Eur. J. Biochem. 208,83 - 90 (1992)
0FEBS 1992
The polygalacturonases of Aspergilks niger are encoded by a family of diverged genes Hendrik J. D. BUSSINK', Frank P. BUXTON', Bartholomeus A. FRAAYE', Leo H. de GRAAFF' and Jaap VISSER' Section of Molecular Gcnetics, Department of Genetics, Wageningen Agricultural University, Thc Netherlands Ciba-Gcigy AG, Biotechnology Department, Basel, Switzerland (Received March 6/May 11, 1992) - EJB 920311
Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases T and I1 (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lEMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergilh nidulans transformants on the basis ofactivity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A . niduluns transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A . nidulans transformed with the subcloned pguC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intronlexon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A . niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to rcpresent a binding site for a regulatory protein as it shows a high similarity to the yeast CYCl upstream activation site recognized by the HAP2/3/4 activation complex.
Polygalacturonases are involved in the degradation orpectin, a complex polysaccharide that is primarily found in the middle lamella and primary cell wall of higher plants [I]. Polygalacturonases as well as other pectolytic enzymes are produced by many organisms (reviewed in [2]),and microbial pectolytic enzymes have been extensively studied in relation to plant pathogenesis. The molecular biology of the bacterial enzymes, especially the pectate lyases of Erwiniu, is well developed compared to that of the pectolytic enzymes of fungal pathogens [3, 41. Recently, genomic or cDNA clones coding for pectinesterase [5], pectin lyases [6, 71, pectate lyase [8] and polygalacturonases [9 - 121 have been obtained from Aspei-gillus species, which are considered to be saprophytes. The availability of commercial pectinase preparations derived from Aspergillus niger has contributed to the rapid progress in gene Correspondence to J. Visser, Section of Molccular Genetics, Department of Genetics, Wageningen Agricultural University, Dreyenlaan 2, NL-6703 HA Wageningen, The Netherlands Fax: +31 837083146.
Abbreviation. PG, polygalacturonase. Enzymes. Endopolygalacturonase (EC 3.2.1 .I 5 ) ; exopolygalacturonase (EC 3.2.1.67); pectin lyase (EC 4.2.2.10); pectate lyase (EC 4.2.2.2); pcctinesterase (EC 3.1.1.11). Note. The nucleotide sequence reported in this paper appears in the EMBL, GenBank and DDRJ nucleotide-sequence databases undcr the accession number X64356.
isolation, as well as to studies on plant-cell-wall structure [I 31. A polygalacturonase-inhibiting protein from french bean, that inhibits the polygalacturonases of fungal pathogens similar to an A. niger polygalacturonase and enhances the production of oligogalacturonides that are active as elicitors of defense reactions in plants [14], has also been isolated by affinity chromatography using an A . niger polygalacturonase [15]. Many fungi are known to secrete multiple forms of polygalacturonase, and there are indications that, in some cases, these may be encoded by several genes [16], although the maize pathogen, Cochliobolus carbonurn, probably has only a single endopolygalacturonase gene [17]. Interestingly, its product shows a high similarity to A . niger PGI and PGII, which are encoded by two diverged genes. In addition to these enzymcs, which represent most of the endopolygalacturonase activity from A . niger, three other endopolygalacturonases which resemble PGI in their physicocheniical properties and also react with an antibody raised against PGI have been isolated from a single commercial A . niger pectinase preparation [18]. The role of the individual polygalacturonases in the degradation of plant-cell walls is not understood and has been difficult to address, as this requires substantial amounts of enzyme free of contaminating pectolytic activity. This could be obtained, for example, by expression of the structural polygalacturonase genes from a strong glycolytic A . niger
84 promoter [19].Also, the isolation of the putative genes coding for the less abundant polygalacturonases will contribute to a better understanding of polygalacturonase-gene expression. Here we show that the A . niger polygalacturonases are encoded by a gene family and also characterize the structure of the third member.
Table 1. Classification of recombinant phages hybridizing with thepgaZ2 gene. Phage DNA was restricted with H i d 1 and H i d , electrophoresed in a 1.0% agarose gel, blotted onto nitrocellulose and hybridized with the radiolabelled 1.2-kb BamHT - Bgnl fragment of pGW1800, with washes of 2 x NaCl/Cit. at 62°C. On the basis of ethidium-bromide-stained gels, phages 1I and 12 could be identical and phages 17 and 27 probably have overlapping inserts. Phages numbered 1- 30 were isolated in the first screening of the library at 60°C and phages with a higher number were isolated at 62°C. n.d.,
EXPERIMENTAL PROCEDURES
not determined.
Strains
Gene class
A . niduluns G191 (pahaAl, pyrG89,jwAl, uaY9) [20] was used for transformation. A . niger cotransformants overproduced PGII and PGI from plasmid pGWlS00 and pGW1900, respectively [9, 111. Plasmids were propagated in Escherichia coli JM109 or E. coli DH5aF' and i, phages were plated on E. coli LE392. Manipulation of DNA Preparation of plaque lifts, nick translation, isolation of DNA, Southern-blot analysis and subcloning were performed essentially as described in Maniatis et al. [21]. The gene library of A . niger N400 (CBS 120.49) in the phage4 replacementvector EMBL4 [7] was screened using a hybridization solution that consisted of 1 M NaCI, 50 mM Tris/HCl, pH 7.5, 20 mM EDTA, 0.5% SDS, lox Denhardt's solution [21], 0.1 mg/ml sheared and denatured herring sperm DNA and 0.1 % sodium pyrophosphate. Hybridization was for 40 h for the primary screen (or overnight for subsequent screens), at a temperature of 60°C or 62"C, and filters were washed to 2x NaCl/Cit. (NaCliCit., 0.15 M sodium chloride and 0.015 M trisodium citrate, pH 7.0), 0.1% SDS and 0.1 YOsodium pyrophosphate at the hybridization temperature. Two plasmids were constructed from phage AC20 DNA, pGW1910 and a smaller subclone, pGW1911, that was obtained by ligating the hybridizing 1.3-kb HincII - KpnI fragment into HincIIIKpnIdigested pEMBL18. Subclones of these plasmids were produced in the vector pTZ1SR and sequenced from denatured double-stranded DNA using the Sequenase DNA sequencing kit as recommended (United States Biochemical Corporation) using either universal primer, reverse primer or synthetic oligonucleotide primers to yield the complete sequence from both strands. Nucleotide and amino acid sequences were analyzed using the programs of Devereux et al. [22] and Higgins and Sharp [23].
