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Appl Environ Microbiol, May 1998, p. 1766-1772, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Analysis of a Laccase Gene from the White
Rot Fungus Pycnoporus cinnabarinus
Claudia
Eggert,1,*
Peter R.
LaFayette,2
Ulrike
Temp,1
Karl-Erik L.
Eriksson,3 and
Jeffrey
F. D.
Dean2
Institute of General and Microbial Genetics,
Friedrich-Schiller University of Jena, 07743 Jena,
Germany,1 and
Department of Biochemistry
and Molecular Biology, Center for Biological Resource
Recovery,3 and
Warnell School of
Forest Resources,2 University of Georgia,
Athens, Georgia 30602
Received 1 December 1997/Accepted 20 February 1998
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ABSTRACT |
It was recently shown that the white rot basidiomycete
Pycnoporus cinnabarinus secretes an unusual set of
phenoloxidases when it is grown under conditions that stimulate
ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl.
Environ. Microbiol. 62:1151-1158, 1996). In this report we describe
the results of a cloning and structural analysis of the
laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The
coding region of the genomic laccase sequence, which is preceded by the
eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp.
The corresponding laccase cDNA was identical to the genomic sequence
except for 10 introns that were 50 to 60 bp long. A sequence analysis
indicated that the P. cinnabarinus lcc3-1 product has a Phe
residue at a position likely to influence the reduction-oxidation
potential of the enzyme's type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence
encoding a laccase from Coriolus hirsutus (level of
similarity, 84%).
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INTRODUCTION |
By definition, laccases
(p-diphenol:O2 oxidoreductase; EC 1.10.3.2)
catalyze the oxidation of p-diphenols and the concurrent reduction of dioxygen to water, although the actual substrate specificities of laccases are often quite broad and vary with the
enzyme source (11, 29). Laccases are members of the blue copper oxidase enzyme family characterized by having four cupric (Cu2+) ions coordinated such that each of the known
magnetic species (type 1, type 2, and type 3) is associated with a
single polypeptide chain. The Cu2+-binding domains are
highly conserved in the blue copper oxidases, and the crystallographic
structure of ascorbate oxidase, another member of this enzyme class,
has provided a good model for the structure of the laccase active site
(30, 31). This model has been supported by the results of
numerous studies of the electron transfer reactions that occur between
cupric ions during catalysis (35, 39, 40).
In contrast to our understanding of the electron transfer reactions
that occur in laccases, relatively little is known about the
physiological functions of these enzymes. Laccases have been implicated
in pigmentation (1, 9), fruiting body formation (26), and pathogenicity (7, 45), as well as in
lignin degradation (41) and biosynthesis (27).
Very few of these functions have been experimentally proven, and only
because of the availability of multiple gene sequences and
crystallographic data has it been possible to speculate about how
structure-function relationships may be important in the specific roles
played by these enzymes (46). Some of this speculation has
involved attempts to address the apparent contradictory functions of
laccases in the synthesis and breakdown of lignin (3, 11).
To better understand the role of laccases in lignin degradation by
white rot fungi, we studied the ligninolytic system of Pycnoporus
cinnabarinus, a basidiomycete that produces an unusual set of
ligninolytic enzymes. Just a single isoform of laccase, but no lignin
peroxidase (LiP) or manganese peroxidase (MnP), was produced by this
organism under conditions that stimulated lignin degradation
(13). We wanted to determine more completely the pattern of
phenoloxidase production in P. cinnabarinus, so the primary
objective of this study was to analyze the structure of the P. cinnabarinus laccase gene and determine whether there are multiple
laccase genes in the P. cinnabarinus genome.
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MATERIALS AND METHODS |
Organisms and reagents.
