Genome, transcriptome, and secretome analysis
of wood decay fungus Postia placenta supports
unique mechanisms of lignocellulose conversion
Diego Martineza,b, Jean Challacombea, Ingo Morgensternc, David Hibbettc, Monika Schmolld, Christian P. Kubicekd,
Patricia Ferreirae, Francisco J. Ruiz-Duenase, Angel T. Martineze, Phil Kerstenf, Kenneth E. Hammelf,
Amber Vanden Wymelenbergg, Jill Gaskellf, Erika Lindquisth, Grzegorz Sabati, Sandra Splinter BonDuranti,
Luis F. Larrondoj, Paulo Canessaj, Rafael Vicunaj, Jagjit Yadavk, Harshavardhan Doddapanenik,
Venkataramanan Subramaniank, Antonio G. Pisabarrol, Jose ´ L. Lavínl, Jose ´ A. Oguizal, Emma Masterm,
Bernard Henrissatn, Pedro M. Coutinhon, Paul Harriso, Jon Karl Magnusonp, Scott E. Bakerp, Kenneth Brunop,
William Kenealyq, Patrik J. Hoeggerr, Ursula Ku ¨esr, Preethi Ramaiyao, Susan Lucash, Asaf Salamovh, Harris Shapiroh,
Hank Tuh, Christine L. Cheeb, Monica Misraa, Gary Xiea, Sarah Tetero, Debbie Yavero, Tim Jamess, Martin Mokrejst,
Martin Pospisekt, Igor V. Grigorievh, Thomas Brettina, Dan Rokhsarh, Randy Berkao, and Dan Cullenf,1
aLos Alamos National Laboratory/Joint Genome Institute, P.O. Box 1663, Los Alamos, NM 87545;bDepartment of Biology, University of New Mexico, Albuquerque,
NM 87131;cBiology Department, Clark University, Worcester, MA 01610;dResearch Area Gene Technology and Applied Biochemistry, Institute of Chemical
Engineering, Technische Universitat Wien, Getreidemarkt 9/166, A-1060 Vienna, Austria;eCentro de Investigaciones Biolo ´gicas, Consejo Superior de Investigaciones
Cientificas , Ramiro de Maeztu 9, E-28040 Madrid, Spain;fForest Products Laboratory, Madison, WI 53726;gDepartment of Bacteriology, University of Wisconsin,
Madison, WI 53706;hU.S. Department of Energy Joint Genome Institute, 2800 Mitchell Avenue, Walnut Creek, CA 94598;iUniversity of Wisconsin Biotechnology
Center, Madison, WI 53706;jDepartamento de Gene ´tica Molecular y Microbiología, Facultad de Ciencias Biolo ´gicas, Millennium Institute for Fundamental and
Applied Biology, Pontifica Universidad Cato ´lica de Chile, Casilla 114-D, Santiago 833-1010, Chile;kDepartment of Environmental Health, University of Cincinnati,
Cincinnati, OH 45267;lGenetics and Microbiology Research Group, Public University of Navarre, 31006 Pamplona, Spain;mChemical Engineering, University of
Toronto, Toronto, ON, Canada M5S 3E5;nArchitecture et Fonction des Macromole ´cules Biologiques, Unite ´ Mixte de Recherche 6098, Centre National de la
Recherche Scientifique, Universite ´s d’Aix-Marseille I and II, Case 932, 163 Avenue de Luminy, 13288 Marseille, France;oNovozymes Inc., 1445 Drew Avenue, Davis,
CA 95618;pPacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352;qMascoma Inc., Lebanon, NH 03766;rMolecular Wood Biotechnology and
Technical Mycology, Bu ¨sgen-Institute, Georg-August-University Go ¨ttingen, Bu ¨sgenweg 2, D-37077 Go ¨ttingen, Germany;sDepartment of Ecology and Evolutionary
Biology, University of Michigan, Ann Arbor, MI 48109; andtFaculty of Science, Charles University, Vinicna 5, 12844 Prague, Czech Republic
Edited by Richard A. Dixon, The Samuel Roberts Noble Foundation, Ardmore, OK, and approved December 15, 2008 (received for review
September 24, 2008)
Brown-rot fungi such as Postia placenta are common inhabitants of
forest ecosystems and are also largely responsible for the destructive
decay of wooden structures. Rapid depolymerization of cellulose is a
and underlying genetics are poorly understood. Systematic exami-
nation of the P. placenta genome, transcriptome, and secretome
revealed unique extracellular enzyme systems, including an unusual
repertoire of extracellular glycoside hydrolases. Genes encoding exo-
cellobiohydrolases and cellulose-binding domains, typical of cellulo-
lytic microbes, are absent in this efficient cellulose-degrading fungus.