Transformation and analysis of polygalacturonase production A . nidulclns GI93 was transformed as described [24], using 1 pg pGW635 [25] and 20-40 pg cotransforming DNA. Phage DNA for transformation was obtained from phages 1Al0-43, AB4, ADS-11 and AEh. The polygalacturonase production of Aspergillus strains was analyzed essentially as described before [ll]. In short, A . nidulans was grown at 30°C in medium containing both sugar-beet pulp and pectin as carbon source, ammonium chloride as nitrogen source and a high (1 5 g/l) or a low (1.5 g/l) concentration of monobasic potassium phosphate. Media were supplemented with 1.37 mg/l 4-aminobenzoate and, when appropriate, with 10 mM uridine. Polygalacturonase activity was determined using 50 mM sodium acetate, pH 4.8, containing 0.25% polygalacturonic acid at 30°C; 1 U PG activity was defined as the amount of enzyme that releases 1 pmol reducing end groups (galacturonic acid equivalent)/min. SDS/PAGE was
Phage
Restriction fragment produced with HincII
Hinfl
kb PgaI A B C D
E F G
42' 9, 10, 43 4, 35, 36 1, 16,20, 37 8,11, 12 6, 38 5 17, 27
1.1 1.3 2.4 1.8 1.3; 0.4 1.3; 0.8 2.3 n.d.
1.3; 0.3 0.7; 0.3 1.1 1.3 1.8 0.6 0.6 n.d.
The previously isolated phages PGI-4 and PGI-7 containing the pga1 gene showcd identical fragments performed with the midget-gel system of LKB using Proteintestmischung 4 size markers (Serva).
RESULTS Isolation of sequences related to the pgaZZ gene A . niger N400 DNA was analyzed by Southern blotting, using the 1.2-kb BumHI-BglII fragment of plasmid pGWl800 as a probe and employing hybridization conditions of moderate stringency (at 60"C, with washes of 2 x NaCl/ Cit.). This probe contains most of the structural pgall gene and approximately 200 bp of the 5' upstream region. In addition to a strongly hybridizing fragment with thepgall gene, several other more weakly hybridizing fragments were observed (data not shown). These pgaZZ-related sequences were cloned by screening an A . niger N400 gene library in the phageA replacement-vector EMBL4. In order to identify phages which contain the pgall gene, a duplicate plaque lift was hybridized employing stringent conditions. Thus, among about 10000 phages screened, approximately 30 positive signals were observed on duplicate filters hybridized under conditions of moderate stringency, in addition to seven much stronger signals from phages with thepgall gene. Another set of phages was obtained in a second experiment, which was performed at a slightly higher hybridization temperature (62 "C). After a second screening step, DNA was isolated from a number of these phages and they were classified on the basis of the hybridizing HincII and HinfI fragments observed on Southern blots (Table 1). These frequently cutting restriction enzymes were chosen in order to avoid hybridizing fragments that contain phage4 sequences. Phages containing the pgul gene were also isolated by the same screening procedures and were identified on the basis of hybridization with the 1.S-kb Hind111 fragment of pGWl900 which contains the pgal gene. In addition, one of these phages was further characterized and showed hybridizing fragments identical to those of previously
85 Table 2. Production of polygalacturonaseactivity by A. nidulans G191 transformed with the pyrA gene and by A . niduiuns G191 cotransformed with A . nigev polygalacturonasegenes. The samples were identical to those in Figs 1 and 3 (High phosphate medium, collected 46 h after inoculationj .
Gene
Strain
Activity
PYrA
G191(pGW635j2 G191(pGW1900)6 G19 1 (pG W 19 10)3 G191(AA)1 G191(AB)3 G191(AD)1 G191(%E)8
U/ml
Fig. 1. High-level expression of the A. nigevpguZandpguC genes in A . nidulans. Proteins in the culture medium of A . niduluns transformants grown for 46 h on pectin and sugar-beet-pulp medium with a low phosphate or a high phosphate concentration were separated on a 10% SDSjPAGE gel and stained with Coomassie brilliant blue. The transformants shown are included in Table 2; thepgaC transformant was constructed using the plasmid CpGW1910) described in Fig. 2. (A) Lane 1 , pyrA transformant (control)/low phosphate; lane 2, p g a l transformant/low phosphate; lane 3, pgnl transformant/high phosphate; lane 4, pyr A transformant/high phosphate; lane 5 , pgaC transformant/high phosphate. In each lane 11 pl culture medium was loaded. (B) Lane 1, pgaC transformant/high phosphate; lane 2, p g a l transformant/high phosphate; lane 3, PGI purified from a commercial A . niger pectinase. Molecular-mass markcrs (a, 92.5 kDa; b, 67 kDa; c, 45 kDa; d, 29 kDa) are indicated. In lanes 1 and 2, 1.1 p1 culture medium was loaded.
characterized pgal phages but different from those observed for the other classes. As the gene library used was complex, we were able to identify at least five different A. niger sequences related to the pgull gene, termed classes A-E. These classes each contain independent phages and, therefore, do not reflect cloning artefacts. The polygalacturonase gene of class-C phages, which was further characterized, has been designated pguC. Those of the other classes have been tentatively designated pguA, pguB, pguD and pguE, accordingly. The single phage of class F may represent a sixth related sequence but could also contain part of the sequence present in phages of another class or, less likely, it may result from a cloning artefact. The two phages of class G gave a much weaker hybridization signal than phages of the other classes.
Expression of A . niger polygalacturonase genes in A . nidulans From previous results [lo, 11, 181it was expected that the identification of the polygalacturonases that are encoded by the pgall-related sequences, following the introduction of these sequences into the A . niger genome, would not be straightforward. Therefore, we tested A . niduluns, to determine if it could serve as a suitable host. Plasmids pGWl800 and pGW1900 were used to cotransform A . niduluns GI91 to uridine prototrophy, using a plasmid containing the A. niger p y r A gene as the selectable marker. In general, the A . nidukuns pGWl800 and pGWl900 transformants produced higher levels of A. niger polygalacturonase than the corresponding A. niger transformants, whereas the A . nidulans pGW1800 transformants also produced PGII when grown on glucose (data not shown). Strains were obtained that secreted PGI into the culture medium as the most abundant protein, and a further increase in enzyme yield was observed upon using a higher phosphate concentration (Fig. 1 ) as had been pre-
PgaC PgaA PgaB PgaD PgaE
BE S
U I
K H 3 I
I
I
0.72 510 33 0.83 8.7 2.0 1.5
KXS
E
II I
I
I///’/////////,j
+
SpX
,
6
I ,
n 1 kbp
Fig. 2. Restriction map of the 7.8-kb BgnI fragment that contains the A . niger pgaC gene. The protein-coding region (shadcd bar) and the direction of transcription (arrow) arc indicated. In plasmid pGW1910 the 5’ end of the fragment as shown is adjacent to the Sun site in the pUC9 polylinker and this plasmid has no BgnI sites. The insert of pGW1911 extends from the indicated HincII site, which is not unique, to the 3’ KpnI site. B, BgZII; E, EcoRl; S, Smal; K, KpnI; H, HincII; x, XhoI; sp, SphI.
