhoI/KpnI fragment of pkaR from pIM490 was ligated into this construct to get pIM493. The 7·1 kbp EcoRI fragment of this construct was used for transformation of A. niger NW245.

To construct the pkaC disruption plasmid, pKOG10, a SmaI/NcoI fragment of pyrA was ligated into the NsiI (blunt end)/NcoI sites of pkaC in pPKAC1 (Benina et al., 1997 ). A 6·4 kbp EcoRI fragment of the resulting plasmid (pKOG10, see Fig. 1) was used for pkaC disruption by REMI transformation of A. niger strain NW219.

Northern analysis.
Mycelium grown in liquid complete medium (CM) for 24 h was transferred for 1 h into MM medium with 1 or 15% glucose, harvested by filtration, washed briefly with ice-cold 0·05 M potassium phosphate buffer pH 7·0, frozen in liquid nitrogen and ground using a micro dismembrator (Braun Biotech). RNA was extracted using the TRIzol reagent (Life Technologies). After electrophoresis, RNA was transferred to nylon membranes (Hybond-N+; Amersham Pharmacia Biotech) by capillary blotting in 10xSSC. Pre-hybridization and hybridization were performed at 42 °C in formamide hybridization buffer containing 50% (w/v) formamide, 0·75 M NaCl, 50 mM NaH2PO4 (pH 7·4), 10 mM EDTA, 2x Denhardt’s, 0·1% (w/v) SDS and 10% (w/v) dextran sulphate. Northern blots were washed at 65 °C in 4xSSC, 0·5% SDS and 2xSSC, 0·5% SDS. The probes used for Northern analysis were as follows: a 0·8 kbp BglII/XhoI fragment from pIM490 for pkaR mRNA (probe RI, Fig. 1), a 1·2 kbp EcoRI/KpnI fragment from pIM492 for pkaC mRNA (probe C, Fig. 1) and a 0·7 kbp EcoRI fragment of A. niger 18S rRNA. The pkaR and pkaC probes are also depicted in Fig. 1.

Transformation of A. niger.
Mycelium was obtained by growing A. niger for 16–18 h in liquid culture on complete medium with appropriate supplements. Preparation of protoplasts and subsequent transformation of A. niger were performed as described by Kusters-van Someren et al. (1991) . The A. niger pyrA gene and A. nidulans argB gene were used as selection markers. For co-transformation of A. niger NW219, 1 µg pGW635 DNA and 27 µg of the co-transforming plasmid pIM491 (containing pkaR) or pIM492 (containing a copy of both the pkaR and the pkaC genes) were added to 2x107 protoplasts. To disrupt pkaR, 5 µg of the 7·1 kbp EcoRI pkaR fragment from pIM493 was used to transform A. niger NW245. To disrupt pkaC, 5 µg of the 5 kbp EcoRI pkaC fragment from pKOG10 was used to transform A. niger NW219. The transformants were selected and purified by replating at low spore densities on selective medium without uridine or arginine.

Enzyme assays.
Frozen mycelium (0·5 g) obtained as described for RNA isolation, was ground and suspended in 1 ml extraction buffer (350 mM KH2PO4, pH 7·5, 0·1 mM DTT, 10% glycerol). After 15 min extraction, the homogenate was centrifuged at 10000 g for 10 min. Supernatant was used for the enzyme assay. Cell extracts (with protein concentration of approximately 2 mg ml-1) were diluted two- to tenfold before measuring enzyme activity. The activity of PKA was detected by the non-radioactive PepTag test method with dye-labelled Kemptide as a substrate, according to the manufacturer’s protocol (Promega) or by SpinZyme (Pierce), according to Benina et al. (1997) . The incubation time for the enzymic reaction was 30 min at 30 °C. One unit of enzyme activity was defined as the amount of enzyme required to transfer 1 pmol phosphate from ATP to the substrate (Kemptide) per min at 30 °C. The catalytic subunit of bovine heart PKA (Promega) was used as a standard. Protein concentrations in cell-free extracts were determined with the Bicinchoninic-acid protein kit (Sigma), according to the supplier’s instructions, and using BSA fraction V (Roche) as a standard.

RT-PCR.
RT-PCR was performed according to Benina & Legia (1999) . Total RNA (2 µg) and primers PKAC14 [5'-AGAAAGGCGTGAAACCACACAG-3' (Benina et al., 1997 )] and PEPC2 [5'-TATCACGGTGAGAGATACGAGC-3' (Frederick et al., 1993 )] (Ransom Hill Bioscience) were used. cDNA product (2 µl) was transferred to 20 µl PCR reaction mixture containing primers PKAC13 [5'-TGGTCATGGACTTCGTAGAGGG-3' (Benina et al., 1997 )], PKAC14, PEPC1 [5'-CTATCTGGGTCTCAAGAACACC-3' (Frederick et al., 1993 )], PEPC2 (Ransom Hill Bioscience). PCR was performed in a Perkin Elmer thermal cycler GeneAmp PCR System 2400.

