1 Center for Process Biotechnology, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark
2 Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria Pabellon 2 Piso 4, 1428 Buenos Aires, Argentina
3 Department of Fungal Biotechnology, Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark
Correspondence
José Arnau
jar{at}bioteknologisk.dk
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ABSTRACT |
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These authors made equal contributions to this work.
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INTRODUCTION |
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Dimorphism in fungi is also relevant in the field of fungal virulence (Lengeler et al., 2000). Studies in different fungi, including ascomycetes and basidiomycetes, have converged to define two broadly conserved signal transduction cascades that regulate fungal development and virulence. One is a mitogen-activated protein (MAP) kinase cascade that mediates responses to pheromone. The second is a nutrient-sensing receptor-mediated cAMP protein kinase A (PKA) cascade. These pathways function co-ordinately to regulate mating, filamentation and virulence. Particular attention has been given to the cAMP signal transduction pathway and fungal morphology (Kronstad et al., 1998
; D'Souza & Heitman, 2001
). This second messenger regulates the activity of PKA by binding to the regulatory subunit (PKAR) and releasing the catalytic subunits (PKAC) from the tetrameric inactive holoenzyme to initiate a phosphorylation cascade (Taylor et al., 1992
).
Mucor circinelloides is a dimorphic zygomycete that displays multipolar yeast or filamentous morphology in response to a number of environmental conditions, including the gas atmosphere and the level of nutrients (Orlowski, 1991; McIntyre et al., 2002
; Wolff et al., 2002
). Typically, the yeast morphology is favoured during anaerobic growth in the presence of a fermentable carbon source, while the filamentous form is triggered in the presence of oxygen or upon nutritional challenge. The participation of cAMP in the morphogenetic process of Mucor has been shown for both Mucor rouxii and Mucor racemosus (Orlowski, 1991
) and analysed further particularly for M. rouxii (Pereyra et al., 1992
, 2000
). However, no molecular genetic approach had been attempted until recently with this species in order to analyse the role of PKA in Mucor morphology.
The role of PKA in the control of morphology and hyphal branching has been shown recently for Aspergillus niger (Saudohar et al., 2002) using genetic approaches. While a null mutation in the A. niger pkaR gene led to a small colony phenotype and lack of conidiation, an increase in pkaR expression did not result in any measurable phenotypic difference (Saudohar et al., 2002
). The pkaR and pkaC genes of M. circinelloides have been cloned recently. Expression of pkaR was observed during anaerobic yeast growth and during the shift from yeast to filamentous growth. In addition, overexpression of pkaR resulted in an increase in pkaC expression during anaerobic growth. A constructed strain, KFA121, overexpressing the pkaR gene from the promoter of the gpd1 gene (gpd1P) showed a multibranching colony phenotype during growth on plates (Wolff et al., 2002
). Here, we confirm that the multibranching phenotype in strain KFA121 is a consequence of the overexpression of PKAR and present a characterization of the biochemical and morphological properties associated with this phenotype. A role for PKA in the control of branching in M. circinelloides is presented.
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METHODS |
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During the morphological analysis in the flow-through cell, all strains were cultivated in liquid YNB medium [1·5 g (NH4)2SO4 l-1, 1·5 g glutamic acid l-1, 0·5 g yeast nitrogen base l-1] adjusted to pH 4·5 with 1 M H2SO4 and supplemented with 1 mg thiamine chloride l-1 and 1 mg niacin amide l-1. The nature and concentration of the carbon source were varied as indicated. Cultures were grown at 28 °C in either YNB or rich medium (YPG; yeast, peptone, glucose) as indicated for PKA measurements. Glucose concentration was variable as indicated.
Flow-through cell experiments and image analysis.
Spores were immobilized in a temperature-controlled flow-through cell mounted on a motorized stage with continuous medium flow, to assess fungal growth and branching in a quantitative way. The construction and set-up of the flow-through cell has been described in detail previously (Spohr et al., 1998). The cell consisted of two microscope slides (26x76 mm) separated by a Parafilm M spacer clamped into a steel frame equipped with tubes for feed addition and waste withdrawal. To sterilize both the cell and the tubing, 70 % ethanol was injected through a filter (0·45 µm); this was followed by addition of distilled water after 20 min to remove the ethanol. One millilitre of 0·1 % poly-D-lysine (Sigma) was then filtered into the cell to mediate spore fixation. The cell was inoculated with spores to gain a final number of approximately 20 spores. Medium was continuously added and removed from the cell at a flow rate of 3 ml h-1.