P. cinnabarinus PB (= ATCC
200478), an isolate recovered from decaying pine wood in the vicinity
of Sydney, New South Wales, Australia, was maintained as described
previously (13). Escherichia coli INV
F' (One
Shot competent cells) and the pCR2.1 vector used for direct cloning of
PCR products were purchased from Invitrogen (San Diego, Calif.). Unless
otherwise indicated, the enzymes used to manipulate DNA or RNA were
obtained from Boehringer Mannheim (Indianapolis, Ind.), New England
Biolabs (Beverly, Mass.), or Invitrogen (T4 DNA ligase) and were used
according to the manufacturers' instructions. All chemicals and
reagents were at least analytical grade.
Oligonucleotides, probes, and primers.
The sequences of most
oligonucleotide primers used in this study are shown in Fig.
1; the exceptions are the sequences of the oligonucleotides used to isolate the P. cinnabarinus
lcc3-1 promoter. A digoxigenin-labeled laccase probe was prepared
by using primers P3 and P6 (Fig. 1). The AP oligonucleotide primer was
purchased from Life Technologies (Bethesda, Md.). Other primers were
synthesized at the Molecular Genetics Instrumentation Facility of the
University of Georgia.
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FIG. 1.
Strategy used for PCR cloning of the laccase-encoding
cDNA from P. cinnabarinus and oligonucleotide primer
sequences. The boxes indicate the regions encoding the N terminus of
the mature protein and the Cu(II)-binding regions of the laccase that
are highly conserved in blue copper oxidases. (A) Results of
laccase-specific reverse transcription of P. cinnabarinus
RNA and anchor-ligated PCR used to amplify the 450-bp fragment of the
P. cinnabarinus laccase cDNA on which the gene-specific
primer P6 was based. (B) After primer AP-primed reverse transcription
of full-length P. cinnabarinus mRNAs, primer P5 was used in
conjunction with primer P6 to amplify the laccase-encoding sequence.
Primers P6 and P7 were used to clone the genomic laccase sequence.
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RNA isolation.
P. cinnabarinus cultures grown for 3 days in modified Dodson medium (13) at 30°C on a rotary
shaker (135 rpm) were induced with 2,5-xylidine (10 µM) as described
previously (8). Longer cultivation times led to increased
production of extracellular polysaccharides, which strongly interfered
with RNA isolation. Fungal mycelia were collected by filtration, and
then they were washed twice in sterile phosphate buffer (20 mM, pH 7.0)
and frozen in liquid nitrogen before RNA was isolated by the method of
Chomczynski and Sacchi (8). For Northern analyses, total RNA
(10 µg) was separated on a 1.4% (wt/vol) agarose gel
(32), transferred to Nytran-Plus membranes (Schleicher & Schuell, Keene, N.H.), and hybridized with labeled probes under
high-stringency conditions as described below for the Southern blot
analysis.
Genomic DNA isolation.
Mycelia from P. cinnabarinus grown in 250 ml of malt extract medium (15 g/liter,
pH 5.0) at 30°C for 4 days were harvested, washed, and frozen in
liquid N2 as described above. High-molecular-weight genomic
DNA was isolated from frozen mycelia after grinding by using a plant
DNA isolation kit (Boehringer) as recommended by the manufacturer.
cDNA synthesis, 5' anchor ligation PCR, and PCR cloning.
Ligation-anchored PCR (42) was used to obtain full-length
P. cinnabarinus laccase cDNA. Total RNA (1.0 µg) was
primed by using a degenerate oligonucleotide primer (primer P1)
designed to complement the third (from the amino terminus)
copper-binding domain (domain III) that is conserved in laccases and
other blue copper oxidases (Fig. 1A). Subsequent reverse transcription
was performed with Superscript reverse transcriptase (Life
Technologies), and an oligonucleotide anchor was ligated to the 5' end
of the resultant cDNA. Primer P2, which was complementary to the anchor sequence, was used in combination with two degenerate primers, primers
P3 and P4, which were synthesized to match the second copper-binding
domain (domain II) conserved in laccases, to amplify a 450-bp fragment
of the P. cinnabarinus laccase gene. For PCR amplification
of the anchor-ligated cDNA with primers P2 and P3, an aliquot (2 µl)
of the ligation mixture was used as the template in a 25-µl reaction
mixture containing Tfl polymerase (Epicentre Technologies,
Madison, Wis.). For half-nested amplification of the first PCR product,
a template (1 µl) was added to a reaction mixture containing primers
P2 and P4 and Expand high-fidelity polymerase (Boehringer Mannheim).