Twenty microlitres of the PCR products were separated by 1% (w/v) agarose gel electrophoresis, transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) and subsequently the biotin-labelled fragments were detected by the avidin alkaline phosphatase conjugate (Tropix) using CDP-Star (Tropix) as a substrate. Signals were determined by imaging film X-OMATAR (Eastman Kodak). The expected sizes of the PCR products were 230 and 420 bp. The intensity of the signals on the film was measured with an Imaging Densitometer model GS-670 (Bio-Rad) and analysed by Molecular Analyst Software (Bio-Rad).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequence analysis of A. niger pkaR
The A. niger pkaR gene, encoding PKA-R, was cloned from an A. niger N400 genomic library in {lambda}EMBL4 using Mag. grisea pkaR (Adachi & Hamer, 1998 ) and A. nidulans pkaR [accession no. AF043231 (gi: 3170247)] as probes. Six phages were isolated with the first probe and four phages with the second. Restriction patterns of these phages were similar except for minor differences, indicating that the two different probes hybridized to the same gene. An 8 kbp EcoRI fragment from one of the phages originally obtained with the A. nidulans probe was cloned in pEMBL19, resulting in plasmid pIM490 (Fig. 1).

The nucleotide sequence of a 3330 bp fragment of pIM490 was determined on both strands. The sequence from position -319 to -326 could not be identified unambiguously due to high GC content. The nucleotide sequence was deposited in the EMBL database under the accession no. AJ296317. In addition, a 2002 bp cDNA fragment of pkaR was isolated from an A. niger cDNA library using stringent hybridization conditions and a 0·6 kbp BamHI/SalI fragment of pIM490 as a probe.

By comparing the cDNA sequence with the sequence of the genomic clone, a single intron of 700 bp was found from position -112 to -811 with borders 5'-GTAAGG and 3'-CAG. Three putative CCAAT boxes were found upstream from the start codon at positions -361, -426 and -473. Analysis of the promoter region by the MATINSPECTOR v2.2 software package (core and matrix similarity parameters: 1·0 and 0·9) (Quandt et al., 1995 ) revealed several putative transcription elements, including stress response elements (Stre), a binding site for heat-shock factors (Hsp), a target site for an activator of nitrogen-regulated genes (Nit1), the positive transcriptional regulator Gcr1, the transcriptional activator for control of development, AbaA, and the regulator of asexual reproduction and differentiation of hyphae, StuA. In addition, the promoter region contains several E-boxes (CANNTG) (Murre et al., 1989 ) and nine C4T or reverse complementary (AGGGG) sequences (Treger et al., 1998 ), five of which are inside the intron. No obvious TATA sequence was found in the 5' non-coding region.

The cDNA contained an ORF of 1233 nt encoding a protein of 411 aa with a calculated molecular mass of 44527 Da, which is fairly similar to the apparent molecular mass determined for the purified protein, 48 kDa (Legia & Benina, 1994 ). The coding sequence showed a strong bias for A in the third position of the codon, which was also found in the A. niger pkaC gene (Benina et al., 1997 ). Three putative structural features were present within PKA-R (Leon et al., 1997 ; Su et al., 1995 ). First, a dimerization domain located at the N-terminal third of the protein. Second, a site for interaction between the regulatory and catalytic subunits [(117)RRTSVSAE] containing an autophosphorylation site (Ser-120). Third, two cAMP-binding sites: (227)VgSvgpGGSFGELALmYnaPRAATV and (348)VksykrgDYFGElALLddkPRAAsI (conserved amino acid residues are underlined). The deduced amino acid sequence suggests that the PKA-R of A. niger is most closely related to type II PKA-R, showing an overall identity of 75% with A. nidulans pkaR [accession no. AF043231 (gi: 3170247)], 69% with N. crassa (Bruno et al., 1996 ) and 66% with Mag. grisea (Adachi & Hamer, 1998 ).

Downstream of the stop codon the mRNA still contains 459 nt. Putative poyladenylation signals, ATAAA, were found at 16 and 366 nt downstream of the stop codon.

Construction of strains modified in pkaR and pkaC
To understand the role of the PKA-R in cell physiology, transformants containing multiple copies of pkaR and/or pkaC, and strains in which these genes were disrupted were prepared.

Multicopy pkaR strains (mcR) were isolated after co-transforming A. niger with pIM491 using pyrA as a selection marker. Similarly, transformants with increased copy numbers of both pkaC and pkaR (mcRC strains) were produced using pIM492.

Several transformants were analysed for copy number of pkaR and pkaR/pkaC by Southern analysis. Four transformants were found with multiple copies of pkaR (strain 10mcR, one extra copy; strain 38mcR, approximately 10 copies; strains 42mcR and 39mcR contained more than 20 copies). Five transformants contained an increased number of copies for both genes (strain 35mcRC, one extra copy for each gene; strain 7mcRC, 4–5 copies; strain 44mcRC, approximately 10 copies; strains 1mcRC and 18mcRC had at least 20 copies). Increased copy numbers of pkaC and pkaR resulted in overexpression of the two genes (Fig. 2).



