School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK1
Centre for Phytotechnology, Institute for Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 Al Leiden, The Netherlands2
Department of Genetics and Microbiology, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK3
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, Utrechtseweg 48, PO Box 360, 3700 AJ Zeist, The Netherlands4
Department of Cell Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK5
Author for correspondence: Geoffrey D. Robson. Tel: +44 161 275 5048. Fax: +44 161 275 5656. e-mail: Geoff.Robson{at}man.ac.uk
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ABSTRACT |
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Keywords: Aspergillus niger, protein secretion, glucoamylase, green fluorescent protein (GFP), heterologous protein production
Abbreviations: ER, endoplasmic reticulum; GFP, green fluorescent protein; sGFP, synthetic GFP(S65T); GLA, glucoamylase
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INTRODUCTION |
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In filamentous fungi, protein secretion is thought to occur at growing hyphal apices where released proteins can pass by bulk flow through the newly synthesized cell wall (Wessels, 1994 ). Wösten et al. (1991)
demonstrated indirectly that secretion was coupled to hyphal growth and that glucoamylase appeared to be secreted primarily at the apices of Aspergillus niger hyphae, although subapical localization of glucoamylase in A. niger (A. Keizer-Gunnink, personal communication) and lignin peroxidase in Phanerochaete chrysosporium (Moukha et al., 1993
) has also been reported.
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been used as a reporter for monitoring gene expression and protein localization studies in many organisms (Chalfie, 1995 ). Red-shifted variants of GFP have been developed, in which the excitation maximum peak is shifted to 490 nm, resulting in much brighter fluorescence than the wild-type (Heim et al., 1995
). A synthetic version of one of these variants, S65T (sGFP), was developed for use in plants (Chiu et al., 1996
; Haas et al., 1996
) and has been expressed successfully in a number of fungi, including Candida albicans, Aspergillus nidulans, Aureobasidium pullulans and Ustilago maydis, (Spellig et al., 1996
; Cormack et al., 1997
; Suelmann et al., 1997
; van den Wymelenberg et al., 1997
; Fernández-Ábalos et al., 1998
; Siedenberg et al., 1999
).
In this study, we describe the expression of a glucoamylase (GLA)::sGFP (GLA::sGFP) fusion protein in A. niger as an in vivo reporter of protein secretion in growing hyphae. The results demonstrate the use of GLA::sGFP fusion proteins as in vivo reporters of protein trafficking and secretion in A. niger and as a tool in the isolation of mutants in the general secretory pathway.
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METHODS |
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Growth and culture conditions.
A. niger cytoplasmic sGFP and GLA514 fusion strains were grown on BenneH/Lasure media, whereas the GLA499 fusion strains were grown on Vogels medium (Vogel, 1956 ) (modified by substitution of 10 g maltodextrin or glucose l-1 for sucrose), solidified with 1·5% (w/v) agar when required. For microscopy, spores of the cytoplasmic sGFP and GLA514 fusion strains were adhered to coverslips and grown under a thin liquid layer in a Petri dish (Harris et al., 1994
). A modified version of soya milk medium (MacKenzie et al., 1994
) was also used. Media for A. niger pyrG strains contained 10 mM uridine. Conidia for spore inocula were grown on potato dextrose agar (PDA), harvested after 56 d with 0·1% (w/v) Tween 20 and washed twice with distilled water. All cultures were grown at 30 °C and agitated at 250 r.p.m. when necessary.
Construction of gene fusions and transformant strains.
