FEMS Microbiology Letters 175 (1999) 143^148
Candida albicans exoglucanase as a reporter gene in Schizosaccharomyces pombe G. Molero, V.J. Cid, C. Vivar, C. Nombela, M. Saènchez-Peèrez * Departamento de Microbiolog|èa II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain Received 8 February 1999; received in revised form 26 March 1999; accepted 6 April 1999
Abstract The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37³C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Candida albicans exoglucanase; Flow cytometry ; Reporter gene ; Schizosaccharomyces pombe
1. Introduction Schizosaccharomyces pombe is a very valuable model for studying the basic biological problems that are keys to our understanding of the more complex behaviour of higher eukaryotic cells. The development of new experimental tools to study gene expression, such as reporter gene systems, is therefore a priority. Over the past decade, yeast exoglucanases have been extensively characterised, both biochemical and genetically. Three non-essential genes encoding exo-1,3-L-glucanases have been reported in Saccharomyces cerevisiae (reviewed in [1,2]), and one in * Corresponding author. Tel.: +34 (1) 3941744; Fax: +34 (1) 3941745; E-mail:
[email protected]
Candida albicans (XOG1) [3], but no such activity has been detected in S. pombe [4]. The XOG1 gene from C. albicans is highly homologous (58% identity and 85% similarity) to the EXG1 gene from S. cerevisiae [3]. Xog1p shows serological [3] and biochemical properties similar to those of Exg1p [5^7] and is able to restore exoglucanase activity when expressed in S. cerevisiae exg1 mutant strains [8]; it has been described as a readily detectable reporter gene in these exoglucanase-defective strains of S. cerevisiae [8]. The XOG1 gene has been disrupted in C. albicans and its usefulness as a homologous reporter gene in the xog1-null strain as recipient has been described as well [9]. In order to test the exoglucanase system in the ¢ssion yeast, the ORF from the XOG1 gene was set under the control of the nmt1 thiamine-repressi-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 8 6 - X
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ble promoter developed by Maundrell [10,11]. Expression of the heterologous enzyme should occur only in the absence of thiamine.
2. Materials and methods 2.1. Plasmids and strains A XhoI-SalI fragment carrying the XOG1 gene from vector pXC-ADH [8] was ligated in the SalI site of pREP-3X vector [10], generating the pREPXG plasmid (Fig. 1). S. pombe hÿs ura 4-D18 leu132, S. pombe hÿn ura 4-D18 leu1-32 and S. pombe hÿs leu1-32 strains (provided by Dr S. Moreno) were transformed with pREP-XG plasmid. As a control, cells transformed with pREP-3X (lacking a reporter gene) were tested in parallel. Transformation procedures were done according to [12]. 2.2. Exoglucanase activity measurement To detect L-glucanase activity in solid cultures, transformants were grown on selective media [12]
with (repressing conditions) or without (non-repressing conditions) 5 Wg ml31 of thiamine [13] at both 32³C and 37³C for 2^3 days. Then, colonies were covered with an overlay prepared by mixing equal volumes of melted agar (10 g l31 ) and a solution of 0.3 g l31 of the substrate 4-methyl-umbelliferyl-L-Dglucoside (MUG, Sigma), both in 0.05 M acetate bu¡er, pH 5.3, and allowed to solidify at room temperature. The plates were then incubated at 30³C for 30 min in darkness and observed under UV light (V = 260 nm). Positive colonies (exoglucanase-producing) showed a white luminescence [8]. Quantitative measurements of exoglucanase activity were based on the release of glucose residues from laminarin [5,9] and these were measured with glucose oxidase coupled to peroxidase [14]. Enzymatic units are expressed in Wg of glucose residues released per hour per 107 cells (in the case of washed cells) or per ml of culture supernatant at 0.6 units of OD at 600 nm (approximately 1U107 cells ml31 ). Samples were prepared as follows: transformed cells from plasmids pREP-XG and pREP-3X were grown for 16 or 24 h at 32³C or 37³C in liquid minimal medium [12] with or without 5 Wg ml31 of thiamine. Appropriate dilutions with 0.05 M acetate bu¡er, pH 5.3, were made to adjust the OD at 600 nm to 0.6 U (1U107 cells ml31 ) and 1 ml of this dilution was spun. The supernatant and resulting pellet (washed twice with the same bu¡er and resuspended in 1 ml of bu¡er) were kept for activity determinations. 125 Wl of cell suspension or culture supernatant were added to 125 Wl of 0.5% laminarin in the same bu¡er. The mixture was incubated at 37³C for 2 h and the corresponding control was incubated without laminarin. All samples were treated with a mixture of peroxidase (type II, Sigma) and o-dianisidine (Sigma), incubated for 30 min at 30³C, and the reaction was stopped by adding 6 N HCl [14]. Colour intensity was measured at 540 nm. Values were referred to a control curve of di¡erent glucose concentrations (10^180 Wg ml31 ). 2.3. Flow cytometric analysis of exoglucanase activity
Fig. 1. A XhoI-SalI fragment from plasmid pXG-ADH [8] carrying the XOG1 gene from C. albicans (CaXOG1) was cloned in the SalI site of plasmid pREP-3X [10] under the control of the nmt1 thiamine-repressible promoter (Pnmt1). Only relevant restriction sites are shown.