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2. PKA activities and Northern analysis of selected A. niger pkaR and pkaR pkaC transformants. The following strains were analysed: A395, wild-type (wt); 10mcR, 38mcR, 39mcR, 42mcR, strains containing multiple copies of pkaR; 35mcRC, 7mcRC, 1mcRC, 44mcRC, 18mcRC, strains containing multiple copies of both pkaR and pkaC; 13mcC, multicopy pkaC; {Delta}C, pkaC deletion strain NW258; 9{Delta}R, pkaR deletion strain NW275; 9{Delta}Rsp, pseudorevertant of strain 9{Delta}R capable of conidiation. Cell-free extracts for PKA activity and total RNA were isolated from mycelium that was pre-grown on CM with 1% glucose for 24 h and then transferred to MM with 1 ({blacksquare}) or 15% ({square}) glucose. After 1 h of incubation, mycelium was harvested and used for the above-mentioned analysis. PKA activities shown in the graph are means from three independent determinations. The probes used for Northern analysis are described in Methods and indicated in Fig. 1. For detection of the pkaR transcript, probe RI (Fig. 1) was used. 18S was used as a loading control. In the Northerns two lanes are shown for each strain, showing expression of the genes in mycelium incubated with 1 and 15% glucose.

 
Additionally, transformants with disrupted pkaR ({Delta}R) or pkaC ({Delta}C) were constructed. For disruption of the pkaR gene, plasmid pIM493 was constructed in which a 5' part of the pkaR coding region and part of the promoter was removed and replaced by the A. nidulans argB gene. To obtain a pkaC knock-out strain plasmid pKOG10 was constructed in which A. niger pyrA was inserted into pkaC after removing a small part of the coding region. Five pkaR disruption strains (9{Delta}R, 26{Delta}R, 29{Delta}R, 38{Delta}R, 47{Delta}R) and one pkaC disruption strain ({Delta}C) were obtained. Gene replacements in strains {Delta}C and 9{Delta}R were verified by Southern analysis (Fig. 3). In strain {Delta}C the presence of a 7·5 kbp EcoRI fragment instead of a 5 kbp fragment indicated proper gene replacement. In {Delta}R strains replacement of a 3 kbp BglII fragment by a 4·2 kbp fragment established gene replacement. In addition, removal of a 1·2 kbp BglII/XhoI fragment was confirmed in {Delta}R strains. All five pkaR disruptants had very compact colonies and did not sporulate (see below). Strain 9{Delta}R was analysed in more detail.



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 3. Disruption of pkaC and pkaR in A. niger. Southern analysis was performed with EcoRI-digested genomic DNA of pkaC disruption strains (top panels) or BglII-digested genomic DNA of pkaR disruption strains (middle and lower panels). The following strains were analysed: wt, wild-type strain N400; {Delta}R, pkaR disruptant NW275; {Delta}C, pkaC disruptant NW258; {Delta}R{Delta}C, pkaR pkaC double disruptant NW277. Blots were probed with a 1·2 kbp EcoRI/KpnI pkaC fragment (probe C; Fig. 1) to detect gene replacement for pkaC (top panels), a 1·6 kbp SstI/BglII pkaR fragment (probe RII; Fig. 1) to detect gene replacement for pkaR (middle panels) or a 0·8 kbp BglII/XhoI pkaR fragment (probe RI; Fig. 1) to show deletion of part of the pkaR gene (lower panels). Molecular size markers are indicated on the left.

 
To construct a strain in which both pkaR and pkaC were disrupted, two consecutive mitotic recombination steps were performed. Linkage analysis on the resulting recombinants allowed chromosomal localization of pkaC and pkaR. In the first step, strain NW258 (pyrA6, leuA1{Delta}pkaC::pyrA nicA1) was recombined with strain NW274 (fwnA24 {Delta}argB::pyrA goxC17 bioA1 prtF28) (Table 1). From NW258//NW274, 97 descendants were randomly picked and analysed. Considering {Delta}pkaC ({Delta}C) morphology (see below) and uridine and leucine requirement we found 21 descendants which were {Delta}pkaC leuA1, 1 pyrA6 leuA1, 15 pyrA6 and 60 pkaC+ leuA+ pyrA+ strains. Since only one leuA1 pkaC+ recombinant was found, linkage of pkaC with leuA1 is 99% and pkaC is therefore located on chromosome IV. Subsequently, one of the {Delta}pkaC::pyrA descendants (NW276) was recombined with NW275 ({Delta}argB::pyrA pyrA6 leuA1 nicA1 {Delta}pkaR::argB). The diploid of NW275//NW276 showed segregation of all three morphology types (see below) on benomyl plates. Many wild-type recombinants were obtained, some recombinants showing {Delta}C morphology and a few recombinants with {Delta}pkaR ({Delta}R) morphology. Since it was rather difficult to isolate and characterize {Delta}R recombinants, we purified 39 recombinants with {Delta}C morphology and analysed these in more detail, in particular for the presence of the pkaR disruption. In this case the relation between phenotype and genotype was less obvious (Table 2). All recombinants carry the argB disruption on chromosome I, but only some have the pkaR disruption with the intact argB gene. The perfect linkage between arg+ and nicA1 proves that pkaR is located on chromosome V. All the recombinants with {Delta}C morphology are expected to contain the pkaC disruption. This can be derived from their pyr+ phenotype. Although all recombinants carry the pyrA6 mutation, they can be pyr+ due to either one of the two disruptions carrying pyrA; argB::pyrA linked to black spore colour and pkaC::pyrA. All 26 fawn recombinants were pyr+, proving the presence of the pkaC disruption. Assuming that black recombinants with {Delta}C morphology also carried the pkaC disruption, we selected one black double mutant, NW277, which was used for further experiments. The presence of the pkaC and pkaR disruptions in NW277 was confirmed by Southern analysis (Fig. 3).


