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Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth1
FEMS Microbiology Letters 149 (1997) 279^284
Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth Kenji Tatsuno a , Hisafumi Yamada-Okabe a , Masamichi Takagi b , Mikio Arisawa a , Masayuki Sudoh a * ;
a
Department of Mycology, Nippon Roche Research Center, Kamakura-shi, Kanagawa 247, Japan
b
Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received 31 January 1997; revised 20 February 1997; accepted 20 February 1997
Abstract
A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid. The biochemical characteristics of chitin synthase B (ChsB) expressed in S. cerevisiae were examined. The chitin synthase B produced in galactose medium showed zymogenicity due to activation by trypsin treatment and required Mg2 ion to exert maximal activity. It was competitively inhibited by polyoxin D. The Ki value of the inhibitor was 10 WM, and the Km for the substrate was 1.6 mM. The activity was enhanced by the addition of N-acetylglucosamine. The optimal pH is 7.5 when Mg2 is used. These characteristics are the same as those of other chitin synthases. Keywords : Aspergillus nidulans
;
Saccharomyces cerevisiae
; Chitin synthase gene; Polymerase chain reaction
1. Introduction
Chitin, a L(1,4)-linked polymer of N-acetylglucosamine (GlcNAc), is a major component of the cell wall of most ¢lamentous fungi [1] and plays a major role in the determination of cell morphology. Its synthesis constitutes a model for morphogenesis and is useful for us to de¢ne potential targets for the discovery of antifungals. In the ¢lamentous fungus, As-
* Corresponding author. Tel.: +81 (467) 47 2242; fax: +81 (467) 46 5320; e-mail: [email protected] The accession number for the cDNA of is D83216.
chsB
Aspergillus nidulans
pergillus nidulans, four chitin synthase genes, chsA, chsB, chsC and chsD, have been recently cloned and characterized [2^4]. The chsB gene was isolated with the S. cerevisiae CHS2 gene as a Southern probe; the chsB gene encodes a 916-residue polypeptide, whose amino acid sequence is 36.3% identical with the sequence of Saccharomyces cerevisiae [2]. Disruption of chsA, chsC or chsD causes no defects in cell growth or morphology during the asexual cycle, while that of chsB is lethal. On the other hand, in S. cerevisiae, three chitin synthase genes, CHS1, CHS2 and CAL1 were isolated [5^7] and were characterized genetically and biochemically. Chitin synthase 1 (Chs1) is a repair enzyme [8] counterbalancing the activity of the chi-
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 0 8 8 - 8
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tinase; chitin synthase 2 (Chs2) participates in the formation of the chitin ring that completes the primary septum during cytokinesis [9]; and chitin synthase 3 (Cal1) is responsible for chitin in the ring at bud emergence as well as in the cell wall [10]. Some of the characteristics of these three enzymes are also di¡erent; they prefer di¡erent cations and might be activated by di¡erent mechanisms. The only sophisticated biochemical work on the characterization of a chitin synthase was of that isolated from Absidia glauca, a typical Zygomycete [11]; at present, similar information is not available for A. nidulans chitin synthases, probably due to the di¤culties in isolating each enzyme from the organism. Among ¢lamentous fungi, A. nidulans chsB is the ¢rst gene demonstrated to be an essential gene for hyphal growth [2]. Here, we report on the expression of the chsB cDNA in S. cerevisiae with the S. cerevisiae GAL1 promoter. The expressed chitin synthase B (ChsB) was characterized by biochemical and molecular biological methods. 2. Materials and methods
2.1. Strains, media and cultures