Molecular methods for plasmid isolation, restriction enzyme analysis, ligation of DNA fragments and transformation of Escherichia coli were essentially as described by Sambrook et al. (1989) . Constructs are shown in Fig. 1
. For the GLA499::sGFP-encoding construct, a full-length 742 bp sgfp fragment was generated by PCR using pMCB30 as a template. Primers used were (1) 5'-CGATATCTAGAATGGTGGCAAGGGCGAGGA, and (2) 5'-TGACTTCTAGATTACTTGTACAGCTCGTCCA. The sgfp fragments were digested and ligated into the XbaI cloning site of pIGF between the truncated glaA gene and the glaA terminator. The GLA514::sGFP-encoding gene fusion was generated by cloning sgfp as an NcoI (blunt)BamHI fragment from pBluescript-sGFP-TyG-nos-hs (Sheen et al., 1995
) between the EheI and BglII (partial) sites of pAN 56-2Not, resulting in the vector pAN56-2sGFP. For the GLA514::sGFP-HDEL-encoding gene fusion, pBluescript-sGFP-TyG-nos-hs (Sheen et al., 1995
) was used as the template. Primers used were (1) reverse primer on pBluescript 5'-GGAAACAGCTATGACCATG, and (2) sGFP-HDEL-reverse primer 5'-CGGGATCCTTACAGCTCGTCGTGCTTGTACAGCTCGTCCATGCC. The 882 base pair sGFP-HDEL fragment was digested with NcoI and BamHI and cloned into pAN56-2 to give pAN56-2(sGFP-HDEL). All constructs were verified by sequencing. The cytoplasmic sGFP was constructed as described by Siedenberg et al. (1999)
.
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Microscopy.
Fluorescence microscopy with the A. niger cytoplasmic sGFP and GLA514 fusion strains was conducted using a Zeiss axioplan microscope. Standard FITC filters were used. Fixation of mycelia for electron microscopy was carried out overnight at room temperature with 2·5% (v/v) glutaraldehyde in 0·05 M sodium cacodylate with 0·05% (v/v) Nonidet P40. Dehydration was achieved with an ethanol series with progressive cooling to -35 °C. Fungal tissue was embedded in LR white resin containing 0·05% (w/v) benzoin methyl ether in a low-temperature box and polymerization mediated by UV light at -20 °C. Sections, on gold grids, were blocked with 1% (w/v) acetylated BSA (Aurion BSA-C) with 0·01% (v/v) Tween 20 for 1 h. Grids were incubated in a 1:15 dilution of anti-sGFP antibodies (Clontech polyclonal anti-sGFP) overnight at 4 °C. After washing three times in PBS, sections were incubated in 10 nm gold-conjugated goat anti-rabbit antibody (Biocell) for 1 h at room temperature. Both primary and secondary antibodies were cross-absorbed using an acetone powder of A. niger at 10 mg ml-1 for 30 min just prior to use. After washing in PBS, sections were fixed in 1% glutaraldehyde in PBS for 15 min and washed four times in water before being stained with uranyl acetate/lead citrate and observed in a JEOL 1200EX electron microscope.
Analytical methods.
Growth was measured by biomass dry weight, with mycelia filtered through filter papers (Whatman no. 1) and dried to constant weight. Extracellular culture supernatant samples were filtered through a 0·2 µm membrane and stored at -20 °C. For relative fluorescence measurements in liquid culture, 1·5 ml samples were excited at 490 nm using a Hitachi 2000 fluorescence spectrophotometer with a 508 nm emission filter. Recombinant red-shifted GFP (Clontech) was used as a control. SDS-PAGE was conducted using standard protocols (Sambrook et al., 1989 ). Electroblotting of proteins onto nitrocellulose (Hybond-C; Amersham) was conducted as described by Towbin et al. (1979)
. Immunodetection of sGFP was achieved with a 1:500 dilution of rabbit anti-sGFP antibodies (Clontech), and of glucoamylase with a 1:5000 dilution of rabbit anti-glucoamylase antibodies (kindly provided by Novo Nordisk) using an anti-rabbit antibody peroxidase linked system (ECL; Amersham). To study proteolytic degradation of sGFP, 7 µg recombinant sGFP protein (Clontech) was incubated for up to 6 h with 140 µl culture filtrates from the end of the exponential phase of A. niger AB4.1 grown in soya milk or Vogels medium. Samples were taken at regular intervals and subjected to Western blotting.
Mutagenesis and screening for mutants in the general secretory pathway.