viously observed with A . niger [26]. However, A . niger and A . nidulans showed a somewhat different response, since with A . nidulans, the high phosphate concentration used (1 10 mM) was effective in preventing a large drop in the pH of the growth medium and did not adversely affect the formation of fungal biomass. Optimal PGI levels were observed around two days after inoculation, after which the decreased intensity of the major band on Coomassie-brilliant-blue-stained SDS/ PAGE gels and the occurrence of minor bands of lower molecular mass, that were also present earlier in the fermentation (Fig. 1) and could be seen on Western blots, indicated some proteolytic degradation of PGI. The PGI produced by A . niduluns transformants, at a level of approximately 1 g/1 in batch culture, was enzymically active (Table 2) and showed the same apparent molecular mass as the PGI isolated from A . niger (Fig. 1). The 7.8-kb BglII fragment of phage IC20 that hybridized to the pguIl-coding region from pGWl800, was inserted into the BuwlHI site of pUC9 and the resulting plasmid, pGW1910, was further characterized (Fig. 2 ) . The A . niduluns cotransformants constructed with pGW1910 produced a novel protein (Fig. l), which could also be detected on Western blots probed with polyclonal antibodies raised against PGI and PGII. This polygalacturonase, designated PGC, has an apparent molecular mass of 61 kDa, as estimated by SDSjPAGE with 10% polyacrylamide gels. The polygalacturonase activity of culture filtrates of the PGC-producing transformants (Table 2) was much lower than that of filtrates which contained a very similar amount of PGI, indicating that these polygalacturonases have different enzymic properties. As 50% of the transformants analyzed produced very similar amounts of PGC to the strain shown in Fig. 1, it would appear that genes coding for less abundant A . niger polygalacturonases can be efficiently expressed in A . niduluns. Thus, other phage DNA species were used to cotransform A . niduluns and novel
86 nucleotide sequence was determined on the basis of the similarity of the deduced amino acid sequence to those of other fungal polygalacturonases. The presence of three introns in the pguC gene was inferred by the presence of introns at the corresponding positions in other fungal polygalacturonase genes, the shifting of the reading frame (introns B and C) and the occurrence of in-frame stop codons in all three introns. The sequences found at the splice sites and the conserved internal sequence CTRAC, which in the pguC introns starts 15 or 16 nucleotides before the 3' splice site, are identical to or closely resemble those in other fungal introns [27]. The deduced amino acid sequence of PGC has an Nterminal extension of 40 amino acids before the putative start of the mature protein. Thus, the PGC precursor is probably Fig. 3. Expression of the A. nigerpgaA, pgaB,pgaD andpgaE genes in cleaved after a pair of basic amino acids as is also the case for A . nidulans. Transformants were grown in high phosphate medium as some other secreted fungal proteins [28], including the PGI described in the legend to Fig. 1 and the culture media were analyzcd precursor. In addition, a possible signal-peptidase-cleavage by Western blotting, using a polyclonal antiserum raised against PG1 site is also found after Ala16 [29], suggesting that PGC is and an alkaline-phosphatase-conjugated secondary antibody. The synthesized as a prepro-protein. The molecular mass of thc transformants shown are included in Table 2. Lane 1, A . nidulans G191; lanc 2, p y r A transformant; lane 3, pguA transformant; lane 4, mature PGC, calculated from the deduced amino acid sequence, namely 36.2 kDa, is much lower than the value estipguB transformant; lanes 5 and 7 , p g a D transformant; lanes 6 and 8, pgaE transformant. The samples were collected 46 h (lanes 1-6) or mated from SDS/PAGE, as has also been observed for PGI 61 h (lanes 7 and 8) after inoculation. Thc positions of PGI (I) and [111. PGII (11) are indicated by arrows. The bands below the PGII marker, The codon usage of the pguC gene, in which 55 different in lanes 7 and 8, represent an A . nidulans protein that was also sense codons occur, is much less biased than that of the pgal obscrvcd in the 61-h samples of the other transformants. gene and more closely resembles the codon usage in the pgdZ gene. polygalacturonases were detected upon Western-blot analysis of the culture medium of strains transformed with DNA of the phage classes A, B, D and E (Fig. 3). In most cases, these strains also produced a considerably higher total polygalacturonase activity than a strain transformed with the pyrA gene alone (Table 2) or the host A . niduluns (3191. Southern-blot analysis of DNA isolated from these strains, using the pguII probe under hybridization conditions ofmoderate stringency, showed the presence of newly acquired hybridizing sequences, which gave stronger signals than the pgaZI-related sequences of A . nidulans (3191 (data not shown). Upon probing with 1 DNA, phage4 sequences were also detected in the DNA of the transformants. The expression of the A . nigerpgaA,pguD andpgaE genes in A . niduluns resulted in products which showed an electrophoretic mobility similar to that of PGI, and thus these polygalacturonases may correspond to polygalacturonases isolated from a commercial enzyme preparation [18]. The PGE consisted of two major bands with a small difference in apparent molecular mass, similar to the products of the A . niger pelA gene expressed in A . nidulans that have been shown to differ in the extent of glycosylation [24]. The electrophoretic pattern of the putative products of the pguD gene was more complex, as several forms with a considerable difference in apparent molecular mass were detected, and it can be seen that their ratios changed during the fermentation. It is also possible that AD phages contain more than one polygalacturonase gene. PGB resembles PGII in its apparent molecular mass but was not detected with a PGIIspecific monoclonal antibody and gave a much stronger signal than the PGII control upon probing with a polyclonal antibody raised against PGI, which has a low affinity for PGII [181.