View this table:
[in this window]
[in a new window]
 
Table 2. Phenotype, frequency and genotype of descendants from recombinant NW275 ({Delta}R)//NW276 ({Delta}C)

 
Expression of pkaC and pkaR and determination of PKA activity in A. niger wild-type and various transformants
The level of expression of pkaR and pkaC in various transformants was determined in mycelium grown on 1% glucose medium for 24 h and subsequently incubated for 1 h in medium with either 1 or 15% glucose. The high glucose concentration, 15%, was used since it has been shown that pkaC expression and PKA activity change during growth at high sugar concentration (Benina & Legia, 2000 ). Our results, however, show that the levels of pkaC- and pkaR-specific mRNA were similar under these two conditions (Fig. 2), indicating that incubation of mycelium at high sugar concentration did not affect the level of expression of pkaC and pkaR.

Specific PKA activities were measured to correlate it to modifications in the copy number of pkaC and pkaR. PKA activity was significantly higher in transformants with multiple copies of both pkaR and pkaC (Fig. 2). The activity correlated qualitatively to copy number and mRNA level (Fig. 2), e.g. transformant 44mcRC had about 10 copies of both genes and 0·6 mU mg-1 (wild-type activity was 0·1 mU mg-1). In transformants with an increased copy number of only pkaR, PKA activity remained at the wild-type level (Fig. 2). PKA activity was measured in one pkaR disruption strain, 9{Delta}R, and was found to be similar to wild-type activity (0·175 mU mg-1). Finally, in the pkaC disruptant PKA activity was below the detection level (Fig. 2).

Growth and morphology of A. niger strains modified in pkaR and pkaC
The role of PKA activity in morphology and hyphal development was monitored by observing the phenotypic features of individual transformants growing on solid medium and in liquid cultures. By comparing growth of wild-type, various multicopy transformants and the different disruptants on solid medium, three morphological classes were identified on the basis of colony size and the ability to form spores (Fig. 4). Strains in the first group were similar to wild-type and included a pkaR multicopy transformant (38mcR), a pkaC multicopy transformant (13mcC with a three- to fourfold increased PKA-C level) and a strain with an increased copy number of both genes (44mcCR). The second morphological class included strains with reduced or abolished PKA activity. Transformants in which pkaC ({Delta}C) or both pkaC and pkaR ({Delta}R{Delta}C) were disrupted formed colonies with a two- to threefold smaller diameter than the wild-type (Fig. 4). A pkaR disruptant (9{Delta}R) formed a third morphological class and developed colonies of only about half the size of wild-type and no sporulation could be observed after 3 days growth on agar plates.



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 4. Morphological characteristics of A. niger wild-type and pka transformants and mutants. (a) Colony morphology and size of conidiospores. Pictures and spore sizes are from 3-day-old colonies grown on solid minimal medium with 2% glucose at 30 °C. Spore diameter is given with standard deviation in parentheses (n>=10). (b) Hyphal morphology during submerged growth. Cells were grown for 24 h in minimal medium containing 2% glucose at 30 °C. Bar, 10 µm. Strain designations: wt, wild-type strain A395; 44mcRC, strain with multiple copies of pkaR and pkaC; 38mcR, multicopy pkaR strain; 13mcC, multicopy pkaC strain; 9{Delta}R, pkaR disruptant NW275; {Delta}R{Delta}C, pkaR pkaC double disruptant NW277; {Delta}C, pkaC disruptant NW258; 9{Delta}Rsp pseudorevertant of strain 9{Delta}R.