A conidial suspension of Aspergillus nidulans FGSC89 (argB2 biA1) from Fungal Genetics Stock Center (Kansas, USA) was cultured in 1 liter of complete medium (2% malt extract, 0.1% peptone, 2% glucose, 0.2 Wg biotin/ml and 0.2 mg arginine hydrochloride/ml) in a 3-l Erlenmeyer £ask for 3 days at 30³C. Transformation of S. cerevisiae RRA400-1U (MATa his3 trp1 ura3 leu2 chs1: :URA3 chs3: :HIS3)and 4992H (MATa his3 trp1 ura3 leu2 lys2 ade2 chs2: :HIS3) was done by electroporation as described by Delome [12]. Transformants were grown in 0.7% yeast nitrogen base and 2% dextrose (YNBD) supplemented with appropriate amino acids [13]. Plasmids were ampli¢ed in Escherichia coli JM109. E. coli was grown in LB medium and transformation was done by the standard method [14]. 2.2. Poly(A) RNA preparation
Mycelium of A. nidulans was harvested by ¢ltra-
tion and washed with 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE) bu¡er, frozen in liquid nitrogen, and ground in liquid nitrogen with a mortar. The powdered mycelium was then freeze-dried. Total RNA was extracted from the dried mycelial powder in a guanidinium thiocyanate bu¡er [14], and puri¢ed by centrifugation on a cesium chloride cushion. Poly(A) RNA was isolated using Oligotex-dT30 (Takara). 2.3. cDNA preparation of chitin synthase B and plasmid construction
To prepare cDNA for A. nidulans chsB we used an RNA PCR kit (Takara) with A. nidulans poly(A) RNA as the template with the primers E15, 5PACTACCAGTTCTAGAATGGCCTACCACGGCTCTGG-3P and E63, 5P-AGGCAAAGCTCTAGATTACCGACGGGCGAAGCAGC-3P. Then, a full length cDNA (2.8 kb) was ampli¢ed and cloned into the TA vector (Invitrogen). The sequence was con¢rmed by comparison with that of the genomic DNA sequence [2]. For expression in S. cerevisiae, the cDNA was digested with XbaI and cloned into the XbaI site of YpGALx [12] to generate YpGALchsB in which the chsB transcription was under the control of the S. cerevisiae GAL1 promoter (Fig. 1A). 2.4. Sequencing
DNA sequencing was done by the method of Sanger et al. [15]. The reactions were carried out using the autosequencer core kit (Toyobo). The reaction products were analyzed with an A.L.F. DNA sequencer (Pharmacia-Biotech). 2.5. Preparation of mixed membrane fraction S. cerevisiae transformant cells induced with galactose were harvested and washed twice with TE bu¡er and suspended in bu¡er containing 100 mM Tris-HCl, 20 mM MgCl2 , pH 7.5 (Tris-Mg bu¡er), 0.25 mM phenylmethylsulfonyl £uoride, 2 Wg/ml chymostatin, 1.5 Wg/ml leupeptin, 1 Wg/ml pepstatin, and 5 Wg/ml antipain. Cells were lysed with glass beads (0.5 mm in diameter) with a Braun homogenizer (B. Braun), and cell debris was removed by low speed
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centrifugation (3000Ug). The supernatant fraction was then sedimented at 300 000Ug for 40 min at 4³C, and washed once with Tris-Mg bu¡er. The ultracentrifugation was repeated and then the pellet was resuspended in the Tris-MgCl bu¡er containing 33% glycerol. The membrane fractions were diluted to 20 mg/ml and stored at 380³C until use. The protein concentration was measured with the DC Protein assay kit (BioRad).
281
2.6. Chitin synthase assay
The assay of chitin synthase was carried out according to the method of Archer [16]: incubation with 1% (w/w) of trypsin (Sigma) for 15 min at 30³C activated mixed membrane fraction; the reaction was stopped by the addition of a 1.5-fold excess of soybean trypsin inhibitor (Sigma). The standard assay was carried out in 50 Wl of a standard reaction mixture containing 127 mM Tris-HCl, 10 mM MgCl2 , 100 mM GlcNAc, 1 mM UDP-[3 H]GlcNAc (480 000 dpm), 20 Wg of protein of trypsinized membrane, and 13% glycerol at 30³C for 60 min. Reactions were stopped by the addition of 0.5 ml of cold 10% (w/v) trichloroacetic acid. The insoluble material was collected by ¢ltration on a glass ¢ber disk (Whatman). Data are means of duplicate determinations.
Table 1 E¡ect of cations on synthesis of chitin Addition Radioactivity incorporated (dpm/min/mg protein) EDTA 20 1200 MgCl2 MnCl2 730 CaCl2 360 Co(CH3 COO)2 230 CuSO4 33 ZnCl2 30 Data are expressed as radioactivity incorporated into chitin from 1 mM UDP-GlcNAc (480 000 dpm) in 1 h at 30³C. Metal salts were added to a concentration of 8 mM to the reaction mixture. EDTA was added at 4 mM in place of the other metals.