The resultant product was subcloned into the pCR2.1 vector
(Invitrogen), and two clones were sequenced. From the resulting
sequence, primer P6, an oligonucleotide primer whose sequence exactly
matched the sequence of the 5' untranslated region of the P. cinnabarinus laccase mRNA, was synthesized. After reverse
transcription of total P. cinnabarinus RNA with primer AP,
primer P5, which complemented a portion of the primer AP sequence, was
used in combination with primer P6 and Expand polymerase to amplify the
full-length P. cinnabarinus laccase cDNA.
To isolate genomic laccase sequences, exact-match primers P6 and P7
(the sequence of P7 corresponded to the sequence immediately downstream
of the stop codon) were used in PCR mixtures (volume, 25 µl)
containing genomic DNA (1 µg) and Expand polymerase. Amplified products that were approximately 2,100 bp long were cloned into the
pCR2.1 vector, and two of the resultant clones were sequenced on both
strands.
Isolation of the P. cinnabarinus lcc3-1 promoter
region.
P. cinnabarinus genomic DNA was digested with
KpnI, which cuts at positions 1050 and 1296 in the
lcc3-1 gene. The cleavage products were circularized by
ligation with T4 DNA ligase. The putative promoter region upstream of
the laccase coding sequence was amplified by a two-step inverse PCR
process by using primers PA
(5'-CCACAGCGGCAAGAGAGACG-3') and PB
(5'-GAGGACAAAGGAGAGGAGAGATTGG-3') (which were directed in
the 5' direction from nucleotides 343 and 319, respectively) and primer
PC (5'-GATCACCCCCGCTCCTCTCA-3') (which was
directed in the 3' direction from nucleotide 457). Sequential PCR
amplifications were performed by starting with an aliquot (2 µl) of
the ligation product in a 25-µl reaction mixture containing Expand
polymerase. The resultant 1,400-bp fragment was subcloned into the
pCR2.1 vector, and two of the resultant clones were sequenced on both
strands.
DNA sequencing.
Nucleotide sequences were determined by
using Taq polymerase cycle sequencing and an automated DNA
sequencer (model ABI 377; Perkin-Elmer Corp., Foster City, Calif.) at
the Molecular Genetics Instrumentation Facility on the University of
Georgia campus. All cloned DNAs were sequenced on both strands, and the
encoded amino acid sequences were predicted by using Gene Runner
(Hastings Software, Hastings-on-the-Hudson, N.Y.). Sequences were
aligned by using the CLUSTAL V algorithm (MegAlign; DNASTAR, Madison, Wis.).
Southern blot analysis.
Restriction endonuclease-digested
DNA samples (10 µg) were separated on a 0.8% agarose gel and
transferred to Nytran-Plus nylon membranes (Schleicher & Schuell) by
using the procedure of Zhou et al. (49). When
high-stringency conditions were used, hybridization was performed at
45°C with a DIG Easy Hyb solution (Boehringer Mannheim), the filters
were washed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.0.15 M sodium
citrate)-0.5% sodium dodecyl sulfate (SDS) at 24°C and then in
0.1× SSC-0.5% SDS at 68°C. Unless indicated otherwise, when
low-stringency conditions were used, hybridization was performed at
42°C with the same hybridization solution, and the filters were
washed at 24°C in 2× SSC-0.5% SDS and then at 49°C in 0.1×
SSC-0.5% SDS. Blots were developed by following the manufacturers'
instructions for chemiluminescent detection of digoxigenin-labeled
probes with alkaline phosphatase-antibody conjugates (Boehringer
Mannheim). Digoxigenin-labeled probes for the P. cinnabarinus laccase gene were prepared by using primers P2 and P3
to amplify a 450-bp fragment from the cloned cDNA.