 
No major changes in hyphal morphology could be observed for most of the strains after 24 h submerged growth in minimal medium (data not shown). Exceptions were strains with increased pkaC copy number or disruption of pkaR. Young mycelium of a pkaC multicopy transformant (13mcC) exhibited hyphae with a 30% increased diameter (Fig. 4), but in a later stage (typically after 28 h growth) newly formed hyphae were similar to the wild-type. To study the morphology of the pkaR disruptant (9{Delta}R) in submerged cultures, peripheral hyphae from a surface-growing colony were taken as an inoculum, since no spores were available. After 24 h incubation remarkable morphological changes were observed. There was a distinct change from polar to apolar growth and enlarged bulbous cells were formed on the tips of filamentous hyphae (Fig. 4).

Germination kinetics were similar for the wild-type strain and strains overexpressing pkaR or both pkaR and pkaC (Fig. 5). Germination of conidiospores from the pkaC disruption strain ({Delta}C) and the pkaC pkaR double disruptant ({Delta}R{Delta}C) was slightly delayed (30 min to 1 h), but conidia from strain 13mcC, overproducing PKA-C , germinated considerably slower than the wild-type strain (1 to 2 h). Like the results obtained for colony morphology, strains {Delta}C and {Delta}R{Delta}C behaved similarly in germination experiments, indicating that disruption of pkaC was epistatic to deletion of pkaR.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5. Germination kinetics of A. niger wild-type, pkaC and pkaR multicopy transformants and {Delta}pkaC mutants. Germination was monitored as described in Methods using minimal medium containing 2% (w/v) glucose at 30 °C. The following strains were analysed: {circ}, wild-type strain A395; {blacksquare}, 44mcRC, a strain with multiple copies of pkaR and pkaC; {bullet}, 38mcR, multicopy pkaR strain; {square}, 13mcC, multicopy pkaC strain; {triangleup}, {Delta}C, pkaC disruptant NW258; {blacktriangleup}, {Delta}R{Delta}C, pkaR pkaC double disruptant NW277. Results are means from three independent determinations; bars represent SD.

 
Suppression of the pkaR mutant phenotype
During propagation of strain 9{Delta}R on solid medium, sectors appeared in the colonies exhibiting altered morphology and growth rate. One such strain was designated 9{Delta}Rsp (Fig. 4). Interestingly, the morphology of this strain on solid medium resembled that of the strain lacking pkaC ({Delta}C), i.e. compact colonies (Fig. 4). During submerged growth in minimal medium, bulbous cells, typical for strain 9{Delta}R, were not observed. Instead morphology was very similar to wild-type and the {Delta}C transformant. The morphological resemblance between the 9{Delta}Rsp and {Delta}C strains suggested that, due to a suppressor mutation in strain 9{Delta}R, pkaC was inactivated or transcription of pkaC was decreased. By RT-PCR, the pkaC mRNA level in strain 9{Delta}R was found to be 103±24% (n=3) of the wild-type level, while in strain 9{Delta}Rsp pkaC expression was only 40±9% (n=3). Moreover, the specific activity of PKA in strain 9{Delta}Rsp was below the detection level (Fig. 2). The absence of PKA activity may indicate a mutation in the pkaC coding region, resulting in an inactive protein and perhaps reduced mRNA stability, but this has to be analysed further.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this paper we describe cloning of the Aspergillus niger pkaR gene, encoding PKA-R. The following data imply that we cloned pkaR. First, the deduced PKA-R amino acid sequence is very similar to PKA-R proteins from related fungi, in particular those of A. nidulans, N. crassa and Mag. grisea (Bruno et al., 1996 ; Adachi & Hamer, 1998 ). Second, three putative domains involved in PKA-R function are present in the sequence: a dimerization domain, a site for interaction with PKA-C containing an autophosphorylation site (Ser-120) (Kreegipuu et al., 1999 ) and two cAMP binding sites. Given the similarity to other PKA-R proteins and the structural features of the protein, A. niger PKA-R most likely belongs to type II PKA regulatory subunits. The structure of the A. niger PKA holoenzyme is most likely tetrameric (Taylor et al., 1992 ).

The presence of an intron within the 5' non-coding region of A. niger pkaR has also been reported for the PKA-R genes of B. emersonii (Marques & Gomes, 1992 ), N. crassa (Bruno et al., 1996 ) and Mag. grisea (Adachi & Hamer, 1998 ). Similar to intron I of B. emersonii, the A. niger pkaR intron (700 bp) is unusually large compared to the normal size (50–100 bp) of fungal introns (Unkles, 1992 ). Its role in the promoter region of pkaR is not clear, but it might play a role in the expression of the gene, as reported for rat cytochrome c (Evans & Scarpulla, 1988 ). Analysis of the A. niger pkaR promoter region revealed no typical GC sites or binding sites for transcription factor Sp1 as could be found in the promoter of A. niger pkaC (Benina et al., 1997 ) and the pkaR promoter of B. emersonii (Marques & Gomes, 1992 ). However, the promoter regions of the pkaC and pkaR genes of A. niger bear some similarities which are indicative of common transcription control mechanisms. Both promoters contain stress response elements (C4T) and E-motifs. Such elements have been found in the multistress response genes of Sac. cerevisiae (Treger et al., 1998 ).