All experiments were carried out at 28 °C. Images of the hyphal elements were obtained at time intervals of 15 min on a Nikon Optiphot 2 microscope equipped with a CCD camera (Bischke CCD-5230P) connected to an image analysis system (QUANTIMET 600S; Leica Cambridge). Automatic image analysis was applied in the detection of hyphal elements and measurements of hyphal length (Spohr et al., 1998). The hyphal tips were counted manually. These values were used to calculate the hyphal growth unit length (HGUL) (total hyphal length of a mycelium divided by its tip number, µm tips-1) (Caldwell & Trinci, 1973
).
Crude extract preparation for PKA studies.
The cells were harvested by centrifugation at 7000 g during 15 min and processed immediately. Crude extracts were prepared by vortexing the cells four times for 1 min at 4 °C with glass beads (460600 µm diameter) and buffer A (25 mM Tris pH 8, 5 mM EDTA, 3 mM EGTA, 10 mM 2-mercaptoethanol, supplemented with the indicated concentration of the Roche complete EDTA-free protease inhibitor cocktail). Protein concentration was determined by the method of Bradford (1976).
cAMP-binding assay.
Aliquots from crude extracts, containing 510 µg protein, were incubated for 30 min at 30 °C or overnight at 4 °C in a final volume of 70 µl with 0·3 µM [3H]cAMP (62 000 d.p.m. pmol-1) in 0·5 M NaCl in buffer A, filtered through nitrocellulose filters, washed with 30 ml water, dried and counted in liquid scintillation mixture.
PKA assay.
Aliquots from crude extracts, containing 0·050·5 µg protein, were incubated for 10 min at 30 °C in 70 µl of the following incubation mixture: 50 mM Tris/HCl pH 7·2, 15 mM MgCl2, 200 µM kemptide, 0·1 mM [-32P]ATP (specific activity 1300 c.p.m. pmol-1), and 10 µM cAMP, when added. Samples (50 µl) were loaded onto phosphocellulose paper squares and processed according to Roskoski (1983)
. As opposed to the cAMP-binding assay, which is linear to the protein concentration over a large range of concentrations and can be used directly with crude extracts, linearity is often compromised when measuring PKA activity (Sorol et al., 2001
). Low amounts of protein have to be used to enable quantification of PKA levels. In each case, conditions to attain a linear response of activity versus crude extract protein concentration have to be settled.
One unit of protein kinase activity is defined as picomoles of phosphate incorporated into kemptide per 10 minutes under the standard assay conditions.
Sucrose gradient centrifugation.
Crude extracts containing 3 mg protein were loaded onto 4·5 ml 520 % sucrose gradients in buffer A, and centrifuged for 15 h at 35 000 r.p.m. in a SW 55 Ti rotor of a Beckman ultracentrifuge. Fractions were collected from bottom to top, and in each fraction cAMP-binding and PKA activities were assayed by the standard assays indicated above. Catalase (11·3 S) and horseradish peroxidase (3·5 S) were used as sedimentation markers.
Western blot analysis.
Samples of crude extracts were analysed by 10 % SDS-PAGE and blotted onto nitrocellulose membranes using 25 mM Tris, 192 mM glycine, 20 % (v/v) methanol buffer in a Transphor apparatus. Blots were blocked with 5 % non-fat milk, 0·05 % Tween 20 in Tris-buffered saline. Membranes were incubated overnight at 4 °C with a 1 : 20 000 dilution of an antibody developed in rabbit against M. rouxii PKAR purified via cAMPagarose affinity chromatography (Rossi et al., 1992; V. González Polo, S. Rossi & S. Moreno, unpublished data). Secondary anti-rabbit IgG peroxidase-conjugated antibody was used at 1 : 10 000. After three washes the blots were developed with Chemiluminescence Luminol reagent, and immunoreactive bands were visualized by digital imaging. A duplicate gel was stained with Coomassie brilliant blue to estimate protein loading.
Kemptide and anti-rabbit IgG peroxidase-conjugated antibody were from Sigma Chemical. [-32P]ATP and [3H]cAMP were from Perkin Elmer Life Sciences. Phosphocellulose paper was from Whatman. Nitrocellulose membrane ECL Western Blotting Detection Reagent and Roche complete EDTA-free protease inhibitor cocktail tablets were from Amersham Biosciences.