Fig. 1. Restriction and physical map of the S. cerevisiae plasmid YpGALchsB and expression of A. nidulans chitin synthase. A: Pgal, chsB and Tgap mean GAL1 promoter, the open reading frame of A. nidulans chitin synthase B and GAP terminator, respectively. B: Chitin synthase activity of A. nidulans chsB expressed in S. cerevisiae RRA 400-1U. 3. Results and discussion
3.1. Expression of A. nidulans chsB cDNA in S. cerevisiae
Since A. nidulans contains at least four genetically distinct chitin synthases, it seemed very di¤cult to attempt to characterize each chitin synthase directly. In addition, in the yeast host-vector system, no foreign gene containing introns is expressed due to splicing errors, because there is a stringent recognition of the conserved sequence of introns [17,18].
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282
reading frame, and then constructed an expression
S. cerevisiae (Fig. 1A). We expressed A. nidulans chitin synthase B gene in S. cerevisiae under the regulation of the galactose inducible promoter, GAL1 ; YpGALchsB was then transformed into S. cerevisiae RRA400plasmid in
the cDNA of the
1U. The transformant cells were cultured in galactose medium to induce the expression of the gene.
Cells
harboring
the
YpGALchsB
chsB
plasmid
showed a signi¢cantly higher chitin synthase activity whereas those harboring the YpGALx vector did not, demonstrating that the induced chitin synthase activity is derived from the
chsB
gene (Fig.
1B). Bowen et al. [19] classi¢ed all chitin synthases into three classes according to their amino acid sequences. Furthermore, computer analysis allowed us to divide chitin synthases into ¢ve classes, I, II, III,
chsB
IV and V, and showed that
belongs to class
III [2,4]. Interestingly, class III type chitin synthases have been identi¢ed from the ¢lamentous fungi Fig. 2. pH activity pro¢le of
revisiae.
A. nidulans chsB
expressed in
S. ce-
After trypsin treatment, the activated membrane fraction
was assayed at pH values of 6.5^8.3 with 50 mM Tris-maleate bu¡er [24] containing 10 mM MgCl2 , 100 mM GlcNAc and 1 mM UDP-GlcNAc.
nidulans
A. fumigatus
A.
[20]. Complementation
chs2-de¢S. cerevisiae 4992H combination was tested. In contrast to Candida CHS1A [21] encoding a class II chitin synthase, A. nidulans ChsB could not rescue analysis of the YpGALchsB plasmid and
cient
the
A. nidulans chitin synthase B, for A. nidulans chsB and ampli-
[4] and
abnormal
morphology
(unpublished
data).
Thus, to characterize
Therefore, the evolutionary diversity of chitin syn-
we prepared cDNA
thase genes might be re£ected in the failure of func-
¢ed cDNA using the 5P and 3P primer set of the open
Fig. 3. Trypsin treatment of
cerevisiae.
A. nidulans
ChsB expressed in
S.
tional complementation.
Fig. 4. E¡ect of thase B activity.
FEMSLE 7516 1-5-97
N-acetylglucosamine
on
A. nidulans
chitin syn-
K. Tatsuno et al. / FEMS Microbiology Letters 149 (1997) 279^284
Fig. 5. Chitin synthase B of A. nidulans is competitively inhibited by polyoxin D. Polyoxin D concentrations are indicated in the ¢gure.