Aliquots (20 ml) of a spore suspension (1x107 spores ml-1) of A. niger N402 containing the GLA499::sGFP construct was subjected to UV mutagenesis (UVP; model R-52G) for 40 s, which killed between 95 and 99% of spores. Mutagenized spores were spread onto modified Vogels agar medium (containing 0·1% (w/v) glucose and 0·05% (v/v) Triton X-100) at a spore density sufficient to give rise to 3050 colonies after incubation for 3 d at 25 °C. Triton X-100 was included in the medium to restrict colony growth. Molten modified Vogels starch medium [containing 1% (w/v) corn starch] (10 ml) was poured carefully over the colonies and plates were incubated at 42 °C for 24 h. Previous studies had shown that non-mutagenized colonies would produce a visible halo in the starch-containing medium after between 6 and 14 h. Colonies which did not form a halo after 24 h were subcultured and rescreened on starch-containing medium at both 25 and 42 °C. Spore suspensions or mycelial plugs (4 mm diam.) of mutants from the secondary screen were used to inoculate Vogels gelatin medium [containing 1% glucose, 1% (w/v) gelatin, 0·05% Triton X-100 and lacking ammonium sulphate) and incubated at 25 and 42 °C for 4 and 2 d, respectively. Previous studies had shown that non-mutagenized colonies would produce a clearing zone around the colonies after 1824 and 614 h at 25 and 42 °C, respectively.
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RESULTS |
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DISCUSSION |
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In a series of gene fusions we replaced the starch-binding domain of glucoamylase with sGFP to create fluorescent markers for the study of the secretion process in A. niger. Three glucoamylase sGFP fusions were made. Two of them were designed for secretion of the fusion protein and employed two different lengths of the glucoamylase protein (GLA499 and GLA514), although both constructs lacked the starch-binding domain. This approach has been used successfully to secrete heterologous proteins in both cases. The third construct included a C-terminal HDEL motif designed to retain the fusion protein within the lumen of the ER. The different lengths of glucoamylase in the secretory fusions were found to have no significant effect as both GLA499::sGFP and GLA514::sGFP produced the same patterns of fluorescence in A. niger, and were distinct from that of the ER-retained GLA514::sGFP-HDEL and the cytoplasmic-sGFP-containing strains. In young mycelia expressing the GLA::sGFP fusion protein, bright fluorescence was observed in the hyphal walls indicating that the fusion protein was secreted, but retained within the cell wall. The presence of the fusion protein in the cell wall was confirmed by immuno-gold labelling. Retention of extracellular proteins in the hyphal wall has been reported previously for glucose oxidase in A. niger (Witteveen et al., 1992 ) and invertase in N. crassa (Trevithick & Metezenberg, 1966
) and for a variety of secreted proteins in S. cerevisiae (de Nobel & Barnett, 1991
). Using sGFP, we were able to directly observe in vivo the presence of the fusion protein within the cell wall, which would have been difficult based solely on Western analysis. We developed a simple extraction method that released the fusion protein from the cell wall. It is likely that in previous studies in which glucoamylasegene fusions have been used for heterologous protein secretion, cell wall localization was not recognized because the extraction method used did not discriminate between cell-wall-bound and intracellular proteins (Ward et al., 1990
; Broekhuijsen et al., 1993
; Archer et al., 1994
; Gouka et al., 1997b
).
The more intense fluorescence observed at hyphal apices supports the hypothesis that secretion of the fusion protein takes place at the hyphal tips (Wösten et al., 1991 ). However, as subapical regions of the cell wall were also fluorescent it appears that at least some of the GLA::sGFP fusion protein is retained in the hyphal wall following secretion at the hyphal apex. Unexpectedly, septa were also brightly fluorescent, indicating the presence of GLA::sGFP fusion protein. Since the formation of septa takes place independently of apical growth, the question arises of how septa become fluorescent. One explanation might be that the GLA::sGFP fusion protein in the cell wall is trapped but freely diffusible within the extracellular matrix. Alternatively, the GLA::sGFP fusion protein might be secreted during the formation of the septum. It is also possible that not all the secretion of the fusion protein is correlated with cell growth and that secretion also occurs in subapical cells without cell wall expansion. The effect of extracellular pH on fluorescence of the GLA::sGFP-expressing strain compared to the GLA::sGFP-HDEL strain also provides further evidence for the extracellular localization of GLA::sGFP in the hyphal wall. Fluorescence of GFP has been shown to be sensitive to low pH, and below pH 5·0 loss of fluorescence may be irreversible (Kneen et al., 1998
). The extracellular pH values of media of shake-flask cultures of A. niger have been shown to decrease to as low as 2·0 during growth (Archer et al., 1990
). Low pH induces the production of proteases that are known to affect yields of heterologous proteins (Archer & Peberdy, 1997