To analyse the involvement of PKA in morphogenesis, growth and morphology of strains with increased pkaC and/or pkaR expression as well as strains lacking PKA-C and/or PKA-R were examined on solid and in liquid medium. Strains overexpressing pkaR or both pkaC and pkaR showed higher mRNA levels as expected, but the strains could otherwise not be distinguished from the wild-type, suggesting that regulation of PKA-C activity is normal in these strains. With simultaneous overexpression of pkaC and pkaR, one might expect a relatively high PKA-C activity upon binding of cAMP to PKA-R and release of PKA-C, but this does not appear to interfere with regulation of processes which determine morphology. These data suggest that an increased level of PKA-C is not harmful to the cells as long as its activity is properly regulated by PKA-R.

A three- to fourfold increase in the PKA-C level in pkaC transformant 13mcC did not affect surface growth, but in submerged cultures, young mycelium of this transformant had thicker hyphae. In a later stage, however, newly formed hyphae were like wild-type. Apparently, overproduction of PKA-C only affects morphology at an early growth stage. This observation agrees with the finding that pkaC transcription in A. niger significantly decreased between 19 and 28 h after inoculation (Benina & Legia, 2000 ). Thus, if overproduction of PKA-C results in increased hyphal diameter, a decrease in pkaC transcription might get the PKA-C/PKA-R balance back to normal, resulting in proper regulation of PKA activity and consequently in a normal hyphal diameter. Alternative mechanisms to control PKA-C level or activity in pkaC multicopy transformants may be at the translational level or by inducing the synthesis of PKA-R, but we have no proof that such mechanisms work in A. niger.

A pkaC disruption strain lacked PKA activity and developed smaller colonies than the reference strain on plates. Germination of {Delta}C conidiospores was slightly delayed, but hyphal morphology in liquid culture and sporulation was normal. As expected, silencing of pkaR, in addition to pkaC, did not change the phenotype. Clearly, PKA activity is required for optimal growth and metabolism.

Disruption of pkaR gave the most dramatic phenotype, i.e. very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Compact colony morphology and inhibition of conidiation was also observed for a wild-type strain grown in the presence of 5 mM cAMP (Benina et al., 1997 ). The morphological and developmental effects of both disruption of pkaR and continuous presence of cAMP are presumably due to unrestrained PKA activity. We did not, however, provide actual evidence that PKA activity is not regulated at all in the {Delta}pkaR strain. We cannot, for example, exclude the possibility that A. niger possesses another PKA-R-type protein that may, at least partially, control PKA activity.

Loss of growth polarity and absence of sporulation has also been described for the N. crassa mcb mutant, which is deficient in PKA-R (Bruno et al., 1996 ). Likewise, a key role for cAMP and PKA is reported for morphogenesis in dimorphic fungi (Borges-Walmsley & Walmsley, 2000 ). For example, in U. maydis disruption of ubc1, encoding PKA-R, caused a multiple budding phenotype (Gold et al., 1994 ). Thus, elevated or unrestrained PKA activity gives a budding growth in dimorphic fungi and loss of growth polarity in filamentous fungi. Filamentous fungi have a short period of isotropic growth during spore germination and PKA appears to be important for the switch of isotropic to polarized growth (d’Enfert, 1997 ). Interestingly, the A. niger {Delta}pkaR strain is able to form hyphae during growth on plate, although to a very limited extent. Surface growth may trigger a signal transduction pathway, also involved in hyphal extension, which is to some extent independent of the PKA pathway.

In summary, disruption of pkaR interferes with proper growth and development of A. niger. Our data show that cAMP-dependent protein phosphorylation in A. niger is involved in developmental processes such as growth polarity in germinating conidia and asexual reproduction, i.e. formation of conidiospores. The availability of a {Delta}pkaR mutant will hopefully enable a study aiming to identify the PKA targets directly involved in growth and morphology.


   ACKNOWLEDGEMENTS
 
We acknowledge the Ministry of Education, Science and Sport from the Republic of Slovenia for financial support of M. taudohar and M. Benina. We are grateful to Drs Ralph Dean and Christophe d’Enfert for providing the pkaR genes from Mag. grisea and A. nidulans respectively.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adachi, K. & Hamer, J. E. (1998). Divergent cAMP signalling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea. Plant Cell 10, 1361-1373.[Abstract/Free Full Text]

Benina, M. & Legia, M. (1999). Non-radioactive multiple reverse transcription-PCR method used for low abundance mRNA quantification. Biotechnol Tech 13, 865-869.