Nucleotide sequence accession number.
The nucleotide
sequence of P. cinnabarinus lcc3-1 reported in this paper
has been deposited in the EMBL/GenBank database under accession no.
AF025481.
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RESULTS |
Isolation of the laccase cDNA and characterization of the deduced
protein.
The longest cDNA clone of the P. cinnabarinus
laccase gene was 1,828 bp long without the poly(A) tail and contained a
1,554-bp open reading frame. Blots of the transcription products
obtained from 3-day-old mycelium probed with a 450-bp
digoxigenin-labeled fragment from the cDNA clone revealed that a single
transcript about 1,800 bp long was produced (data not shown). Thus, the
transcript size was consistent with the length predicted from the cDNA
sequence.
G and C accounted for 58% of the nucleotides in the coding sequence,
and the proportion of G and C in degenerate codon positions
was high
(66% GC, with C [46%]
A [6%]). A similar or even more
pronounced preference for pyrimidine bases has been found in other
fungal genes (
22,
36). Some examples of extreme codon bias
in the
P. cinnabarinus laccase are Leu (87% C/UUG/C versus
13%
CUU), Val (80% GUG/C versus 20% GUA/U), and Phe (90% UUC versus
10% UUU).
The 21-amino-acid N-terminal sequence of the purified
P. cinnabarinus laccase (
13) was identical to residues 22 to 42 predicted
from the open reading frame of the cDNA (Fig.
2). The putative
21-amino-acid signal
sequence (Fig.
2) was followed by a sequence
that could act as a
peptidase recognition site as determined by
the (
3,
1)-rule
(
43), which predicts that there is a small,
uncharged amino
acid residue (Ala) at position
1 relative to
the cleavage site. Thus,
the cleavage site sequence had the most
common pattern, A-X-A, found in
the C termini of signal peptides.
The core region of the signal peptide
is predominantly hydrophobic,
which is typical for eukaryotic signal
sequences (
33).
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FIG. 2.
Nucleotide sequence and deduced amino acid sequence of
the P. cinnabarinus lcc3-1 gene. Putative CAAT and TATA
boxes are indicated by open boxes. The N-terminal sequence of the
purified laccase from P. cinnabarinus (13) is
indicated by a shaded box. Possible N-glycosylation sites are
underlined. Residues involved in binding Cu(II) ions are marked by
single boxes. The numbers in italic type (1, 2,
and 3) indicate with which of the three Cu(II) types the
residues coordinate. The putative polyadenylation signal is underlined
with a dotted line. Recognition sites for restriction endonucleases
KpnI, HindIII, and BamHI, as well
as the most prominent transcriptional start site (tsp), are
indicated.
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It was predicted that the mature laccase polypeptide secreted by
P. cinnabarinus contained 497 amino acids and had a
composite
molecular mass of 53,871 Da.
P. cinnabarinus
laccase purified
from culture supernatants was previously shown to have
an apparent
Mr of ca. 76,000 (as determined by
SDS-polyacrylamide gel electrophoresis)
or 81,000 (as determined by gel
filtration) (
13); thus, the
observed and predicted
Mr values for the deduced protein differed
by
about 30%. Glycosylation is one form of posttranslational processing
that is probably responsible for at least some of the difference
(
38). The laccase contains six potential N-glycosylation
sites
(Asn-Xxx-Ser/Thr), at positions 72, 75, 229, 354, 362, and 455
of
the deduced protein (Fig.
2), although for steric reasons,
it seems
unlikely that the sites at positions 72 and 75 are glycosylated
simultaneously. On the other hand, the carbohydrate content reported
previously for the secreted protein (
13) (9%) is not
sufficient
to completely explain the differences between the predicted
and
observed molecular weights. The isoelectric point calculated for
the cloned gene product (pI 4.5) also differs from the experimentally
determined isoelectric point for the purified laccase (pI 3.7)
(
13).