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RESULTS |
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For each hyphal element, the total hyphal length (the length of the skeletonized mycelium after image processing) and the number of branches were determined (Fig. 1). Dividing the total hyphal length of a mycelium by its tip number defines HGUL (Caldwell & Trinci, 1973
). HGUL constitutes a useful parameter for comparing branching characteristics between different strains or for a particular strain cultivated under varying conditions. It attains a fairly constant and thus characteristic value during exponential growth, when new branches are initiated at a constant rate. The specific growth rate (µ) was obtained from a semi-logarithmic plot of the hyphal length versus time, where the slope of the resulting curve is equivalent to µ. Values of HGUL and µ for KFA121 and ATCC 1216b for the different media tested are presented in Fig. 2
. Each data point constitutes a meaned result for 915 individual hyphal elements. For the reference strain ATCC 1216b, HGUL decreased with increasing glucose concentration, i.e. more branches were formed, reaching 70 and 43 % of the maximum HGUL (98 µm tips-1) in medium with a high glucose concentration (1 and 10 g l-1, respectively, Fig. 2a
). This is also coherent with the way fungi would grow in nature. In a nutrient-rich environment, energy is easily available so new branches can be initiated rapidly. However, if the nutritional state of the immediate surroundings is poor, growth without formation of excess branches is advantageous, as this would allow the maximum possible spatial extension, towards a potential new nutrient source. Comparing the branching frequencies of M. circinelloides ATCC 1216b during growth with different carbon sources provided at the same concentration showed that mycelia were more densely branched during growth with glucose than with xylose. The transformant strain KFA121, on the other hand, displayed a branching pattern different from that of ATCC 1216b. At elevated glucose concentrations (1 and 10 g l-1), the relative reduction in HGUL compared to the values at low glucose concentration (130 µm tips-1) was about fivefold (Fig. 2a
). This means that KFA121 formed an increased number of branches in high glucose where the gpd1P is induced, confirming the previous observations during growth on plates (Wolff et al., 2002
). During growth in the presence of xylose, KFA121 formed significantly fewer branches compared to growth with glucose at the same concentration, reaching similar values to growth on low glucose (Fig. 2a
). No significant differences were observed in µ for ATCC 1216b and KFA121 in each growth condition (Fig. 2b
). The highest values were obtained in 0·2 g glucose l-1 (0·5 µ) and were similar during growth in 1 and 10 g glucose l-1 (around 0·4 µ). These results established that the differences in HGUL were not due to differences in µ (Fig. 2
).
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To address whether the observed phenotype in strain KFA121 resulted from an up-regulation or a down-regulation of PKA, it was important to establish the ratio between PKAR and PKAC subunits and also if there was free PKAR in excess of the PKAR associated with PKAC as a holoenzyme. The measurement of activities in crude extracts does not provide an accurate measurement since both subunits seem to increase in the KFA121 strain, and reproducibly the increase in PKAR in each independent experiment is around 1·5-fold greater than the increase in PKAC. To have an independent measure, crude extracts from strains KFA89 and KFA121 grown for 7·5 h were ultracentrifuged on 520 % sucrose gradients to separate the species according to their sedimentation coefficients. The phosphorylating activity of PKAC and the cAMP-binding activity of PKAR were measured in fractions derived from the gradient (Fig. 5). The results showed unambiguously that, in strain KFA121, there was an excess of free PKAR at a lower sedimentation coefficient than the holoenzyme, i.e. not associated with PKAC activity. A relative increase in PKAR and PKAC activities could be deduced from the areas under the gradient peaks, indicating an overall increase of almost threefold in cAMP-binding activity, when comparing KFA121 (Fig. 5b
) to KFA89 (Fig. 5a
) extracts. For PKAC activity, the estimated increase was only around 1·5-fold. The PKAR-to-PKAC ratio within the holoenzyme peak in both gradients was maintained, and the excess of PKAR activity in KFA121 extracts was detected in fractions free from PKAC activity and with a much lower sedimentation coefficient than the holoenzyme. The observed increase in PKAC in strain KFA121 was consistent with the previously reported increase in pkaC expression in this strain (Wolff et al., 2002
).
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DISCUSSION |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 August 2003;
revised 24 September 2003;
accepted 2 October 2003.
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