Cloning and characterization of preferentially expressed genes in an

FEMS Microbiology Letters 230 (2004) 137^142

www.fems-microbiology.org

Cloning and characterization of preferentially expressed genes in an aluminum-tolerant mutant derived from Penicillium chrysogenum IFO4626 Manabu Sugimoto , Yuji Saiki, Demin Zhang, Fusako Kawai Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan Received 25 September 2003 ; received in revised form 9 November 2003 ; accepted 17 November 2003 First published online 6 December 2003

Abstract cDNAs expressed preferentially in an Al-tolerant microorganism were isolated by subtraction hybridization with cDNAs of Al-sensitive Penicillium chrysogenum IFO4626 as driver cDNA and cDNAs of the Al-tolerant mutant derived from the wild cells by UV irradiation as tester cDNA. Northern blot analysis revealed that mRNA levels of six genes were increased significantly in the Al-tolerant mutant after exposure to Al stress when compared with the wild cells. Two genes accumulated in both the presence and absence of Al stress and four genes were induced by Al stress in the Al-tolerant mutant. cDNA fragments were amplified by rapid amplification of cDNA ends and sequenced to obtain full-length cDNAs of the six genes. Two genes were novel or predicted ones and the others showed significant homology to known genes, ADP/ATP translocase, enolase, cysteine synthase, and glucoamylase, which are induced by environmental stresses in prokaryotic and eukaryotic cells. These enzyme activities increased in the Al-tolerant mutant when compared to those in the wild cells, showing that not only the levels of gene expression but also the levels of enzyme activities increased in the Al-tolerant mutant. : 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Penicillium chrysogenum ; Aluminum tolerance; ADP/ATP translocase ; Enolase ; Cysteine synthase; Glucoamylase

1. Introduction Aluminum (Al) is solubilized into the trivalent cation Al3þ , which leads to growth inhibition of microorganisms and plants, under acidic conditions [1^4]. The amount of Al ion increases in acidic soil caused by acidic rain and natural processes, and Al toxicity has become a major limiting factor of plant growth and productivity. Microorganisms in the soil are necessary to improve the soil for agriculture productivity, and Al-tolerant microorganisms may help plants to grow in acidic soil as well as Al-tolerant plants. The mechanisms of Al tolerance in plants include the accumulation or excretion of organic acids such as citrate and malate, which form a complex with Al to detoxify Al3þ [5^9]. Studies on Al tolerance at the molec-

* Corresponding author. Tel. : +81 (86) 424-1661; Fax : +81 (86) 434-1249. E-mail address : [email protected] (M. Sugimoto).

ular level in plants, especially cloning the genes induced by Al stress, have been reported [10^14]. On the other hand, there are few reports about the mechanisms of Al tolerance in microorganisms and speci¢cally expressed genes under Al stress in Al-tolerant microorganisms [15^20]. We have isolated Al-tolerant microorganisms and shown that they have di¡erent strategies to protect themselves from Al toxicity, suggesting a variety of Al-tolerant mechanisms in microorganisms [21,22]. To investigate the mechanisms of Al tolerance, we prepared mRNAs from Penicillium chrysogenum IFO4626 that is sensitive to Al stress and from an Al-tolerant mutant derived from P. chrysogenum IFO4626 by UV irradiation to use as a source for identifying di¡erentially expressed genes. In this paper, we identi¢ed six genes expressed preferentially in the Al-tolerant mutant. The nucleotide and deduced amino acid sequences identi¢ed both novel and known genes, some of which are intermediates for detoxifying oxidative stress and glycolytic and alcohol fermentation enzymes which supply su⁄cient energy to cells for having the tolerance to stress conditions.