When P. placenta was grown in medium containing cellulose as sole
carbon source, transcripts corresponding to many hemicellulases and
to a single putative ?-1–4 endoglucanase were expressed at high
levels relative to glucose-grown cultures. These transcript profiles
were confirmed by direct identification of peptides by liquid
chromatography-tandem mass spectrometry (LC-MS/MS). Also up-
regulated during growth on cellulose medium were putative iron
reductases, quinone reductase, and structurally divergent oxidases
potentially involved in extracellular generation of Fe(II) and H2O2.
These observations are consistent with a biodegradative role for
Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl
for investigating such unusual mechanisms of cellulose conversion.
More broadly, the genome offers insight into the diversification of
lignocellulose degrading mechanisms in fungi. Comparisons with the
closely related white-rot fungus Phanerochaete chrysosporium sup-
port an evolutionary shift from white-rot to brown-rot during which
the capacity for efficient depolymerization of lignin was lost.
cellulose ? fenton ? lignin ? cellulase ? brown-rot
for fixed global carbon and is increasingly eyed as a potential
in lignocellulose to monomeric components (2). The principal
exceptions are basidiomycetes, which attack wood through 2 main
decay types called white-rot and brown-rot. Wood-decaying basid-
and brown-rot fungi are additionally important because they are a
major cause of failure in wooden structures. White-rot fungi
degrade all components of plant cell walls, including cellulose,
hemicellulose, and lignin. Although they cannot grow on lignin
alone, they have the unique ability to degrade a large proportion of
the structural polysaccharides of plant cell walls, thus making them
susceptible to hydrolysis by cellulases and hemicellulases. Brown-
rot fungi employ a different approach; although they modify lignin
extensively, the products remain in situ as a polymeric residue (3,
4). Given the incomplete ligninolysis that occurs during brown-rot,
it remains unclear how these fungi gain access to plant cell wall
polysaccharides. However, it seems probable that the 2 decay types
Author contributions: D.M., J.C., D.H., E.L., S.S.B., I.V.G., T.B., D.R., R.B., and D.C. designed
research; A.V.W., J.G., G.S., and D.C. performed research; D.M., J.C., I.M., D.H., M.S., C.P.K.,
P.F., F.J.R.-D., A.T.M., P.K., K.E.H., A.V.W., J.G., E.L., G.S., S.S.B., L.F.L., P.C., R.V., J.Y., H.D.,
V.S., A.G.P., J.L.L., J.A.O., E.M., B.H., P.M.C., P.H., J.K.M., S.E.B., K.B., W.K., P.J.H., U.K., P.R.,
S.L., A.S., H.S., H.T., C.L.C., M. Misra, G.X., S.T., D.Y., T.J., M. Mokrejs, M.P., I.V.G., R.B., and
D.C. analyzed data; and D.M., P.K., K.E.H., R.B., and D.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The annotated genome is available on an interactive web portal at
http://www.jgi.doe.gov/Postia. The sequences reported in this paper have been deposited
in the GenBank database (accession nos. ABWF00000000 and FL595400-FL633513). The
model and microarray results reported in this paper have been deposited in the Gene
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
February 10, 2009 ?
vol. 106 ?
share at least some mechanisms, because molecular phylogeny,
morphological considerations, and substrate preference suggest
that brown-rot fungi have repeatedly evolved from white-rot fungi
(5). Indeed, the 2 major experimental organisms for studies of
brown-rot, Postia placenta and Gloeophyllum trabeum, are distantly
related species that represent independent origins of brown-rot (5).
Any similarities in their decay mechanisms must represent either
general mechanisms of wood decay common to white-rot and
brown-rot species, or convergently evolved brown-rot mechanisms.
Moreover, P. placenta is closely related to the model white-rot
fungus, Phanerochaete chrysosporium, so comparisons between
these species may provide insight into the mechanistic basis of
transitions from white-rot to brown-rot.