Similarity of fungal polygalacturonases The deduced mature amino acid sequence of PGC shows a high degree of sequence similarity with those of other fungal polygalacturonases, namely 60.9% with PGI, 55.9% with PGII and 57.9% with the polygalacturonase of C. carboizum [I71 and amongst all these proteins 36.5% of the amino acids are conserved. The sequence of mature PGC is eight amino acids longer than that ofPGII, whereas the mature PGI, PGII and C. carbonum polygalacturonase sequences only differ in length by, at most, two amino acids. The two single amino acid insertions which are found in the PGI sequence with respect to that of PGII, are also present in the PGC sequence. In addition, at five different positions in the PGC sequence, insertions of 1- 3 amino acids have occurred, including the extension of two amino acids at the C-terminus. Also, a single deletion of two amino acids is observed with respect to the sequences of the three other fungal polygalacturonases (data not shown). Alignment of the N-terminal amino acid sequences of these polygalacturonases with those of the major polygalacturonases of Colletotrichum lindeniuthiunum and Sclerotiniu sclerotiorunz [30] is shown in Fig. 5. The occurrence of insertions or deletions at two positions in this region underscores the intermediate position of the PGI sequence with respect to the PGC and PGII scquences. It can be seen that the N-terminal amino acid sequences of the C. carbonum and C. lindenzuthiumm polygalacturonases closely resemble the A . niger PGII sequence, as opposed to the corresponding S. sclerotiorum sequence which shows the same insertions or deletions as the PGC sequcnce. Tntronlexon organization of polygalacturonase genes
The nucleotide sequence of thepgaC gene and the deduced amino acid sequence The nucleotide sequence of the pguC gene was determined from both strands of DNA (Fig. 4) and the PGC-encoding
The pgaC gene contains three introns, the highest number found for a fungal polygalacturonase gene so far. The introns in the other two sequenced A . niger genes occur at the same position and in the same phase of the reading frame as introns
87 -601
CCATGGCTGAAACTTTGCCCCTGACCTAGTCCATTTCATTTTATATAATGCTGATGAGATAT GGTTCTTGCACAACTATGCTTTTCCTCGGTCGGTGCGTAATGTATAGTTCGTGGGTGGTAAGGAAATTCACCGACAGCAACTCCCAGCATAACATGGAGAAGAACCGATGAATGAGCAGC
-481
GACAAACATGGTACCACAATTCTTGAATAAGCATTTAGACTCCGGCTTGGTTTTTTCCGTTGCATTGGCGCTGGTGTTGTTCCTGCGCACGGTGCAACCGTTATCTCCACCAATGATTAC -361
+++++++++++
CTGCAGAAAGCCCATGGCTGCAACCACCCAGCCGACTTGAGTCCACCTAACAGCATACTCAGCATAC~CGATGCTGGGAATTACTTCAGGTATGGTCGGCTGAATATGCAGTCGATTTCC
-241
GGCGCCAGGAACAGTCCGGCCCCCTCACCACGGACATAGACCCGCCACCAAAACGTCGATAGCGGCCTCGCTTCGTCAACGACCATTTTGCCCAC~CACCCAGGAAAGACGTCTT
-121
CCAATAACAATATAAAAGGGCGAGTCTTGTCCTCTCACTTTCCGTCTTTCGAGTATGCCTTGCCAGTTCCCAACTTGACTTGAGACCTCCCATTTCGGTCATTTGTCACCCGCTATAAGA
-1
ATG GTC CGT CAG C T T A T C CTG ATC AGC AGT CTG CTG GCA GCT GTT GCT GTG CGC GCG CCT GCC GAT CCG GCT CAT CCC ATG GTT ACG GAA M V R 0 L I L I S S L L A A V A n V R A P A D P A H P M V T E
90 30
GCG CCT GAC GTC AAT TTG GTT GAA AAG AGG GCG ACT ACT TGC ACC TTC TCG GGC TCC GAA GGT GCA TCC AAG GCC AGC AAG TCG AAA ACC A P D V N L V E K R n A T T C T F S G S E G A S K A S K S K T
180 60
TCT TGC TCC ACA A T C TAC CTG TCC GAC GTG GCC GTC CCA TCT GGC ACA ACC CTT GAT CTC TCT GAC CTG AAT GAT GGA ACC CAC W S C S T I Y L S D V A V P S G T T L D L S D L N D G T H
272
*******
T
C
88
C C C G G G T G A T T C A T G T C T T A T T C T C A C A C C C A A T C G C G T C A A T A G T A C T G G ~ A G A T G T A C ~GTG ATC TTC CAG GGA GAA ACC ACT TTT GGA TAC GAG GAA intron A
E
G
F
Q
G
E
D
S
G
Y
E
E
378 101
GTG CAG GTG T T C AGC ATC GAT GGC TCC ACT GAT CTT ACC ATG ACT GAT ATC ACG GTG GAT AAC ACG GAT GGT GAC D
648 191
GGT GCC GAA ATC TAC AAC CAA GAT G A E I Y N Q D
738 221
TTC AGT GCC
839 236
V
Q
V
F
S
L
A
A
N
T
D
G
l
D
G
S
T
D
L
T
M
T
D
l
F
D
I
G
E
S
T
Y
I
T
I
T
T
V
D
N
T
A T 1 TAT ~ G T C C C C T G G C A C T G A T T G A G A G G A G A G G C G C C T T G ~ G A T C T G G AAC ~ intron 8 N I Y
GAC TGC GTT GCC ATC AAT TCT GGA GAG D C V A I N S G E AGT
F
558 161
ACG GAC GAC CTC GCC GCC AAT ACG GAT GGC TTC GAC A T C GGG GAA AGC ACC TAT ATC ACG A T C ACA
D
T
GGT GGC AAT GGT GGT AAA ACA AAG CCC AAG TTC TTC TAT GCC CAT GAC TTG ACC TCT TCC ACC ATC AAG AGC ATC TAC G G N G G K T K P K F F Y A H D L T S S T I K S I Y
L
ATC GAG AAC TCT CCT I E N S P
T
T
468
P
TGG GAT GGA GAG W D G E
I
GTG CGT GTT TCT GGA ACT GAT ATC ACG GTC GAG GGG GAG AGC GAC GCG GTG CTC AAT GGC GAT GGC AGC CGC TGG V R V S G T D I T V E G E S D A V L N G D G S R W
TGG GAA GGG CCT CTT
W
V
D
F
G
S
A
GTG TGT TCT GGT GGT CAC GGC CTG TCT A T 1 GGC TCT GTT GGT GGT CGG GAT GAT AAC ACT GTC AAG AAC GTG ACG T T T TAT GAT GTC V C S G G H G L S I G S V G G R D D N T V K N V T F Y D V
131
929 266
#
G T A A G G G A A A G C A T T T G A A C C A T C C G T T T G C C G T C C T ~ G T A T G C T TCA ~ ATC CGT ATC AAG ACC A T C TAC GGC intron C A I R l K T l Y G
1030
GAC ACT GGC TCC GTC AGC GAA GTC ACT TAC CAT GAG A T 1 GCC TTT TCG GAC GCT ACT GAT TAC GGC A T T GTC A T 1 GAG CAG AAC T A T GAT D T G S V S E V T Y H E I A F S D A T D Y G I V I E Q N Y D
1120 312
TGT ACC C T
1210 342
GAA GTA TAC A T 1 GCC TGC GGT GAT GGC AGC TGC TCG GAT TGG ACT TGG ACT GGC GTG AGC GTG ACT GGC GGC AGT GTC AGT GAC GAC TGC
1300 372
AAT GTT CTC AAG TCC CAG CAA G N V L K S P P
GAC ACC TCC AAG ACC CCT ACT ACC GGG GTA CCG ATC ACG GAT T T T GTG CTC GAG AAC ATC GTT GGA ACG TGT GAA GAT GAT GAT
D
T
E
V
C T T AAT L N
S Y
K I
T A
P C
T G
T D
G G
V S
P C
I S
T D
O W
F T
V W
L T
E G
N V
I S
V V
G T
T G
C G
E S
D V
D S
D D
D
C
GTT CCC TCC GGG A T 1 AGC TGC GAT TTG TAG GCCGTTGTGATTGGGGGAGATGTTCCCGGGTACTCTGGTCGTTCGTTTAGCCAGCCCTTTCTGTTGAGTGGC V P S G I S C D L - - -
282
1408 383
CTGTTCTTCTATACTGTCGATTGTGTGTGAGATTACTACCTTGCCCGAGGAAAGAGCAGTGCATCCTATCTTTTATTCTTTGCCGGTCGGGGTTGGCCTACTAGTACATTGTACCCGAAG
1528
AGTCAACGGTGAAAGTAAAGGGCTTACAGAATTAGTCCAGGAGCAAAGACAACTTTTATCAGAGCGCATCGGTCACGTCATGTTGAAAGTATTGATCAATGAAGATAACCTTGG
1642
Fig.4. Nucleotide and deduced amino acid sequences of the A . niger N400pgaC gene. The putativc start of the mature protein ($-), a potential signal peptidase cleavage site (
0FEBS 1992
The polygalacturonases of Aspergilks niger are encoded by a family of diverged genes Hendrik J. D. BUSSINK', Frank P. BUXTON', Bartholomeus A. FRAAYE', Leo H. de GRAAFF' and Jaap VISSER' Section of Molecular Gcnetics, Department of Genetics, Wageningen Agricultural University, Thc Netherlands Ciba-Gcigy AG, Biotechnology Department, Basel, Switzerland (Received March 6/May 11, 1992) - EJB 920311
Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases T and I1 (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lEMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergilh nidulans transformants on the basis ofactivity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A . niduluns transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A . nidulans transformed with the subcloned pguC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intronlexon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A . niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to rcpresent a binding site for a regulatory protein as it shows a high similarity to the yeast CYCl upstream activation site recognized by the HAP2/3/4 activation complex.