The amino acid residues that act as Cu
2+ ligands are highly
conserved in all blue copper oxidases, including laccases
(
31).
All of the expected Cu
2+ ligands (10 His
residues and one Cys residue) were present in
the
lcc3-1
coding sequence and are numbered in Fig.
2 on the basis
of whether they
coordinate with the type 1, type 2, or type 3
Cu
2+ centers.
Another residue (Phe), which is numbered in Fig.
2 to
indicate
interaction with the type 1 copper center and is located
nearest the
carboxyl terminus of the protein, varies in laccases
from different
sources and is considered a residue that is probably
important in
governing the reduction-oxidation potential of type
1 copper centers
(
46).
Isolation and structural analysis of the laccase genomic
sequence.
A portion of the laccase gene was amplified from
P. cinnabarinus DNA by using oligonucleotide primers whose
sequences correspond to sequences identified in the 5' and 3'
untranslated regions of the mRNA (Fig. 2). The 2,390-bp coding region
was 526 bp longer than the corresponding cDNA sequence, and a
comparison of the sequences revealed 10 short introns (length, 50 to 60 bp) in the genomic sequence.
Inverse PCR was used to isolate the laccase gene promoter. Circularized
KpnI digests of
P. cinnabarinus genomic DNA were
amplified
with primers based on the genomic laccase sequence (primers
P
A [nucleotide 343], P
B [nucleotide 319],
and P
C [nucleotide 457]).
This approach yielded an
additional 1,400 bp of upstream sequence,
and 240 bp of this sequence,
including putative CAAT and TATA
promoter elements, is shown in Fig.
2.
An analysis of 5' RACE
(random amplification of cDNA ends)-amplified
cDNAs strongly suggested
that transcription of the laccase gene starts
68 bp upstream of
the translational start site (nucleotide 240). Thus,
the location
of the putative TATA element in this promoter is similar
to the
locations found in several other fungal genes, in which the TATA
box is generally located 30 to 60 nucleotides upstream from the
transcriptional start site (
20). Paired TATA and CAAT
elements
have been identified in other fungal laccase promoters (Tvi
[
21,
22], Po [
19], Ch
[
24], and Pa [
17]); however, although
the order of these motifs is conserved, their absolute positions
vary.
In genes of filamentous fungi, pyrimidine-rich sequences
often directly
precede the transcriptional start site, particularly
in highly
expressed genes (
2,
20); however, such sequences
were not
found in the
P. cinnabarinus laccase promoter. A putative
polyadenylation signal, AATAA, which is a slight variation of
the
consensus polyadenylation signal sequence AATAAA
(
37), was
found 167 bp downstream of the laccase stop
codon.
The 10 introns in the laccase gene were in good agreement with respect
to the consensus sequence predicted for the 5' splice
sites of
eukaryotic genes, GT(a/g)NG(c/t) (
2). Only introns
6 (T at
position 3) and 3 (T at position 5) exhibited slight variations.
The
consensus sequence for 3' splice sites, (c/t)N(c/t)AG, also
matched,
except for position 1 in introns 5 and 7 (A) and introns
6 and 9 (G).
The overall exon-intron structure of the
lcc3-1 gene
was
very similar to the structure determined for the
Coriolus hirsutus laccase gene (
24).
Laccase gene family.
Southern blot analysis was used to
estimate the number of laccase genes in the P. cinnabarinus
genome. Genomic DNA that had been digested with EcoRI,
BamHI, and HindIIII was hybridized to a 450-bp
digoxigenin-labeled cDNA probe spanning the region from the start of
the open reading frame to the second Cu-binding domain. None of these
restriction enzymes cut in the probe sequence. In HindIII digests, the laccase probe hybridized to a
single band of approximately 8.6 kb (Fig.
3), but three EcoRI fragments
(4.1, 5.5, and 6.7 kb) and four BamHI fragments (3.7, 3.9, 6.1, and 8.1 kb) were detected by the probe. Each of the fragments in
the latter digests was large enough to contain a complete copy of the
laccase gene.