0378-1097 / 03 / $22.00 : 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00886-3

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M. Sugimoto et al. / FEMS Microbiology Letters 230 (2004) 137^142

2. Materials and methods

2.5. Virtual Northern hybridization

2.1. Microorganism and media

One microgram of cDNA was electrophoresed on 1% agarose gel and transferred onto Hybond-Nþ nylon membrane (Amersham Biosciences). The membrane was hybridized with a fragment of the cDNA labeled with digoxigenin-11-dUTP using the PCR DIG labeling kit (Boehringer Mannheim) with the PCR primer. Hybridization was performed at 4‡C for 16 h in 50% formamide, 5Usodium saline citrate (SSC), 0.25% non-fat milk powder, 0.1% sodium dodecyl sulfate (SDS), 5UDenhardt’s reagent. The membrane was washed twice with 1USSC, 0.1% SDS at room temperature, and twice with 0.1USSC, 0.1% SDS at 68‡C.

P. chrysogenum IFO4626 was purchased from the Institute for Fermentation, Osaka (IFO). The synthetic medium (SM) consisted of 20 g of glucose, 5 g of NaNO3 , 0.14 g of KH2 PO4 , 0.25 g of MgSO4 W7H2 O, 0.1 g of NaCl, 0.1 g of CaCl2 , and 1 ml of trace element solution (500 mg of H3 BO4 , 40 mg of CuSO4 W5H2 O, 100 mg of KI, 200 mg of Fe3 Cl3 W6H2 O, 400 mg of MnSO4 W5H2 O, 200 mg of Na2 MoO4 W2H2 O, and 400 mg of ZnSO4 W7H2 O in 1 l) in 1 l. The pH of the medium was adjusted to 3.0 and sterile AlCl3 W6H2 O was added to the autoclaved medium to make an adequate concentration. The medium (PDB/SM) for screening Al-tolerant mutant consisted of 1 volume of potato dextrose broth (Eiken Chemicals) and 2 volumes of SM. 2.2. Mutation and isolation Spores suspended in SM were irradiated by UV lamp (GL 15, Toshiba) for 5 min and inoculated into SM containing 100 WM Al. After incubation at 30‡C for 5^7 days, viable cells were inoculated into PDB/SM and cultivated. The fungal cells were then inoculated into PDB/SM containing various concentrations of Al (350, 500, 600, 750 WM) and incubated at 30‡C for 5^7 days to show Al tolerance at 750 WM. Tolerant cells were grown on PDB/SM and then on PDB/SM containing various concen trations of Al (500, 750 WM) at 30‡C for 5^7 days, respectively. Finally, the Al-tolerant cells grown on PDB/SM containing 750 WM Al were spread on PDB/SM agar plate containing 750 WM Al and used as the Al-tolerant mutant. 2.3. Cultivation Spores collected from a 9-cm plate were suspended in 10 ml of PDB/SM and 2.5 ml of the suspension was inoculated into 50 ml of PDB/SM either including 750 WM Al for the Al-tolerant mutant or without Al for the wild strain in a £ask at 30‡C with shaking. The cells were harvested by vacuum ¢ltration and washed with saline. 2.4. RNA and cDNA preparation Total RNA was isolated by the guanidinium isothiocyanate method. cDNA for subtraction hybridization and virtual Northern blot [23] was synthesized and ampli¢ed using the SMART PCR cDNA Synthesis Kit (Clontech). Poly(A)þ RNA was reverse-transcribed at 42‡C for 1 h with cDNA synthesis primer and SMART II oligonucleotide. The reaction mixture was ampli¢ed with the polymerase chain reaction (PCR) primer supplied in the kit according to the manufacturer’s instruction.