Benina, M. & Legia, M. (2000). Stress mediated changes in expression of the pkaC gene, encoding the catalytic subunit of cAMP-dependent protein kinase, in Aspergillus niger. Pflueg Arch 439, R76-R78.[Medline]

Benina, M., Panneman, H., Ruijter, G. J. G., Legia, M. & Visser, J. (1997). Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit. Microbiology 143, 1211-1220.[Abstract]

Borges-Walmsley, M. I. & Walmsley, A. R. (2000). cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity. Trends Microbiol 8, 133-141.[Medline]

Bos, C. J., Debets, A. J. M., Swart, K., Huybers, A., Kobus, G. & Slakhorst, S. M. (1988). Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 14, 437-443.[Medline]

Boy-Marcotte, E., Perrot, M., Bussereau, F., Boucherie, H. & Jacquet, M. (1998). Msn2p and Msn4p control large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae. J Bacteriol 180, 1044-1052.[Abstract/Free Full Text]

Bruno, K. S., Aramayo, R., Minke, P. F., Metzenberg, R. L. & Plamann, M. (1996). Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase. EMBO J 15, 5772-5782.[Abstract]

Carlson, G. L. & Nelson, D. L. (1996). The 44-kDa regulatory subunit of the Paramecium cAMP-dependent protein kinase lacks a dimerization domain and may have a unique autophosphorylation site sequence. J Eukaryot Microbiol 43, 347-356.[Medline]

d’Enfert, C. (1997). Fungal spore germination: insights from the molecular genetics of Aspergillus nidulans and Neurospora crassa. Fungal Genet Biol 21, 163-172.

d’Enfert, C., Bonini, B. M., Zapella, P. D., Fontaine, T., da Silva, A. M. & Terenzi, H. F. (1999). Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa. Mol Microbiol 32, 471-483.[Medline]

Daniel, P. B., Walker, W. H. & Habener, J. F. (1998). Cyclic AMP signalling and gene regulation. Annu Rev Nutr 18, 353-383.[Medline]

de Graaff, L., van den Broeck, H. & Visser, J. (1988). Isolation and expression of the Aspergillus nidulans pyruvate kinase gene. Curr Genet 13, 315-321.[Medline]

de Oliveira, J. C. F., Borges, A. C. C., Marques, M. V. & Gomes, S. L. (1997). Cloning and characterisation of the gene for the catalytic subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Eur J Biochem 219, 555-562.[Abstract]

de Voti, J., Seydoux, G., Beach, D. & McLeod, M. (1991). Interaction between ran1+ protein kinase and cAMP dependent protein kinase as negative regulators of fission yeast meiosis. EMBO J 10, 3759-3768.[Abstract]

Evans, M. J. & Scarpulla, R. C. (1988). Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells. Mol Cell Biol 8, 35-41.[Medline]

Fernandez, M., Cao, J. & Villamarin, J. A. (1998). In vivo phosphorylation of phosphofructokinase from the bivalve mollusc Mytilus galloprovincialis. Arch Biochem Biophys 353, 251-256.[Medline]

Frederick, G. D., Rombouts, P. & Buxton, F. P. (1993). Cloning and characterisation of pepC, a gene encoding a serine protease from Aspergillus niger. Gene 125, 57-64.[Medline]

Gancedo, J. M., Mazon, M. J. & Eraso, P. (1985). Biological role of cyclic AMP: similarities and differences between organisms. Trends Biochem Sci 10, 210-212.

Gold, S., Duncan, G., Barrett, K. & Kronstad, J. (1994). cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Gen Dev 8, 2805-2816.[Abstract]

Goncalves, P. M., Griffioen, G., Bebelman, J. P. & Planta, R. J. (1997). Signalling pathway leading to transcriptional regulation of genes involved in the activation of glycolysis in yeast. Mol Microbiol 25, 483-493.[Medline]

Goosen, T., Bloemheuvel, G., Gysler, C., de Bie, D. A., van den Broek, H. W. & Swart, K. (1987). Transformation of Aspergillus niger using the homologous orotidine-5'-phosphate-decarboxylase gene. Curr Genet 11, 499-503.[Medline]

Gradinik-Grapulin, M. & Legia, M. (1997). A spontaneous change in the intracellular cyclic AMP level in Aspergillus niger is influenced by the sucrose concentration in the medium and by light. Appl Environ Microbiol 63, 2844-2849.[Abstract]

Jiang, Y., Davis, C. & Broach, J. R. (1998). Efficient transition to growth on fermentable carbon source in Saccharomyces cerevisiae requires signalling through the Ras pathway. EMBO J 17, 6942-6951.[Abstract/Free Full Text]

Johnstone, I. L., Hughes, J. & Clutterbuck, A. J. (1985). Cloning an Aspergillus nidulans developmental gene by transformation. EMBO J 4, 1307-1311.[Abstract]

Kreegipuu, A., Blom, N. & Brunak, S. (1999). PhosphoBase, a database of phosphorylation sites: release 2.0. Nucleic Acids Res 7, 237-239.