Polygalacturonases are involved in the degradation orpectin, a complex polysaccharide that is primarily found in the middle lamella and primary cell wall of higher plants [I]. Polygalacturonases as well as other pectolytic enzymes are produced by many organisms (reviewed in [2]),and microbial pectolytic enzymes have been extensively studied in relation to plant pathogenesis. The molecular biology of the bacterial enzymes, especially the pectate lyases of Erwiniu, is well developed compared to that of the pectolytic enzymes of fungal pathogens [3, 41. Recently, genomic or cDNA clones coding for pectinesterase [5], pectin lyases [6, 71, pectate lyase [8] and polygalacturonases [9 - 121 have been obtained from Aspei-gillus species, which are considered to be saprophytes. The availability of commercial pectinase preparations derived from Aspergillus niger has contributed to the rapid progress in gene Correspondence to J. Visser, Section of Molccular Genetics, Department of Genetics, Wageningen Agricultural University, Dreyenlaan 2, NL-6703 HA Wageningen, The Netherlands Fax: +31 837083146.
Abbreviation. PG, polygalacturonase. Enzymes. Endopolygalacturonase (EC 3.2.1 .I 5 ) ; exopolygalacturonase (EC 3.2.1.67); pectin lyase (EC 4.2.2.10); pectate lyase (EC 4.2.2.2); pcctinesterase (EC 3.1.1.11). Note. The nucleotide sequence reported in this paper appears in the EMBL, GenBank and DDRJ nucleotide-sequence databases undcr the accession number X64356.
isolation, as well as to studies on plant-cell-wall structure [I 31. A polygalacturonase-inhibiting protein from french bean, that inhibits the polygalacturonases of fungal pathogens similar to an A. niger polygalacturonase and enhances the production of oligogalacturonides that are active as elicitors of defense reactions in plants [14], has also been isolated by affinity chromatography using an A . niger polygalacturonase [15]. Many fungi are known to secrete multiple forms of polygalacturonase, and there are indications that, in some cases, these may be encoded by several genes [16], although the maize pathogen, Cochliobolus carbonurn, probably has only a single endopolygalacturonase gene [17]. Interestingly, its product shows a high similarity to A . niger PGI and PGII, which are encoded by two diverged genes. In addition to these enzymcs, which represent most of the endopolygalacturonase activity from A . niger, three other endopolygalacturonases which resemble PGI in their physicocheniical properties and also react with an antibody raised against PGI have been isolated from a single commercial A . niger pectinase preparation [18]. The role of the individual polygalacturonases in the degradation of plant-cell walls is not understood and has been difficult to address, as this requires substantial amounts of enzyme free of contaminating pectolytic activity. This could be obtained, for example, by expression of the structural polygalacturonase genes from a strong glycolytic A . niger
84 promoter [19].Also, the isolation of the putative genes coding for the less abundant polygalacturonases will contribute to a better understanding of polygalacturonase-gene expression. Here we show that the A . niger polygalacturonases are encoded by a gene family and also characterize the structure of the third member.
Table 1. Classification of recombinant phages hybridizing with thepgaZ2 gene. Phage DNA was restricted with H i d 1 and H i d , electrophoresed in a 1.0% agarose gel, blotted onto nitrocellulose and hybridized with the radiolabelled 1.2-kb BamHT - Bgnl fragment of pGW1800, with washes of 2 x NaCl/Cit. at 62°C. On the basis of ethidium-bromide-stained gels, phages 1I and 12 could be identical and phages 17 and 27 probably have overlapping inserts. Phages numbered 1- 30 were isolated in the first screening of the library at 60°C and phages with a higher number were isolated at 62°C. n.d.,
EXPERIMENTAL PROCEDURES
not determined.
Strains
Gene class
A . niduluns G191 (pahaAl, pyrG89,jwAl, uaY9) [20] was used for transformation. A . niger cotransformants overproduced PGII and PGI from plasmid pGWlS00 and pGW1900, respectively [9, 111. Plasmids were propagated in Escherichia coli JM109 or E. coli DH5aF' and i, phages were plated on E. coli LE392. Manipulation of DNA Preparation of plaque lifts, nick translation, isolation of DNA, Southern-blot analysis and subcloning were performed essentially as described in Maniatis et al. [21]. The gene library of A . niger N400 (CBS 120.49) in the phage4 replacementvector EMBL4 [7] was screened using a hybridization solution that consisted of 1 M NaCI, 50 mM Tris/HCl, pH 7.5, 20 mM EDTA, 0.5% SDS, lox Denhardt's solution [21], 0.1 mg/ml sheared and denatured herring sperm DNA and 0.1 % sodium pyrophosphate. Hybridization was for 40 h for the primary screen (or overnight for subsequent screens), at a temperature of 60°C or 62"C, and filters were washed to 2x NaCl/Cit. (NaCliCit., 0.15 M sodium chloride and 0.015 M trisodium citrate, pH 7.0), 0.1% SDS and 0.1 YOsodium pyrophosphate at the hybridization temperature. Two plasmids were constructed from phage AC20 DNA, pGW1910 and a smaller subclone, pGW1911, that was obtained by ligating the hybridizing 1.3-kb HincII - KpnI fragment into HincIIIKpnIdigested pEMBL18. Subclones of these plasmids were produced in the vector pTZ1SR and sequenced from denatured double-stranded DNA using the Sequenase DNA sequencing kit as recommended (United States Biochemical Corporation) using either universal primer, reverse primer or synthetic oligonucleotide primers to yield the complete sequence from both strands. Nucleotide and amino acid sequences were analyzed using the programs of Devereux et al. [22] and Higgins and Sharp [23].