2.6. Subtraction hybridization and generation of subtraction library Subtraction hybridization [24,25] was performed using the PCR-Selected1 cDNA subtraction kit (Clontech). Tester and driver cDNAs were prepared from total RNAs isolated from the mutant cells exposed to 750 WM Al for 3 days and the wild cells without Al for 3 days, respectively. The enriched cDNA fragments were cloned into a pGEM-T vector (Promega) and transformed into Escherichia coli XL1-Blue cells to construct the subtraction library of the Al-tolerant mutant. Colonies were hybridized with the driver cDNA labeled with digoxigenin11-dUTP as described above and colonies displaying reduced signal were selected. 2.7. Rapid ampli¢cation of cDNA ends (RACE)-PCR The primers for 3P- and 5P-RACE PCR, 5P-CAGGAAGATCGAAGAAGTGATCGGAAG-3P and 5P-CTTCCTGGTGAAGTCATGATCCTGG-AT-3P for PCALR8, 5PTTCATTGTAGCGGTAGTGCGAAAATT-3P and 5P-TGAAAACCGTCCTTTACTCGACTCTCT-3P for PCALR28, 5P-GTGGTGATGTTAACCAGGCCGAGAAGT3P and 5P-GGTCGAGACGACCCTGCTTGTGCATCT3P for PCALR53, 5P-TGGTAAGGGTGTCCTCAACGCCGTCAA-3P and 5P-TCTTGGTTCCGGCAAGGTCGGAGATGT-3P for PCALR64, 5P-GCCTACTGCACCCGGGTGTCAACAGCC-3P and 5P-TTGTCGAAGGCCACTGCGGTGACCGGG-3P for PCALR70, and 5P-CCGTCGTCAAGTCGAACATCTGGCCCA-3P and 5P-TGGGCCAGATGTTCGACTTGACGACGG-3P for PCALR76, respectively, were synthesized on the basis of the sequence of the gene fragments isolated from the subtraction library. RACE-PCR was carried out on the cDNA and the PCR products were cloned into a pGEM-T vector (Promega). The nucleotide sequence on both strands was determined using a BigDye Thermal Cycle Sequencing FS Ready Reaction kit (Applied Biosystems) with a series of synthetic primers.

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2.8. Computer analysis The handling, analysis, and translation of the nucleotide sequences were performed with GENETYX-Mac (Software Development) and the BLAST program [26,27] using the network service at the National Institute of Genetic, Mishima, Japan (http://www.ddbj.nig.ac.jp/Welcome. html). 2.9. Preparation of the cell-free extract The frozen cells were pulverized in a mortar and pestle under liquid nitrogen. The powdered tissues were suspended in 50 mM sodium phosphate bu¡er (pH 7.0) and incubated at 4‡C for 1 h with stirring. Cell debris was removed by centrifugation at 10 000Ug for 30 min and the supernatant was used as the cell-free extract.

139

agent to determine the cysteine concentration. One unit was de¢ned as the amount of enzyme that converts 1 Wmol of O-acetyl-L-serine into cysteine per minute under these conditions. The glucoamylase activity was assayed by measuring the liberated glucose from soluble starch. The reaction mixture consisted of 0.2 ml of 0.5% soluble starch, 0.1 ml enzyme solution, and 0.1 ml of 0.1 M acetate bu¡er (pH 6.0). The mixture was incubated at 37‡C for 30 min and the reaction was stopped by the addition of 0.5 ml of 0.05 M NaOH. The liberated glucose was assayed by the Tris^glucose oxidase^peroxidase method with a Glucose AR-II Test (Wako Pure Chemical Industries) as described previously [30]. One unit was de¢ned as the amount of enzyme that liberates 1 Wmol of glucose from substrate per minute under these conditions. Protein was measured by the method of Bradford [31] with bovine serum albumin as a standard.

2.10. Enzyme and protein assays 2.11. Accession numbers Enolase activity was assayed by the method of Winstead and Wold [28]. The reaction mixture consisted of 1 mM Dglyceric acid 2-phosphate, 1 mM MgSO4 , 0.4 M KCl, 50 mM imidazole bu¡er (pH 6.7), and the enzyme solution in a total volume of 3 ml. The mixture was incubated at 25‡C for 30 min and the rate of change in the absorbance at 240 nm was measured. One unit was de¢ned as the amount of enzyme that changes the absorbance at 240 nm by 0.1 per minute under these conditions. The cysteine synthase activity was assayed by the method described by Graham et al. [29]. The reaction mixture consisted of 1.5 mM O-acetyl-L-serine, 3 mM Na2 S, 10 mM dithiothreitol, and enzyme solution in a total volume of 2 ml. The mixture was incubated at 26‡C for 10 min and the aliquots were added to the acidic ninhydrin re-

Fig. 1. Growth of the wild cells and the mutant cells of P. chrysogenum IFO4626 in the absence and presence of Al. Cultivation was performed in PDB/SM medium containing 750 WM Al or without Al at 30‡C. a, wild cells without Al ; b, wild cells with Al; O, mutant cells without Al ; R, mutant cells with Al. The data presented are typical of the results from triplicate experiments.