3.2. Characterization of A. nidulans chitin synthase B
chitin synthase B expressed in S. cerewas characterized. The apparent pH optimum of chitin synthase B was 7.5 when Mg2 was used (Fig. 2). As almost all chitin synthases are activated by partial treatment with trypsin, we examined whether A. nidulans ChsB is activated by trypsin treatment. As shown in Fig. 3, when 1% trypsin was used for the mixed membrane fraction, the ChsB activity increased by about 10-fold compared to the non-treated control. Therefore, ChsB is essentially a zymogen. Furthermore, chitin synthase B activity was enhanced to 3-fold by the addition of GlcNAc, a known chitin synthase activator (Fig. 4). Of the various metal ions added to the standard reaction mixture, magnesium was most e¡ective for stimulating ChsB activity (Table 1). Mn2 had little e¡ect on the activity whereas Cu2 and Zn2 were inhibitory. Addition of EDTA completely abolished the ChsB activity, indicating that like other chitin synthases, some metal(s) is essential for the enzyme activity. We also found that A. nidulans chsB expressed in S. cerevisiae was strongly inhibited by polyoxin D. Kinetic measurements in which the substrate concentration was varied showed competitive inhibition of ChsB by the inhibitor (Fig. 5). From the kinetics, the Km value for the substrate was 1.6 mM, and the Ki A. nidulans visiae A. nidulans
283
value of polyoxin D was 10 WM, which is similar to that for the A. fumigatus enzyme [16]. In conclusion, biochemical analyses of ChsB revealed that (1) ChsB enhanced incorporation of the chitin synthase substrate, UDP-GlcNAc, into the acid-insoluble fraction in S. cerevisiae, (2) the activity was competitively inhibited by the chitin synthase inhibitor, polyoxin D, and (3) the enzyme was a zymogen, requiring divalent cations, and activated by GlcNAc. These characteristics are typical of the properties of chitin synthases [22]. We have demonstrated here that polyoxin D inhibited ChsB activity, but the inhibition was not as strong as that for Chs1 of S. cerevisiae [23]: it most likely would not inhibit the growth of mycelial fungi. Since chsB is essential, inhibitors of ChsB would be ideal antifungal agents. All these data con¢rm that S. cerevisiae is a suitable host for the expression of heterologous genes and that the expressed chsB gene from A. nidulans can be compared to other chitin synthases with similar essential biochemical characteristics. Acknowledgments
We are grateful to Dr. S. Machida, Dr. H. Horiuchi and Dr. A Miyazaki for their helpful discussions and critical reading of the manuscript. References [1] S. Bartnick-Garcia, Cell wall chemistry, morphogenesis, and taxonomy of fungi, Annu. Rev. Microbiol. 22 (1986) 87^108. [2] K. Yanai, N. Kojima, N. Takaya, H. Horiuchi, A. Ohta, M. Takagi, Isolation and characterization of two chitin synthase genes from Aspergillus nidulans, Biosci. Biotech. Biochem. 58 (1994) 1828^1835. [3] T. Motoyama, M. Sudoh, H. Horiuchi, A. Ohta, M. Takagi, Isolation and characterization of two chitin synthase genes of Rhizopus oligosporus, Biosci. Biotech. Biochem. 58 (1994) 1685^1693. [4] T. Motoyama, M. Fujiwara, N. Kojima, H. Horiuchi, A. Ohta, M. Takagi, The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation, Mol. Gen. Genet. 251 (1996) 520^528. [5] C.E. Bulawa, M. Slater, E. Cabib, J. Au-Young, A. Sburlati, W.L. Adair Jr., P.W. Robbins, The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo, Cell 46 (1986) 213^225. [6] S.J. Silverman, Similar and di¡erent domains of chitin syn-
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thases 1 and 2 of
S. cerevisiae :
two isozymes with distinct
[7] M.H. Valdivieso, P.C. Mol, J.A. Shaw, E. Cabib, A. Duran, CAL1, a gene required for activity of chitin synthase 3 in
Saccharomyces cerevisiae,
thase 1, an auxiliary enzyme for chitin synthesis in
Aspergillus fumigatus,
Biochem. J. 164 (1977)
653^658. [17] C.J. Langford, F.J. Klinz, C. Donath, D. Gellwitz, Point mutations identify the conserved, intron-contained TACTAAC
J. Cell Biol. 114 (1991) 101^109.
[8] E. Cabib, A. Sburlati, B. Bowers, S.J. Silverman, Chitin syn-
myces cerevisiae,
[16] D.B. Archer, Chitin biosynthesis in protoplasts and subcellular fractions of
functions, Yeast 5 (1989) 459^467.
Saccharo-
box as an essential splicing signal sequence in yeast, Cell 36 (1984) 645^653. [18] M.A. Innis, M.J. Holland, P.C. McCabe, G.E. Cole, V.P.