S. pombe transformants were prepared as in the case of the biochemical measurements and incubated with the £uorogenic substrate £uorescein L-D-glucopyranoside (FDGP) (Sigma), as described for S. ce-
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revisiae [8]. Brie£y, pelleted cells were washed once with 0.05 M acetate bu¡er, pH 5.3, and resuspended in 50 Wl of the same bu¡er. 2.5 Wl of a 1 mg ml31 FDGP solution were added to one of each pair of samples (one was taken as a control). All tubes were incubated at 30³C for 60 min in darkness. 0.5 ml of 0.005% propidium iodide (PI) in phosphate bu¡ered saline (PBS) was added to each sample to verify the cell viability along the assays [15]. Flow cytometric analyses were carried out on a FACScan £ow cytometer (Becton-Dickinson, San Jose, CA, USA). 5000 cells were acquired per sample and the £uorescence emitted was measured on an exponential scale. The data were analysed with CellQuest software (BectonDickinson).
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described in Section 2.2 after 16 or 24 h of incubation. The exoglucanase activity was assayed in washed cells and in supernatants to distinguish between secreted and retained enzyme activity. As shown in Fig. 2, pREP-XG transformants incubated in the absence of thiamine showed an increase in exoglucanase activity under all the conditions analysed. The three strains tested produced similar levels of exoglucanase activity induction. This induction was more marked after 24 h of incubation. Temperature also a¡ected the expression, increasing it at 37³C and also increasing the proportion of secreted enzyme at high incubation temperatures. As can be
3. Results 3.1. Heterologous expression of the C. albicans XOG1 gene The XOG1 gene was set under the control of the nmt1 promoter, generating the pREP-XG plasmid, as described in Section 2.1. Three S. pombe strains (see Section 2.1) were transformed with plasmids pREP-XG and pREP-3X. All the pREP-XG transformants displayed exoglucanase activity only when patched on selective media lacking thiamine (nonrepressing conditions) at 32³C and 37³C (data not shown), showing that the heterologous exoglucanase can be produced as an active enzyme and that it is secreted by the ¢ssion yeast. By contrast, pREP-3X transformants did not show any luminescence under any of the conditions analysed. Viable cells were recovered from the agar plates once the exoglucanase activity reaction with MUG had been performed. No di¡erences were found among the three S. pombe strains. 3.2. Quantitative measurements of exoglucanase activity pREP-XG and pREP-3X transformants from the three S. pombe strains were incubated under repressing (with thiamine) and non-repressing (without thiamine) conditions at both 30³C and 37³C and exoglucanase activity was quantitated biochemically as
Fig. 2. Graphic plot of exoglucanase activity measured by hydrolysis of the substrate laminarin and expressed in EU: Wg of glucose released per h per 1U107 cells or per ml of culture medium at 0.6 U of OD at 600 nm. Results are means þ S.D. of four di¡erent transformants of each S. pombe strain and of three di¡erent experiments under each of the conditions studied. The values for each transformant are the result of subtracting the corresponding control without substrate, and the values obtained for pREP-3X transformants under each condition analysed were taken as controls and subtracted from the values obtained for the pREP-XG transformants. When subtraction was negative, it was expressed as 0.