Sequence data with annotations have been deposited with the DDBJ, EMBL, and GenBank data banks under the accession numbers AB091505, AB091506, AB091507, AB091508, AB091509, and AB091510.

3. Results and discussion 3.1. Isolation of Al-tolerant mutant A variety of genes have been reported to respond to environmental stresses, however, it is important to con¢rm the type of the stress-inducible genes whether the gene product functions in stress tolerance or the gene is just induced without contributing to stress tolerance [32]. The evaluation whether genes are preferentially expressed in stress-tolerant cells rather than stress-sensitive cells is necessary to identify the genes that would possibly play roles in conferring stress tolerance to microorganisms. In order to prepare microorganisms that show a di¡erent sensitivity to Al toxicity, we collected fungi belonging to Penicillium, Aspergillus, and Trichoderma species because of their ability to grow under the acidic conditions we identi¢ed previously [23]. We found P. chrysogenum IFO4626 to be an Al-sensitive microorganism and derived an Al-tolerant mutant from this by UV irradiation. Both the wild cells and the mutant cells could grow very well in the medium at pH 3.0 without Al. In the medium that contained 750 WM Al, the mutant cells showed good growth, whereas the wild cells showed no growth (Fig. 1). 3.2. Screening of preferentially expressed genes The subtraction library was constructed from cDNAs of the mutant cells as tester cDNA and of the wild cells as

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Fig. 2. Con¢rmation of di¡erential expression of the preferentially expressed genes by virtual Northern blot analysis. mRNAs were prepared from the wild cells in the absence of Al for 3 days (Al-S) and from the mutant cells exposed to 750 WM Al for 3 days (Al-R). cDNA fragments of the six genes were used as a probe.

driver cDNA, which was enriched for the cDNAs expressed speci¢cally in the Al-tolerant mutant. A total of 1000 clones from the subtraction library were hybridized with total cDNAs from the wild cells twice, and 84 clones showed negative signals by colony hybridization. The differential expression of the genes was analyzed by Northern hybridization with the insert cDNA fragments from 84 clones as a probe. Our Northern blot detected that eight genes were expressed preferentially in the Al-tolerant cells and the densitometry indicated that the mRNA levels of the genes in total RNA, designated PCALR5, PCALR8, PCALR28, PCALR46, PCALR53, PCALR64, PCALR70, and PCALR76, were increased 5.4, 3.5, 2.9, 5.0, 5.4, 2.9, 2.2, and 2.2 times, respectively, when compared to the wild cells (Fig. 2). Sequence analysis of the eight cDNA fragments showed that PCALR5, PCALR46, and PCALR53 had the same nucleotide sequence, so that ¢nally six independent genes were isolated which were expressed preferentially in the Al-tolerant mutant under Al stress. To investigate the induction of these genes by Al stress, total RNA was isolated from the Al-tolerant mutant cultured in the presence and absence of 750 WM Al for 3 days and hybridized with the cDNA fragments of the six genes (Fig. 3). In the mutant exposed to Al, mRNA levels of PCALR8, PCALR53, PCALR64, and PCALR70 were increased about 2.9, 4.9, 2.6, and 2.3 times, respectively, when compared to those in the absence of Al, whereas mRNA levels of PCALR28 and PCALR76 were the

same as those in the absence of Al and these genes accumulated in both the presence and absence of Al stress. These results suggest that the promoter and promoterrelated factors of these six genes were changed to express the gene signi¢cantly, especially changed to respond to Al stress on PCALR8, PCALR53, PCALR64, and PCALR70. 3.3. Identi¢cation of the cDNAs To obtain the complete nucleotide sequence of the six genes, cDNA fragments ampli¢ed by 5P- and 3P-RACEPCR with the speci¢c primers and cDNA library from the Al-tolerant mutant treated with Al were sequenced. The resulting nucleotide sequences of PCALR8, PCALR28, PCALR53, PCALR64, PCALR70, and PCALR76, except for the poly(A) sequence, were 836, 1600, 1780, 1492, 1192, and 1935 bp in length, respectively, and contained an open reading frame encoding polypeptides of 208, 430, 434, 438, 371, and 488 amino acid residues, respectively (accession numbers AB091505, AB091506, AB091507, AB091508, AB091509, and AB091510). Each cDNA length obtained by sequencing was very close to that estimated by Northern hybridization (data not shown), suggesting that these are the full-length cDNAs. The deduced amino acid sequences of PCALR28, PCALR53, PCALR64, PCALR70, and PCALR76 showed homologies with an ADP/ATP translocase, a deduced sun family pro-