J. Cell Biol. 108 (1989) 1665^1672.
[9] C.E. Bulawa, B.C. Osmond, Chitin synthase I and chitin syn-
Wittman, R. Tal, K.W.K. Watt, D.H. Gelfand, J.P. Holland,
Sac-
J.H. Meade, Expression, glycosylation, and secretion of an
thase II are not required for chitin synthesis in vivo in
charomyces cerevisiae,
Proc. Natl. Acad. Sci. USA 87 (1990)
Aspergillus
glucoamylase by
Saccharomyces cerevisiae,
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228 (1985) 21^26.
7424^7428. [10] J.A. Shaw, P.C. Mol, B. Bowers, S.J. Silverman, M.H. Valdi-
[19] A.R. Bowen, J.L. Chen-Wu, M. Momany, R. Young, P.J.
vieso, A. Duran, E. Cabib, The function of chitin synthases 2
Szaniszlo, P.W. Robbins, Classi¢cation of fungal chitin syn-
and 3 in the
Saccharomyces cerevisiae
cell cycle, J. Cell Biol.
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114 (1991) 111^123. [11] S. Machida, M. Saito, Puri¢cation and characterization of membrane-bound chitin synthase, J. Biol. Chem. 268 (1993)
The
Aspergillus fumigatus chsC
and
chsG
genes encode class
III chitin synthases with di¡erent functions, Mol. Microbiol. 20 (1996) 667^679.
1702^1707. [12] E. Delome, Transformation of
Saccharomyces cerevisiae
by
[21] M. Sudoh, M. Watanabe, T. Mio, S. Nagahashi, H. Yamada-
electroporation, Appl. Environ. Microbiol. 55 (1989) 2242^
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[13] S. Nagahashi, M. Sudoh, N. Ono, R. Sawada, E. Yamaguchi, Y. Uchida, T. Mio, M. Takagi, M. Arisawa, H. YamadaOkabe, Characterization of chitin synthase 2 of
ces cerevisiae :
Saccharomy-
implication of two highly conserved domains as
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Candida albicans,
canCHS1A,
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2673^2679. [22] C.E. Bulawa, Genetics and molecular biology of chitin synthesis in fungi, Annu. Rev. Microbiol. 47 (1993) 505^534. [23] E. Cabib, Di¡erential inhibition of chitin synthases 1 and 2 from
Saccharomyces cerevisiae
by polyoxin D and nikkomy-
cins, Antimicrob. Agents Chemother. 35 (1991) 170^173. [24] J. Au-Young, P.W. Robbins, Isolation of a chitin synthase gene (CHS1) from
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chain-terminating inhibitors, Proc. Natl. Acad. Sci. USA 74 (1977) 5463^5467.
FEMSLE 7516 1-5-97
Properties of yeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth Kenji Tatsuno a , Hisafumi Yamada-Okabe a , Masamichi Takagi b , Mikio Arisawa a , Masayuki Sudoh a * ;
a
Department of Mycology, Nippon Roche Research Center, Kamakura-shi, Kanagawa 247, Japan
b
Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received 31 January 1997; revised 20 February 1997; accepted 20 February 1997
Abstract
A complementary DNA of the Aspergillus nidulans chsB gene encoding chitin synthase, an essential gene for hyphal growth, was obtained by RT-PCR and expressed in Saccharomyces cerevisiae by using the GAL1 promoter in a multicopy plasmid. The biochemical characteristics of chitin synthase B (ChsB) expressed in S. cerevisiae were examined. The chitin synthase B produced in galactose medium showed zymogenicity due to activation by trypsin treatment and required Mg2 ion to exert maximal activity. It was competitively inhibited by polyoxin D. The Ki value of the inhibitor was 10 WM, and the Km for the substrate was 1.6 mM. The activity was enhanced by the addition of N-acetylglucosamine. The optimal pH is 7.5 when Mg2 is used. These characteristics are the same as those of other chitin synthases. Keywords : Aspergillus nidulans
;
Saccharomyces cerevisiae
; Chitin synthase gene; Polymerase chain reaction
1. Introduction
Chitin, a L(1,4)-linked polymer of N-acetylglucosamine (GlcNAc), is a major component of the cell wall of most ¢lamentous fungi [1] and plays a major role in the determination of cell morphology. Its synthesis constitutes a model for morphogenesis and is useful for us to de¢ne potential targets for the discovery of antifungals. In the ¢lamentous fungus, As-
* Corresponding author. Tel.: +81 (467) 47 2242; fax: +81 (467) 46 5320; e-mail: [email protected] The accession number for the cDNA of is D83216.