Transformation and analysis of polygalacturonase production A . nidulclns GI93 was transformed as described [24], using 1 pg pGW635 [25] and 20-40 pg cotransforming DNA. Phage DNA for transformation was obtained from phages 1Al0-43, AB4, ADS-11 and AEh. The polygalacturonase production of Aspergillus strains was analyzed essentially as described before [ll]. In short, A . nidulans was grown at 30°C in medium containing both sugar-beet pulp and pectin as carbon source, ammonium chloride as nitrogen source and a high (1 5 g/l) or a low (1.5 g/l) concentration of monobasic potassium phosphate. Media were supplemented with 1.37 mg/l 4-aminobenzoate and, when appropriate, with 10 mM uridine. Polygalacturonase activity was determined using 50 mM sodium acetate, pH 4.8, containing 0.25% polygalacturonic acid at 30°C; 1 U PG activity was defined as the amount of enzyme that releases 1 pmol reducing end groups (galacturonic acid equivalent)/min. SDS/PAGE was
Phage
Restriction fragment produced with HincII
Hinfl
kb PgaI A B C D
E F G
42' 9, 10, 43 4, 35, 36 1, 16,20, 37 8,11, 12 6, 38 5 17, 27
1.1 1.3 2.4 1.8 1.3; 0.4 1.3; 0.8 2.3 n.d.
1.3; 0.3 0.7; 0.3 1.1 1.3 1.8 0.6 0.6 n.d.
The previously isolated phages PGI-4 and PGI-7 containing the pga1 gene showcd identical fragments performed with the midget-gel system of LKB using Proteintestmischung 4 size markers (Serva).
RESULTS Isolation of sequences related to the pgaZZ gene A . niger N400 DNA was analyzed by Southern blotting, using the 1.2-kb BumHI-BglII fragment of plasmid pGWl800 as a probe and employing hybridization conditions of moderate stringency (at 60"C, with washes of 2 x NaCl/ Cit.). This probe contains most of the structural pgall gene and approximately 200 bp of the 5' upstream region. In addition to a strongly hybridizing fragment with thepgall gene, several other more weakly hybridizing fragments were observed (data not shown). These pgaZZ-related sequences were cloned by screening an A . niger N400 gene library in the phageA replacement-vector EMBL4. In order to identify phages which contain the pgall gene, a duplicate plaque lift was hybridized employing stringent conditions. Thus, among about 10000 phages screened, approximately 30 positive signals were observed on duplicate filters hybridized under conditions of moderate stringency, in addition to seven much stronger signals from phages with thepgall gene. Another set of phages was obtained in a second experiment, which was performed at a slightly higher hybridization temperature (62 "C). After a second screening step, DNA was isolated from a number of these phages and they were classified on the basis of the hybridizing HincII and HinfI fragments observed on Southern blots (Table 1). These frequently cutting restriction enzymes were chosen in order to avoid hybridizing fragments that contain phage4 sequences. Phages containing the pgul gene were also isolated by the same screening procedures and were identified on the basis of hybridization with the 1.S-kb Hind111 fragment of pGWl900 which contains the pgal gene. In addition, one of these phages was further characterized and showed hybridizing fragments identical to those of previously
85 Table 2. Production of polygalacturonaseactivity by A. nidulans G191 transformed with the pyrA gene and by A . niduiuns G191 cotransformed with A . nigev polygalacturonasegenes. The samples were identical to those in Figs 1 and 3 (High phosphate medium, collected 46 h after inoculationj .
Gene
Strain
Activity
PYrA
G191(pGW635j2 G191(pGW1900)6 G19 1 (pG W 19 10)3 G191(AA)1 G191(AB)3 G191(AD)1 G191(%E)8
U/ml
Fig. 1. High-level expression of the A. nigevpguZandpguC genes in A . nidulans. Proteins in the culture medium of A . niduluns transformants grown for 46 h on pectin and sugar-beet-pulp medium with a low phosphate or a high phosphate concentration were separated on a 10% SDSjPAGE gel and stained with Coomassie brilliant blue. The transformants shown are included in Table 2; thepgaC transformant was constructed using the plasmid CpGW1910) described in Fig. 2. (A) Lane 1 , pyrA transformant (control)/low phosphate; lane 2, p g a l transformant/low phosphate; lane 3, pgnl transformant/high phosphate; lane 4, pyr A transformant/high phosphate; lane 5 , pgaC transformant/high phosphate. In each lane 11 pl culture medium was loaded. (B) Lane 1, pgaC transformant/high phosphate; lane 2, p g a l transformant/high phosphate; lane 3, PGI purified from a commercial A . niger pectinase. Molecular-mass markcrs (a, 92.5 kDa; b, 67 kDa; c, 45 kDa; d, 29 kDa) are indicated. In lanes 1 and 2, 1.1 p1 culture medium was loaded.
characterized pgal phages but different from those observed for the other classes. As the gene library used was complex, we were able to identify at least five different A. niger sequences related to the pgull gene, termed classes A-E. These classes each contain independent phages and, therefore, do not reflect cloning artefacts. The polygalacturonase gene of class-C phages, which was further characterized, has been designated pguC. Those of the other classes have been tentatively designated pguA, pguB, pguD and pguE, accordingly. The single phage of class F may represent a sixth related sequence but could also contain part of the sequence present in phages of another class or, less likely, it may result from a cloning artefact. The two phages of class G gave a much weaker hybridization signal than phages of the other classes.
Expression of A . niger polygalacturonase genes in A . nidulans From previous results [lo, 11, 181it was expected that the identification of the polygalacturonases that are encoded by the pgall-related sequences, following the introduction of these sequences into the A . niger genome, would not be straightforward. Therefore, we tested A . niduluns, to determine if it could serve as a suitable host. Plasmids pGWl800 and pGW1900 were used to cotransform A . niduluns GI91 to uridine prototrophy, using a plasmid containing the A. niger p y r A gene as the selectable marker. In general, the A . nidukuns pGWl800 and pGWl900 transformants produced higher levels of A. niger polygalacturonase than the corresponding A. niger transformants, whereas the A . nidulans pGW1800 transformants also produced PGII when grown on glucose (data not shown). Strains were obtained that secreted PGI into the culture medium as the most abundant protein, and a further increase in enzyme yield was observed upon using a higher phosphate concentration (Fig. 1 ) as had been pre-
PgaC PgaA PgaB PgaD PgaE
BE S
U I
K H 3 I
I
I
0.72 510 33 0.83 8.7 2.0 1.5
KXS
E
II I
I
I///’/////////,j
+
SpX
,
6
I ,
n 1 kbp
Fig. 2. Restriction map of the 7.8-kb BgnI fragment that contains the A . niger pgaC gene. The protein-coding region (shadcd bar) and the direction of transcription (arrow) arc indicated. In plasmid pGW1910 the 5’ end of the fragment as shown is adjacent to the Sun site in the pUC9 polylinker and this plasmid has no BgnI sites. The insert of pGW1911 extends from the indicated HincII site, which is not unique, to the 3’ KpnI site. B, BgZII; E, EcoRl; S, Smal; K, KpnI; H, HincII; x, XhoI; sp, SphI.