Fig. 3. Response to Al stress of the preferentially expressed genes in the Al-tolerant mutant. Virtual Northern blots were prepared from mRNAs from the mutant cells cultured in the absence (3Al) and presence (+Al) of 750 WM Al for 3 days. cDNA fragments of the six genes were used as a probe.

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Table 1 Homology of amino acid sequences encoded by preferentially expressed genes in the Al-tolerant mutant Gene (accession number)

Homologous protein (accession number)

Identity (%)

PCALR8 (AB091505) PCALR28 (AB091506)

Novel ADP/ATP translocase from Neurospora crassa X00363) Schizosaccharomyces pombe (Z49974) Sun family protein from Schizosaccharomyces pombe (AL022103) Saccharomyces cerevisiae (Z28267) Enolase from Penicillium citrinum (AF254643) Aspergillus fumigatus (AF284645) Cysteine synthase from Emericella nidulans (U19395) Schizosaccharomyces pombe (AL023589) Glucoamylase from Aspergillus oryzae (AB007825) Talaromyces emersonii (AJ304803)

78 75 46 36 92 91 80 63 60 58

PCALR53 (AB091507) PCALR64 (AB091508) PCALR70 (AB091509) PCALR76 (AB091510)

tein, an enolase, a cysteine synthase, and glucoamylase, respectively, whereas no protein in the databank revealed homology with that of PCALR8 (Table 1). It has been reported that mRNA levels of genes encoding enzymes related to glycolytic and alcohol fermentation pathways increased in response to cold, desiccation, salt, and high temperature stresses in plants and yeast [33,34]. The preferential expression of PCALR64 and PCALR76 encoding enolase and glucoamylase, respectively, in the Al-tolerant cells suggests that these genes are expressed signi¢cantly or induced by Al stress as well as other environmental stresses to supply a su⁄cient degree of energy to cells which should activate the metabolism to resist the environmental stresses, and ADP/ATP translocase encoded by PCALR28, which is the major representative of the mitochondrial membrane proteins, would lead the energy e¡ectively to the stress defense systems. Cysteine synthase encoded by PCALR70 is a key enzyme in cysteine metabolism leading to glutathione synthesis. Induction of PCALR70 would contribute to the intracellular glutathione pool to detoxify oxidative stress derived from environmental stresses [35]. These results suggest that the Altolerant mechanisms may share common features in prokaryotic and eukaryotic cells. 3.4. Enzyme activities To determine whether or not the enzyme activities, which were encoded by the cDNAs expressed preferentially, actually increase in the Al-tolerant mutant, cellfree extracts were prepared from the wild and mutant cells which were cultured in the absence and presence of Al, respectively. The activities of enolase encoded by PCALR64, cysteine synthase encoded by PCALR70, and glucoamylase encoded by PCALR76 were 234, 810, 182 mU mg31 in the wild cells and 419, 1013, 260 mU mg31 in the mutant cells, respectively. Thus, both enzyme activities increased signi¢cantly in the Al-tolerant mutant, showing that not only the levels of gene expression but also the levels of enzyme activity increase in the Al-tolerant mutant.

In the present study we isolated six cDNAs preferentially expressed in the Al-tolerant mutant and identi¢ed those as both novel and known genes. Although it is not clear whether the preferential expression of all these genes together or some of them is necessary to acquire tolerance against Al stress, we have identi¢ed genes that may be good candidates for an anti-Al stress system in microorganisms.

Acknowledgements This research was supported by a grant from the Biooriented Technology Research Advancement Institution (BRAIN), Japan.

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