; Gouka et al., 1997a
; van den Homberg et al., 1997
). Extracellular proteases probably account for the degradation of the GLA::sGFP fusion proteins in the culture supernatant, even in soya milk medium where degradation of sGFP was found to occur at a slower rate than in defined medium. Cleavage of the GLA::sGFP fusion protein appeared to occur initially within the linker region between the glucoamylase and sGFP as cleaved sGFP with an apparent molecular mass of 27 kDa (the expected molecular mass for intact sGFP) was detected by Western analysis after 4 d growth in soya milk medium. Cleavage of the glucoamylase fusion protein at or near to the fusion junction has been reported for other heterologous proteins, even in the absence of a recognized processing site (Roberts et al., 1992
). Further degradation of sGFP in the supernatant was indicated by the loss of detectable amounts of sGFP after 6 d. The D15 mutant, which has a reduced ability to acidify the medium, was able to sustain extracellular wall fluorescence for longer than AB4.1. The data suggest that the GLA::sGFP fusion constructs can be used to monitor protein secretion in fermenters as long as the pH is held above pH 6·0. It may also be possible to use the GLA::sGFP-expressing strain to screen for additional protease-deficient mutants. Taken together, the results indicate that in young mycelia the GLA::sGFP fusion protein is primarily secreted at hyphal tips but partly retained within the cell wall, resulting in wall fluorescence. In older mycelia, extracellular wall fluorescence is lost as a result of the acidification of the culture medium and proteolytic degradation, possibly by acid-induced proteases.
To isolate potential mutants in the general secretory pathway, we developed a two-step screening procedure. Initially, mutants were isolated which were unable to degrade starch on plates and, subsequently, these were screened for their ability to degrade gelatin. It was assumed that mutants unable to degrade either of these substrates were more likely to be affected in the general secretory pathway. As general secretory pathway mutants may lead to a lethal phenotype, screening was initially performed at 42 °C on colonies that had been grown for 3 d at 25 °C so that temperature-sensitive mutants could be selected. However, none of the mutants isolated from either the first starch plate screen or the subsequent gelatin plate screen displayed temperature sensitivity, i.e. the inability to degrade either substrate was displayed at both 25 and 42 °C. Of the 10 putative general secretory mutants isolated, 7 appeared to grow at the same rate as the parental strain but all showed reduced sporulation. None of these mutants when examined by fluorescence microscopy showed intracellular accumulation and all had fusion protein present in the walls and septa. These mutants were therefore disregarded. The lack of halo formation on starch and gelatin media may reflect a reduction in the overall ability of these strains to secrete proteins, resulting in delayed halo formation compared to the parent strain rather than a block in secretion per se. The remaining three putative general secretory pathway mutants all displayed poor growth at both 25 and 42 °C compared to the parental strain, a complete loss of sporulation and abnormal hyphal morphology. When examined under fluorescence microscopy, two mutants (gsp 26 and 29) displayed intracellular accumulation of GLA::sGFP fusion protein with no detectable fluorescence in the walls or septa. The third mutant (gsp 31) showed no accumulation and fluorescence in the wall or septa and was disregarded. The pattern of accumulation of GLA::sGFP fusion protein in gsp 26 and 29 differed significantly from each other. In gsp 26 (Fig. 9a), fluorescence was diffuse and evenly located throughout the hyphae, whereas in gsp 29 (Fig. 9b
), accumulation in circular bodies ranging in size from approximately 0·5 to 2 µm diameter could clearly be seen. Thus, for both gsp 26 and 29, preliminary evidence strongly suggests the presence of defects in the general secretory pathway, leading to intracellular accumulation, and that these defects are likely to be affecting different points within the pathway. Further characterization of both gsp 26 and 29 is currently being undertaken to identify the sites at which accumulation occurs as well as examining the effects of inhibitors of secretion on the dynamics and organization of the secretory pathway. Thus the GLA::sGFP constructs allow the direct visualization of protein secretion and localization in vivo in growing hyphae and have proved invaluable in the characterization of secretory mutants, allowing a quick and reliable method for confirming blocks in the secretory pathway and providing additional visual information on the sites of accumulation within the hyphae.
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ACKNOWLEDGEMENTS |
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The work carried out in the UK was funded by a BBSRC studentship to C.G. and a scholarship from the Pasteur Institute of Iran to V.K.
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Received 9 April 1999;
revised 11 October 1999;
accepted 28 October 1999.