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FIG. 3.
Southern blot of P. cinnabarinus genomic DNA.
Total DNA from P. cinnabarinus was digested with
EcoRI (lane E), BamHI (lane B), or
HindIII (lane H). The resultant DNA fragments (10 µg
per lane) were resolved by agarose gel electrophoresis and blotted onto
a nylon membrane. The filter was probed with a 450-bp
digoxigenin-labeled fragment of P. cinnabarinus lcc3-1. The
relative mobilities of HindIII-restricted lambda DNA
fragments are indicated on the left.
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Similarity to other laccase sequences.
The results of a
comparison of the amino acid sequence of the P. cinnabarinus
laccase (encoded by the P. cinnabarinus lcc3-1 gene) with
all laccase sequences available in databases are shown in Table
1. P. cinnabarinus lcc3-1 is
most closely related to C. hirsutus phe1 (84.0%
similarity), followed by Trametes villosa lcc1 (83.0%
similarity) and lac2 of the unidentified basidiomycete strain CECT 20197 (82.6% similarity). The laccases isolated so far
from the ligninolytic basidiomycetes are highly conserved (>58%
similarity). In general, sequence similarity follows phylogenetic position: basidiomycetous laccases (36 to 84% similarity) > ascomycetous laccases (23 to 25% similarity) > plant laccases (18 to
20% similarity), with the notable exception of the Aspergillus
nidulans laccase (17% similarity).
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DISCUSSION |
Structural similarity of laccases.
A laccase-encoding gene and
its corresponding cDNA were cloned from P. cinnabarinus, and
the gene product was shown to correspond to a laccase previously
isolated from ligninolytic cultures of this organism (13).
The single cysteine residue and 10 histidine residues that bind the
four catalytic cupric ions in all blue copper oxidases, including
laccases, were conserved in the P. cinnabarinus gene.
Studies of type 1 copper centers have shown that an additional residue
10 amino acids downstream of the conserved cysteine can have a major
effect on the redox potential of the cupric ion (6). This
residue was found to be a phenylalanine residue in the P. cinnabarinus laccase, but leucine and methionine residues have
been found in the laccase sequences of other fungi and plants (Fig.
4). The results of site-directed
mutagenesis studies performed with azurin (6, 31), as well
as work done by Xu et al. (46), support the hypothesis that
laccases harboring phenylalanine residues at this position should have
type 1 copper centers with high redox potentials, whereas the copper
centers of laccases with methionines at this position should have low redox potentials. On the basis of the three known possible residues at
the critical position, we categorized the known laccase sequences into
classes 1, 2, and 3 in order of postulated increasing redox potential.
This classification is also coordinated with the proposed Lac1 (Met)-Lac2 (Leu)-Lac3 (Phe)
nomenclature recently submitted by one of us (J.F.D.D.) to the
Commission for Plant Gene Nomenclature (CPGN) for plant laccase genes.
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FIG. 4.
Alignment of the amino acid sequences constituting the
copper-binding domain closest to the carboxyl terminus in laccases from
a variety of sources. The residues in boxes are associated with the
type 1 and type 3 copper centers, as indicated by the numbers at the
bottom. It has been predicted that the residues in the box farthest to
the right (M, L, and F) influence the redox characteristics of the
enzyme, and so these residues provide the basis for assigning laccases
to class 1, 2, or 3. The numbers on either side of the sequences
indicate the positions of the amino acids within the laccase
polypeptide, starting from the translational start site. Annotated
laccase gene sequences that lead to a putative protein sequence with an
anomalous Cu-4 center, including the Cryptococcus neoformans
(45) (GenBank accession no. L22866) and Rhizoctonia
solani lcc4 (44) (GenBank accession no. Z54277)
sequences, are not shown. T. versicolor, Trametes
versicolor; Bas., basidiomycete; P. anserina,
Podospora anserina; L. tulipifera,
Liriodendron tulipifera; N. tabacum,
Nicotiana tabacum; A. pseudoplatanus, Acer
pseudoplatanus.