chsB
Aspergillus nidulans
pergillus nidulans, four chitin synthase genes, chsA, chsB, chsC and chsD, have been recently cloned and characterized [2^4]. The chsB gene was isolated with the S. cerevisiae CHS2 gene as a Southern probe; the chsB gene encodes a 916-residue polypeptide, whose amino acid sequence is 36.3% identical with the sequence of Saccharomyces cerevisiae [2]. Disruption of chsA, chsC or chsD causes no defects in cell growth or morphology during the asexual cycle, while that of chsB is lethal. On the other hand, in S. cerevisiae, three chitin synthase genes, CHS1, CHS2 and CAL1 were isolated [5^7] and were characterized genetically and biochemically. Chitin synthase 1 (Chs1) is a repair enzyme [8] counterbalancing the activity of the chi-
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 0 8 8 - 8
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tinase; chitin synthase 2 (Chs2) participates in the formation of the chitin ring that completes the primary septum during cytokinesis [9]; and chitin synthase 3 (Cal1) is responsible for chitin in the ring at bud emergence as well as in the cell wall [10]. Some of the characteristics of these three enzymes are also di¡erent; they prefer di¡erent cations and might be activated by di¡erent mechanisms. The only sophisticated biochemical work on the characterization of a chitin synthase was of that isolated from Absidia glauca, a typical Zygomycete [11]; at present, similar information is not available for A. nidulans chitin synthases, probably due to the di¤culties in isolating each enzyme from the organism. Among ¢lamentous fungi, A. nidulans chsB is the ¢rst gene demonstrated to be an essential gene for hyphal growth [2]. Here, we report on the expression of the chsB cDNA in S. cerevisiae with the S. cerevisiae GAL1 promoter. The expressed chitin synthase B (ChsB) was characterized by biochemical and molecular biological methods. 2. Materials and methods
2.1. Strains, media and cultures
A conidial suspension of Aspergillus nidulans FGSC89 (argB2 biA1) from Fungal Genetics Stock Center (Kansas, USA) was cultured in 1 liter of complete medium (2% malt extract, 0.1% peptone, 2% glucose, 0.2 Wg biotin/ml and 0.2 mg arginine hydrochloride/ml) in a 3-l Erlenmeyer £ask for 3 days at 30³C. Transformation of S. cerevisiae RRA400-1U (MATa his3 trp1 ura3 leu2 chs1: :URA3 chs3: :HIS3)and 4992H (MATa his3 trp1 ura3 leu2 lys2 ade2 chs2: :HIS3) was done by electroporation as described by Delome [12]. Transformants were grown in 0.7% yeast nitrogen base and 2% dextrose (YNBD) supplemented with appropriate amino acids [13]. Plasmids were ampli¢ed in Escherichia coli JM109. E. coli was grown in LB medium and transformation was done by the standard method [14]. 2.2. Poly(A) RNA preparation
Mycelium of A. nidulans was harvested by ¢ltra-
tion and washed with 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE) bu¡er, frozen in liquid nitrogen, and ground in liquid nitrogen with a mortar. The powdered mycelium was then freeze-dried. Total RNA was extracted from the dried mycelial powder in a guanidinium thiocyanate bu¡er [14], and puri¢ed by centrifugation on a cesium chloride cushion. Poly(A) RNA was isolated using Oligotex-dT30 (Takara). 2.3. cDNA preparation of chitin synthase B and plasmid construction
To prepare cDNA for A. nidulans chsB we used an RNA PCR kit (Takara) with A. nidulans poly(A) RNA as the template with the primers E15, 5PACTACCAGTTCTAGAATGGCCTACCACGGCTCTGG-3P and E63, 5P-AGGCAAAGCTCTAGATTACCGACGGGCGAAGCAGC-3P. Then, a full length cDNA (2.8 kb) was ampli¢ed and cloned into the TA vector (Invitrogen). The sequence was con¢rmed by comparison with that of the genomic DNA sequence [2]. For expression in S. cerevisiae, the cDNA was digested with XbaI and cloned into the XbaI site of YpGALx [12] to generate YpGALchsB in which the chsB transcription was under the control of the S. cerevisiae GAL1 promoter (Fig. 1A). 2.4. Sequencing