Kronstad, J., de Maria, A., Funnell, D., Laidlaw, R. D., Lee, N., de Sa, M. M. & Ramesh, M. (1998). Signalling via cAMP in fungi: interconnections with mitogen-activated protein kinase pathway. Arch Microbiol 170, 395-404.[Medline]

Kunisawa, R., Davis, T. N., Urdea, M. S. & Thorner, J. (1987). Complete nucleotide sequence of the gene encoding the regulatory subunit of 3', 5'-cyclic AMP-dependent protein kinase from the yeast Saccharomyces cerevisiae. Nucleic Acids Res 15, 368-369.[Medline]

Kusters-van Someren, M. A., Harmsen, J. A. M., Kester, H. C. M. & Visser, J. (1991). Structure of the Aspergillus niger pelA gene and its expression in the Aspergillus niger and Aspergillus nidulans. Curr Genet 20, 293-299.[Medline]

Legia, M. & Benina, M. (1994). Evidence for the activation of 6-phosphofructo-1-kinase by cAMP-dependent protein kinase in Aspergillus niger. FEMS Microbiol Lett 118, 327-334.[Medline]

Legia, M. & Gradinik-Grapulin, M. (1995). Sudden substrate dilution induces a higher rate of citric acid production by Aspergillus niger. Appl Environ Microbiol 61, 2732-2737.[Abstract]

Legia, M., Cimerman, A. & Sterle, M. (1981). Germination of Aspergillus niger in a high citric acid yielding medium. FEMS Microbiol Lett 11, 149-152.

Leon, D. A., Herberg, F. W., Banky, P. & Taylor, S. S. (1997). A stable alpha-helical domain at the N terminus of the RIalpha subunits of cAMP-dependent protein kinase is a novel dimerization/docking motif. J Biol Chem 272, 28431-28437.[Abstract/Free Full Text]

Maeda, T., Watanabe, Y., Kunitomo, H. & Yamamoto, M. (1994). Cloning of pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe. J Biol Chem 269, 9632-9637.[Abstract/Free Full Text]

Marques, M. V. & Gomes, L. S. (1992). Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii. J Biol Chem 267, 17201-17207.[Abstract/Free Full Text]

Mitchell, T. K. & Dean, R. A. (1995). The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. Plant Cell 7, 1869-1878.[Abstract/Free Full Text]

Murre, R., Schonheber, P. & Baltimore, D. (1989). A new DNA-binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD and myc proteins. Cell 56, 777-783.[Medline]

Mutzel, R., Lacombe, M.-L., Simon, M.-N., de Gunzburg, J. & Veron, M. (1987). Cloning and cDNA sequence of the regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum. Proc Natl Acad Sci USA 84, 6-10.[Abstract]

Niimi, M. (1996). Dibutyryl cyclic AMP-enhanced germ tube formation in exponentially growing Candida albicans cells. Fungal Genet Biol 20, 79-83.[Medline]

Orlowski, M. (1991). Mucor dimorphism. Microbiol Rev 55, 234-258.

Pontecorvo, G., Roper, J. A., Hemmons, L. J., MacDonald, K. D. & Bufton, A. W. J. (1953). The genetics of Aspergillus nidulans. Adv Genet 5, 141-238.

Quandt, K., Frech, K., Karas, H., Wingender, E. & Werner, T. (1995). MarInd and MatInspector-New fast and versatile tools for determination of consensus matches in nucleotide sequence data. Nucleic Acids Res 23, 4878-4884.[Abstract]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]

Su, Y., Dostmann, W. R., Herberg, F. W., Durick, K., Xuong, N. H., Ten Eyck, L., Taylor, S. S. & Varughese, K. I. (1995). Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains. Science 269, 807-813.[Medline]

Taylor, S. S., Knighton, D. R., Zheng, J., Eyck, L. F. T. & Sowadski, J. M. (1992). Structural framework for the protein kinase family. Annu Rev Cell Biol 8, 429-462.

Treger, J. M., Magee, T. R. & McEntee, K. (1998). Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae. Biochem Biophys Res Commun 243, 13-19.[Medline]

Unkles, S. E. (1992). Gene organisation in industrial filamentous fungi. In Applied Molecular Genetics of Filamentous Fungi , pp. 28-53. Edited by J. R. Kinghorn & G. Turner. London:Blackie.

Van Diepeningen, A. D., Debets, A. J. M. & Hoekstra, R. F. (1998). Intra- and interspecies virus transfer in aspergilli via protoplast fusion. Fungal Genet Biol 25, 171-180.[Medline]

Visniac, W. & Santer, M. (1957). The thiobacili. Bacteriol Rev 21, 195-213.

Zahringer, H., Holzer, H. & Nwaka, S. (1998). Stability of neutral trehalase during heat stress in Saccharomyces cerevisiae is dependent on the activity of the catalytic subunit of cAMP-dependent protein kinase, Tpk1 and Tpk2. Eur J Biochem 255, 544-551.[Abstract]

Received 28 March 2002; accepted 24 April 2002.