viously observed with A . niger [26]. However, A . niger and A . nidulans showed a somewhat different response, since with A . nidulans, the high phosphate concentration used (1 10 mM) was effective in preventing a large drop in the pH of the growth medium and did not adversely affect the formation of fungal biomass. Optimal PGI levels were observed around two days after inoculation, after which the decreased intensity of the major band on Coomassie-brilliant-blue-stained SDS/ PAGE gels and the occurrence of minor bands of lower molecular mass, that were also present earlier in the fermentation (Fig. 1) and could be seen on Western blots, indicated some proteolytic degradation of PGI. The PGI produced by A . niduluns transformants, at a level of approximately 1 g/1 in batch culture, was enzymically active (Table 2) and showed the same apparent molecular mass as the PGI isolated from A . niger (Fig. 1). The 7.8-kb BglII fragment of phage IC20 that hybridized to the pguIl-coding region from pGWl800, was inserted into the BuwlHI site of pUC9 and the resulting plasmid, pGW1910, was further characterized (Fig. 2 ) . The A . niduluns cotransformants constructed with pGW1910 produced a novel protein (Fig. l), which could also be detected on Western blots probed with polyclonal antibodies raised against PGI and PGII. This polygalacturonase, designated PGC, has an apparent molecular mass of 61 kDa, as estimated by SDSjPAGE with 10% polyacrylamide gels. The polygalacturonase activity of culture filtrates of the PGC-producing transformants (Table 2) was much lower than that of filtrates which contained a very similar amount of PGI, indicating that these polygalacturonases have different enzymic properties. As 50% of the transformants analyzed produced very similar amounts of PGC to the strain shown in Fig. 1, it would appear that genes coding for less abundant A . niger polygalacturonases can be efficiently expressed in A . niduluns. Thus, other phage DNA species were used to cotransform A . niduluns and novel
86 nucleotide sequence was determined on the basis of the similarity of the deduced amino acid sequence to those of other fungal polygalacturonases. The presence of three introns in the pguC gene was inferred by the presence of introns at the corresponding positions in other fungal polygalacturonase genes, the shifting of the reading frame (introns B and C) and the occurrence of in-frame stop codons in all three introns. The sequences found at the splice sites and the conserved internal sequence CTRAC, which in the pguC introns starts 15 or 16 nucleotides before the 3' splice site, are identical to or closely resemble those in other fungal introns [27]. The deduced amino acid sequence of PGC has an Nterminal extension of 40 amino acids before the putative start of the mature protein. Thus, the PGC precursor is probably Fig. 3. Expression of the A. nigerpgaA, pgaB,pgaD andpgaE genes in cleaved after a pair of basic amino acids as is also the case for A . nidulans. Transformants were grown in high phosphate medium as some other secreted fungal proteins [28], including the PGI described in the legend to Fig. 1 and the culture media were analyzcd precursor. In addition, a possible signal-peptidase-cleavage by Western blotting, using a polyclonal antiserum raised against PG1 site is also found after Ala16 [29], suggesting that PGC is and an alkaline-phosphatase-conjugated secondary antibody. The synthesized as a prepro-protein. The molecular mass of thc transformants shown are included in Table 2. Lane 1, A . nidulans G191; lanc 2, p y r A transformant; lane 3, pguA transformant; lane 4, mature PGC, calculated from the deduced amino acid sequence, namely 36.2 kDa, is much lower than the value estipguB transformant; lanes 5 and 7 , p g a D transformant; lanes 6 and 8, pgaE transformant. The samples were collected 46 h (lanes 1-6) or mated from SDS/PAGE, as has also been observed for PGI 61 h (lanes 7 and 8) after inoculation. Thc positions of PGI (I) and [111. PGII (11) are indicated by arrows. The bands below the PGII marker, The codon usage of the pguC gene, in which 55 different in lanes 7 and 8, represent an A . nidulans protein that was also sense codons occur, is much less biased than that of the pgal obscrvcd in the 61-h samples of the other transformants. gene and more closely resembles the codon usage in the pgdZ gene. polygalacturonases were detected upon Western-blot analysis of the culture medium of strains transformed with DNA of the phage classes A, B, D and E (Fig. 3). In most cases, these strains also produced a considerably higher total polygalacturonase activity than a strain transformed with the pyrA gene alone (Table 2) or the host A . niduluns (3191. Southern-blot analysis of DNA isolated from these strains, using the pguII probe under hybridization conditions ofmoderate stringency, showed the presence of newly acquired hybridizing sequences, which gave stronger signals than the pgaZI-related sequences of A . nidulans (3191 (data not shown). Upon probing with 1 DNA, phage4 sequences were also detected in the DNA of the transformants. The expression of the A . nigerpgaA,pguD andpgaE genes in A . niduluns resulted in products which showed an electrophoretic mobility similar to that of PGI, and thus these polygalacturonases may correspond to polygalacturonases isolated from a commercial enzyme preparation [18]. The PGE consisted of two major bands with a small difference in apparent molecular mass, similar to the products of the A . niger pelA gene expressed in A . nidulans that have been shown to differ in the extent of glycosylation [24]. The electrophoretic pattern of the putative products of the pguD gene was more complex, as several forms with a considerable difference in apparent molecular mass were detected, and it can be seen that their ratios changed during the fermentation. It is also possible that AD phages contain more than one polygalacturonase gene. PGB resembles PGII in its apparent molecular mass but was not detected with a PGIIspecific monoclonal antibody and gave a much stronger signal than the PGII control upon probing with a polyclonal antibody raised against PGI, which has a low affinity for PGII [181.