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Fig. 3. Flow cytometry analysis of exoglucanase activity. The ordinates show relative cell numbers and the abscissae represent the green £uorescence emission of the hydrolysed substrate. A and B show a representative experiment performed after 24 h of incubation at 32³C. The same kind of plot is seen after 24 h of incubation at 37³C and with the three strains analysed. A: pREP-3X transformants grown under repressing and non-repressing conditions. B: The same with pREP-XG transformants. A clear displacement of the peak can be observed when cells are incubated in the absence of thiamine.
observed, exoglucanase activity was partly retained in washed cells under all the conditions assayed, as previously described for S. cerevisiae [8], allowing the analysis of the expression by £ow cytometry.
(Fig. 2). Propidium iodide staining of the same samples revealed that 99% of the cells remained viable during the assays (data not shown).
3.3. Flow cytometry analyses
4. Discussion
S. pombe transformants from plasmids pREP-XG and pREP-3X (taken as control) were incubated under the same conditions as for the quantitative biochemical measurements. The three strains of S. pombe previously described were used for this purpose, showing similar results. As shown in Fig. 3B, in the absence of thiamine, pREP-XG transformants gave rise to a fairly strong £uorescence signal that was readily detectable by £ow cytometry after 24 h of incubation at both 32³C and 37³C. Repression of exoglucanase expression in the presence of thiamine was clear since the activity observed as the £uorescent signal in cells bearing plasmid pREP-XG was comparable to that of the negative controls (pREP3X transformants) (Fig. 3A,B). On the other hand, this displacement of the peak was less remarkable after 16 h of incubation under non-repressing conditions (data not shown), probably due to the lower level of exoglucanase activity displayed by pREP-XG transformants under these conditions
In contrast to S. cerevisiae and C. albicans, S. pombe lacks exoglucanase activity [4], allowing the use of this reporter system in any given strain of this organism, while in the budding yeast and in C. albicans, exoglucanase-defective backgrounds are required. We further postulate that this reporter gene might be useful in other fungi lacking exoglucanase activity. The great advantage of this system as compared to others currently used, such as Escherichia coli lacZ, is that viable cells can be recovered from the assay, while previous methods require ¢xation of the cells for activity measurements. Furthermore, using cell sorting, the analysis of viable cell populations by £ow cytometry allows us to follow those that achieve a certain level of expression of a reporter gene. However, we have found that the induction levels previously described for the nmt1 promoter, 80U for the cat gene (chloramphenicol acetyltransferase) [11] or 300U for E. coli lacZ [16], are not reached with our
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reporter system (5^10U). Thus, a disadvantage for the exoglucanase-based reporter system might be a low sensitivity when trying to detect subtle variations in the expression. The advent of the green £uorescent protein from Aequorea victoria (GFP) [17,18], which can also be used as a reporter gene in S. pombe [19], together with subsequently modi¢ed proteins [20^23] has allowed some of these problems to be overcome and permits in vivo localisation of proteins. Both exoglucanase and GFP allow expression studies to be made without ¢xation or killing of the cells and neither of them interferes with normal cell growth. Additionally, both can be expressed at di¡erent incubation temperatures in any given strain of S. pombe. Despite this, we postulate that biochemically-detectable secretable proteins, such as exoglucanase, might be useful since they allow the quanti¢cation of expression by a simple reaction, both in cells and in supernatants. We also posit that the availability of two reporter genes that can be expressed simultaneously in the same cell and detected separately might allow parallel studies of two di¡erent promoters under the same environmental conditions to be carried out.
[4]
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
Acknowledgments We thank Dr Sullivan for providing the XOG1 gene and Dr Sergio Moreno for providing S. pombe strains and answering methodological questions. We è lvarez and A. Vaèzquez for their techthank Dr A. A nical support and J. Regidor and Dr A. Mendoza for their help. This work was supported by the Fondo de Investigaciones Sanitarias (FISS) (Spain) with Grant 97/0047-01.
[13]
[14]
[15]
[16] [17]
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