DNA sequencing was done by the method of Sanger et al. [15]. The reactions were carried out using the autosequencer core kit (Toyobo). The reaction products were analyzed with an A.L.F. DNA sequencer (Pharmacia-Biotech). 2.5. Preparation of mixed membrane fraction S. cerevisiae transformant cells induced with galactose were harvested and washed twice with TE bu¡er and suspended in bu¡er containing 100 mM Tris-HCl, 20 mM MgCl2 , pH 7.5 (Tris-Mg bu¡er), 0.25 mM phenylmethylsulfonyl £uoride, 2 Wg/ml chymostatin, 1.5 Wg/ml leupeptin, 1 Wg/ml pepstatin, and 5 Wg/ml antipain. Cells were lysed with glass beads (0.5 mm in diameter) with a Braun homogenizer (B. Braun), and cell debris was removed by low speed
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centrifugation (3000Ug). The supernatant fraction was then sedimented at 300 000Ug for 40 min at 4³C, and washed once with Tris-Mg bu¡er. The ultracentrifugation was repeated and then the pellet was resuspended in the Tris-MgCl bu¡er containing 33% glycerol. The membrane fractions were diluted to 20 mg/ml and stored at 380³C until use. The protein concentration was measured with the DC Protein assay kit (BioRad).
281
2.6. Chitin synthase assay
The assay of chitin synthase was carried out according to the method of Archer [16]: incubation with 1% (w/w) of trypsin (Sigma) for 15 min at 30³C activated mixed membrane fraction; the reaction was stopped by the addition of a 1.5-fold excess of soybean trypsin inhibitor (Sigma). The standard assay was carried out in 50 Wl of a standard reaction mixture containing 127 mM Tris-HCl, 10 mM MgCl2 , 100 mM GlcNAc, 1 mM UDP-[3 H]GlcNAc (480 000 dpm), 20 Wg of protein of trypsinized membrane, and 13% glycerol at 30³C for 60 min. Reactions were stopped by the addition of 0.5 ml of cold 10% (w/v) trichloroacetic acid. The insoluble material was collected by ¢ltration on a glass ¢ber disk (Whatman). Data are means of duplicate determinations.
Table 1 E¡ect of cations on synthesis of chitin Addition Radioactivity incorporated (dpm/min/mg protein) EDTA 20 1200 MgCl2 MnCl2 730 CaCl2 360 Co(CH3 COO)2 230 CuSO4 33 ZnCl2 30 Data are expressed as radioactivity incorporated into chitin from 1 mM UDP-GlcNAc (480 000 dpm) in 1 h at 30³C. Metal salts were added to a concentration of 8 mM to the reaction mixture. EDTA was added at 4 mM in place of the other metals.
Fig. 1. Restriction and physical map of the S. cerevisiae plasmid YpGALchsB and expression of A. nidulans chitin synthase. A: Pgal, chsB and Tgap mean GAL1 promoter, the open reading frame of A. nidulans chitin synthase B and GAP terminator, respectively. B: Chitin synthase activity of A. nidulans chsB expressed in S. cerevisiae RRA 400-1U. 3. Results and discussion
3.1. Expression of A. nidulans chsB cDNA in S. cerevisiae
Since A. nidulans contains at least four genetically distinct chitin synthases, it seemed very di¤cult to attempt to characterize each chitin synthase directly. In addition, in the yeast host-vector system, no foreign gene containing introns is expressed due to splicing errors, because there is a stringent recognition of the conserved sequence of introns [17,18].
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282
reading frame, and then constructed an expression
S. cerevisiae (Fig. 1A). We expressed A. nidulans chitin synthase B gene in S. cerevisiae under the regulation of the galactose inducible promoter, GAL1 ; YpGALchsB was then transformed into S. cerevisiae RRA400plasmid in
the cDNA of the
1U. The transformant cells were cultured in galactose medium to induce the expression of the gene.
Cells
harboring
the
YpGALchsB
chsB
plasmid
showed a signi¢cantly higher chitin synthase activity whereas those harboring the YpGALx vector did not, demonstrating that the induced chitin synthase activity is derived from the
chsB
gene (Fig.