Similarity of fungal polygalacturonases The deduced mature amino acid sequence of PGC shows a high degree of sequence similarity with those of other fungal polygalacturonases, namely 60.9% with PGI, 55.9% with PGII and 57.9% with the polygalacturonase of C. carboizum [I71 and amongst all these proteins 36.5% of the amino acids are conserved. The sequence of mature PGC is eight amino acids longer than that ofPGII, whereas the mature PGI, PGII and C. carbonum polygalacturonase sequences only differ in length by, at most, two amino acids. The two single amino acid insertions which are found in the PGI sequence with respect to that of PGII, are also present in the PGC sequence. In addition, at five different positions in the PGC sequence, insertions of 1- 3 amino acids have occurred, including the extension of two amino acids at the C-terminus. Also, a single deletion of two amino acids is observed with respect to the sequences of the three other fungal polygalacturonases (data not shown). Alignment of the N-terminal amino acid sequences of these polygalacturonases with those of the major polygalacturonases of Colletotrichum lindeniuthiunum and Sclerotiniu sclerotiorunz [30] is shown in Fig. 5. The occurrence of insertions or deletions at two positions in this region underscores the intermediate position of the PGI sequence with respect to the PGC and PGII scquences. It can be seen that the N-terminal amino acid sequences of the C. carbonum and C. lindenzuthiumm polygalacturonases closely resemble the A . niger PGII sequence, as opposed to the corresponding S. sclerotiorum sequence which shows the same insertions or deletions as the PGC sequcnce. Tntronlexon organization of polygalacturonase genes
The nucleotide sequence of thepgaC gene and the deduced amino acid sequence The nucleotide sequence of the pguC gene was determined from both strands of DNA (Fig. 4) and the PGC-encoding
The pgaC gene contains three introns, the highest number found for a fungal polygalacturonase gene so far. The introns in the other two sequenced A . niger genes occur at the same position and in the same phase of the reading frame as introns
87 -601
CCATGGCTGAAACTTTGCCCCTGACCTAGTCCATTTCATTTTATATAATGCTGATGAGATAT GGTTCTTGCACAACTATGCTTTTCCTCGGTCGGTGCGTAATGTATAGTTCGTGGGTGGTAAGGAAATTCACCGACAGCAACTCCCAGCATAACATGGAGAAGAACCGATGAATGAGCAGC
-481
GACAAACATGGTACCACAATTCTTGAATAAGCATTTAGACTCCGGCTTGGTTTTTTCCGTTGCATTGGCGCTGGTGTTGTTCCTGCGCACGGTGCAACCGTTATCTCCACCAATGATTAC -361
+++++++++++
CTGCAGAAAGCCCATGGCTGCAACCACCCAGCCGACTTGAGTCCACCTAACAGCATACTCAGCATAC~CGATGCTGGGAATTACTTCAGGTATGGTCGGCTGAATATGCAGTCGATTTCC
-241
GGCGCCAGGAACAGTCCGGCCCCCTCACCACGGACATAGACCCGCCACCAAAACGTCGATAGCGGCCTCGCTTCGTCAACGACCATTTTGCCCAC~CACCCAGGAAAGACGTCTT
-121
CCAATAACAATATAAAAGGGCGAGTCTTGTCCTCTCACTTTCCGTCTTTCGAGTATGCCTTGCCAGTTCCCAACTTGACTTGAGACCTCCCATTTCGGTCATTTGTCACCCGCTATAAGA
-1
ATG GTC CGT CAG C T T A T C CTG ATC AGC AGT CTG CTG GCA GCT GTT GCT GTG CGC GCG CCT GCC GAT CCG GCT CAT CCC ATG GTT ACG GAA M V R 0 L I L I S S L L A A V A n V R A P A D P A H P M V T E
90 30
GCG CCT GAC GTC AAT TTG GTT GAA AAG AGG GCG ACT ACT TGC ACC TTC TCG GGC TCC GAA GGT GCA TCC AAG GCC AGC AAG TCG AAA ACC A P D V N L V E K R n A T T C T F S G S E G A S K A S K S K T
180 60
TCT TGC TCC ACA A T C TAC CTG TCC GAC GTG GCC GTC CCA TCT GGC ACA ACC CTT GAT CTC TCT GAC CTG AAT GAT GGA ACC CAC W S C S T I Y L S D V A V P S G T T L D L S D L N D G T H
272
*******
T
C
88
C C C G G G T G A T T C A T G T C T T A T T C T C A C A C C C A A T C G C G T C A A T A G T A C T G G ~ A G A T G T A C ~GTG ATC TTC CAG GGA GAA ACC ACT TTT GGA TAC GAG GAA intron A
E
G
F
Q
G
E
D
S
G
Y
E
E
378 101
GTG CAG GTG T T C AGC ATC GAT GGC TCC ACT GAT CTT ACC ATG ACT GAT ATC ACG GTG GAT AAC ACG GAT GGT GAC D
648 191
GGT GCC GAA ATC TAC AAC CAA GAT G A E I Y N Q D
738 221
TTC AGT GCC
839 236
V
Q
V
F
S
L
A
A
N
T
D
G
l
D
G
S
T
D
L
T
M
T
D
l
F
D
I
G
E
S
T
Y
I
T
I
T
T
V
D
N
T
A T 1 TAT ~ G T C C C C T G G C A C T G A T T G A G A G G A G A G G C G C C T T G ~ G A T C T G G AAC ~ intron 8 N I Y
GAC TGC GTT GCC ATC AAT TCT GGA GAG D C V A I N S G E AGT
F
558 161
ACG GAC GAC CTC GCC GCC AAT ACG GAT GGC TTC GAC A T C GGG GAA AGC ACC TAT ATC ACG A T C ACA
D
T
GGT GGC AAT GGT GGT AAA ACA AAG CCC AAG TTC TTC TAT GCC CAT GAC TTG ACC TCT TCC ACC ATC AAG AGC ATC TAC G G N G G K T K P K F F Y A H D L T S S T I K S I Y
L
ATC GAG AAC TCT CCT I E N S P
T
T
468
P
TGG GAT GGA GAG W D G E
I
GTG CGT GTT TCT GGA ACT GAT ATC ACG GTC GAG GGG GAG AGC GAC GCG GTG CTC AAT GGC GAT GGC AGC CGC TGG V R V S G T D I T V E G E S D A V L N G D G S R W
TGG GAA GGG CCT CTT
W
V
D
F
G
S
A
GTG TGT TCT GGT GGT CAC GGC CTG TCT A T 1 GGC TCT GTT GGT GGT CGG GAT GAT AAC ACT GTC AAG AAC GTG ACG T T T TAT GAT GTC V C S G G H G L S I G S V G G R D D N T V K N V T F Y D V
131
929 266
#
G T A A G G G A A A G C A T T T G A A C C A T C C G T T T G C C G T C C T ~ G T A T G C T TCA ~ ATC CGT ATC AAG ACC A T C TAC GGC intron C A I R l K T l Y G
1030
GAC ACT GGC TCC GTC AGC GAA GTC ACT TAC CAT GAG A T 1 GCC TTT TCG GAC GCT ACT GAT TAC GGC A T T GTC A T 1 GAG CAG AAC T A T GAT D T G S V S E V T Y H E I A F S D A T D Y G I V I E Q N Y D
1120 312
TGT ACC C T
1210 342
GAA GTA TAC A T 1 GCC TGC GGT GAT GGC AGC TGC TCG GAT TGG ACT TGG ACT GGC GTG AGC GTG ACT GGC GGC AGT GTC AGT GAC GAC TGC
1300 372
AAT GTT CTC AAG TCC CAG CAA G N V L K S P P
GAC ACC TCC AAG ACC CCT ACT ACC GGG GTA CCG ATC ACG GAT T T T GTG CTC GAG AAC ATC GTT GGA ACG TGT GAA GAT GAT GAT
D
T
E
V
C T T AAT L N
S Y
K I
T A
P C
T G
T D
G G
V S
P C
I S
T D
O W
F T
V W
L T
E G
N V
I S
V V
G T
T G
C G
E S
D V
D S
D D
D
C
GTT CCC TCC GGG A T 1 AGC TGC GAT TTG TAG GCCGTTGTGATTGGGGGAGATGTTCCCGGGTACTCTGGTCGTTCGTTTAGCCAGCCCTTTCTGTTGAGTGGC V P S G I S C D L - - -
282
1408 383
CTGTTCTTCTATACTGTCGATTGTGTGTGAGATTACTACCTTGCCCGAGGAAAGAGCAGTGCATCCTATCTTTTATTCTTTGCCGGTCGGGGTTGGCCTACTAGTACATTGTACCCGAAG
1528
AGTCAACGGTGAAAGTAAAGGGCTTACAGAATTAGTCCAGGAGCAAAGACAACTTTTATCAGAGCGCATCGGTCACGTCATGTTGAAAGTATTGATCAATGAAGATAACCTTGG
1642
Fig.4. Nucleotide and deduced amino acid sequences of the A . niger N400pgaC gene. The putativc start of the mature protein ($-), a potential signal peptidase cleavage site (