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The
A. nidulans (class 1) laccase exhibits very low
levels of similarity to all other known laccases, including those
isolated
from plants. This probably reflects the very specialized
function
of this laccase (i.e., spore morphogenesis) (
1). In
contrast,
the
Botrytis cinerea laccase, whose nucleotide
sequence is not
known, has been implicated in the detoxification of
phytoalexins
(
4). These examples highlight the variety of
specialized physiological
functions for which laccases have evolved in
fungi. It remains
to be seen, however, whether multiple laccases that
have different
physiological functions can be isolated from the same
organism.
Multiple laccase genes in P. cinnabarinus.
When grown in
liquid culture, P. cinnabarinus secreted a single laccase
isozyme (13). Stimulation of laccase secretion by adding
2,5-xylidine, a strategy previously shown to induce laccase production
in a wide variety of basidiomycetes (5), did not result in
production of additional bands in isoelectric focusing gels. Likewise,
reverse transcriptase PCR analyses did not reveal any additional
laccase transcripts when the fungus was exposed to 2,5-xylidine. The
N-terminal sequences deduced for all of the cDNA-encoded laccases
isolated were identical to the sequence previously determined for the
laccase purified from cultures.
On the other hand, Southern analysis revealed the presence of a small
gene family encoding laccases in
P. cinnabarinus. Whereas
the laccase probe hybridized to four
BamHI fragments, it
hybridized
to only a single 8.6-kb
HindIII fragment,
suggesting that there
is a laccase gene cluster in
P. cinnabarinus. As
P. cinnabarinus PB is a dikaryon,
allelic variants of the laccase gene are expected,
and the presence of
four nonallelic laccase genes is highly unlikely
since all of them
would have to be located on the same 8.6-kbp
HindIII
fragment.
The copy numbers of laccase genes vary among fungi. A laccase gene
family in which the genes encoding two of five laccases
were located on
the same chromosome was found in
T. villosa (
47,
48), and three laccase genes were found to be clustered within
approximately 11 kb of each another in
Rhizoctonia solani
(
44).
Pleurotus ostreatus and
Agaricus
bisporus each contain at least
two different laccase genes
(
19,
34), while allelic forms
of a single laccase gene have
been found in
C. hirsutus, as well
as
Neurospora
crassa (
18,
24). Single copies of laccase genes
were
also found in
Phlebia radiata and
Cryphonectria
parasitica (
7,
38).
In
P. cinnabarinus, the laccase purified from liquid
cultures was found to be important not only for lignin degradation
(
14,
16) but also for synthesis of the phenoxazinone
pigments which
give the fruiting bodies a red color (
15). In
fact, a more precise
name for the laccase from
P. cinnabarinus is 3-hydroxyanthranilate:O
2 oxidoreductase. The phenoxazinone pigments and, consequently,
the
laccase activity can also be linked to the antimicrobial activity
of
this organism (
12). Thus, the product of one laccase gene
in
P. cinnabarinus appears to serve two separate, but
interwoven,
functions in this fungus. It remains to be determined under
what
conditions the other laccase genes are expressed and what
physiological
function(s) they perform under those conditions.
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ACKNOWLEDGMENTS |
Alexandria Tristram provided valuable technical advice and
assistance.
This research was supported by a fellowship from the Alexander von
Humboldt-Stiftung (to C.E.) supplemented with funds from the University
of Georgia Research Foundation, as well as by grant RR 50778F from the
National Science Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
General Microbiology and Microbial Genetics, Friedrich-Schiller
University of Jena, Neugasse 24, D-07743 Jena, Germany. Phone and fax:
(49) 3641-949327. E-mail: Claudia.Eggert{at}uni-jena.de.
| |
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Appl Environ Microbiol, May 1998, p. 1766-1772, Vol. 64, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