1B). Bowen et al. [19] classi¢ed all chitin synthases into three classes according to their amino acid sequences. Furthermore, computer analysis allowed us to divide chitin synthases into ¢ve classes, I, II, III,
chsB
IV and V, and showed that
belongs to class
III [2,4]. Interestingly, class III type chitin synthases have been identi¢ed from the ¢lamentous fungi Fig. 2. pH activity pro¢le of
revisiae.
A. nidulans chsB
expressed in
S. ce-
After trypsin treatment, the activated membrane fraction
was assayed at pH values of 6.5^8.3 with 50 mM Tris-maleate bu¡er [24] containing 10 mM MgCl2 , 100 mM GlcNAc and 1 mM UDP-GlcNAc.
nidulans
A. fumigatus
A.
[20]. Complementation
chs2-de¢S. cerevisiae 4992H combination was tested. In contrast to Candida CHS1A [21] encoding a class II chitin synthase, A. nidulans ChsB could not rescue analysis of the YpGALchsB plasmid and
cient
the
A. nidulans chitin synthase B, for A. nidulans chsB and ampli-
[4] and
abnormal
morphology
(unpublished
data).
Thus, to characterize
Therefore, the evolutionary diversity of chitin syn-
we prepared cDNA
thase genes might be re£ected in the failure of func-
¢ed cDNA using the 5P and 3P primer set of the open
Fig. 3. Trypsin treatment of
cerevisiae.
A. nidulans
ChsB expressed in
S.
tional complementation.
Fig. 4. E¡ect of thase B activity.
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N-acetylglucosamine
on
A. nidulans
chitin syn-
K. Tatsuno et al. / FEMS Microbiology Letters 149 (1997) 279^284
Fig. 5. Chitin synthase B of A. nidulans is competitively inhibited by polyoxin D. Polyoxin D concentrations are indicated in the ¢gure.
3.2. Characterization of A. nidulans chitin synthase B
chitin synthase B expressed in S. cerewas characterized. The apparent pH optimum of chitin synthase B was 7.5 when Mg2 was used (Fig. 2). As almost all chitin synthases are activated by partial treatment with trypsin, we examined whether A. nidulans ChsB is activated by trypsin treatment. As shown in Fig. 3, when 1% trypsin was used for the mixed membrane fraction, the ChsB activity increased by about 10-fold compared to the non-treated control. Therefore, ChsB is essentially a zymogen. Furthermore, chitin synthase B activity was enhanced to 3-fold by the addition of GlcNAc, a known chitin synthase activator (Fig. 4). Of the various metal ions added to the standard reaction mixture, magnesium was most e¡ective for stimulating ChsB activity (Table 1). Mn2 had little e¡ect on the activity whereas Cu2 and Zn2 were inhibitory. Addition of EDTA completely abolished the ChsB activity, indicating that like other chitin synthases, some metal(s) is essential for the enzyme activity. We also found that A. nidulans chsB expressed in S. cerevisiae was strongly inhibited by polyoxin D. Kinetic measurements in which the substrate concentration was varied showed competitive inhibition of ChsB by the inhibitor (Fig. 5). From the kinetics, the Km value for the substrate was 1.6 mM, and the Ki A. nidulans visiae A. nidulans
283
value of polyoxin D was 10 WM, which is similar to that for the A. fumigatus enzyme [16]. In conclusion, biochemical analyses of ChsB revealed that (1) ChsB enhanced incorporation of the chitin synthase substrate, UDP-GlcNAc, into the acid-insoluble fraction in S. cerevisiae, (2) the activity was competitively inhibited by the chitin synthase inhibitor, polyoxin D, and (3) the enzyme was a zymogen, requiring divalent cations, and activated by GlcNAc. These characteristics are typical of the properties of chitin synthases [22]. We have demonstrated here that polyoxin D inhibited ChsB activity, but the inhibition was not as strong as that for Chs1 of S. cerevisiae [23]: it most likely would not inhibit the growth of mycelial fungi. Since chsB is essential, inhibitors of ChsB would be ideal antifungal agents. All these data con¢rm that S. cerevisiae is a suitable host for the expression of heterologous genes and that the expressed chsB gene from A. nidulans can be compared to other chitin synthases with similar essential biochemical characteristics. Acknowledgments
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