White-rot fungi produce complex ligninolytic systems that are
thought to depend in part on extracellular oxidative enzymes,
especially peroxidases, laccases, and other oxidases. It remains an
open question whether brown-rot fungi possess any of these ligni-
nolytic components. White-rot fungi also secrete complete, syner-
gistically acting cellulase systems that include both endo- and
exo-acting enzymes. These exocellobiohydrolases and endoglu-
been described in brown-rot fungi (6). It has been long recognized
that cellulose depolymerization appears to occur before the sub-
strate porosity has increased enough to admit cellulases (7), and
more recent studies (8) have shown that the amorphous regions
within cellulose microfibrils are cleaved by P. placenta, resulting in
rapid depolymerization but little weight loss. One possibility con-
sistent with these observations is that brown-rot fungi attack
with a limited set of relatively small cellulases.
The hydroxyl free radical, generated via Fenton chemistry
(H2O2? Fe2?? H?3 H2O ? Fe3?? ?OH), has long been
implicated as 1 of the small oxidants that contributes to polysac-
charide depolymerization during brown-rot. Current models for
hydroxyl radical participation have been reviewed (6) and typically
involve generation of this highly reactive oxidant at or near the
substrate. Key requirements for Fenton systems include mecha-
nisms for extracellular H2O2generation and for reduction of Fe3?
to Fe2?, which might be accomplished by extracellular fungal
as cellobiose dehydrogenase.
with transcript profiles and mass spectrometric identification of
extracellular proteins. Consistent with a unique strategy for cellu-
lose degradation, we observed a dramatic absence of conventional
cellulase genes and most class II fungal peroxidases and a rich
diversity of genes potentially supporting generation of extracellular
reactive oxygen species.
Carbohydrate Active Enzymes. Given the well-known efficiency with
which brown-rot fungi rapidly depolymerize and degrade cellulose,
the P. placenta genome revealed remarkably few, if any, conven-
tional cellulases. Of 17,173 proteins predicted in the dikaryotic
genome, 242 unique genes encode potential carbohydrate-active
These putative CAZY genes include 144 glycoside hydrolases
(GH), 10 carbohydrate esterases (CE), 75 glycosyltransferases
(PL) [complete CAZY list within the National Center for Biotech-
nology Information Gene Expression Omnibus (GEO accession
no. GSE12540 si_table_1.xls)]. In distinct contrast to all cellulolytic
fungal aerobes, exocellobiohydrolases CBH2 (GH6) and CBH1
(GH7), and cellulose-binding endoglucanases are missing in the P.
placenta genome (Fig. 1). Also absent are family 1 carbohydrate
binding modules (CBM1). These highly conserved cellulose-
range of cellulolytic microbes. Surprisingly then, the repertoire of
recognizable cellulolytic enzymes in P. placenta appears limited to
just 2 potential endoglucanases (1,4-?-glucanases) and several
?-glucosidases. In contrast to cellulolytic saprophytes (e.g., Tricho-
derma reesei, Aspergillus spp. or Neurospora crassa) and aggressive
plant pathogens (e.g., Fusarium graminearum or Magnaporthe
grisea), the overall number and distribution of GHs in P. placenta
are similar to those in the ectomycorrhizal symbiont Laccaria
bicolor, the human pathogen Cryptococcus neoformans, and the
biotrophic plant pathogen Ustilago maydis (supporting informa-
tion (SI) Table S1). Phylogenetic analyses of P. placenta and P.
chrysosporium genomes indicate that the transition from white-
rot to brown-rot has been associated with multiple independent
reductions including the GH families 6, 7, 10, 11 and 61 (Figs. 1
and 2; Table S1). Thus, the transition from white-rot to brown-
rot has been associated with multiple independent reductions in
the GH families.
Microarrays representing 12,438 unique alleles were used to
examine P. placenta transcript levels in basal salts medium contain-
ing either glucose or wood-derived microcrystalline cellulose as the
sole carbon sources. In total, 290 gene models showed ?2-fold
transcript accumulation, and of these, 255 increased in cellulose
medium and 35 increased in glucose medium (GEO accession no.
GSE12540 si_table_3.xls). Transcripts of 99 GH-encoding genes
significantly increased (P ? 0.01) in the cellulose medium, and of
these, 18 increased ?2-fold (Fig. 3). Twenty-one GH transcripts
significantly increased in the glucose-containing medium, but none
exceeded a 2-fold change. In addition, shotgun liquid chromatog-
raphy coupled tandem mass spectrometry (LC-MS/MS) identified
26 specific CAZYs in the extracellular fluid of P. placenta grown in
basal salts supplemented with ball-milled aspen wood, microcrys-
talline cellulose, or cotton (Table S2). The CAZY genes expressed
in cellulose included laminarinases, chitinases, and various hemi-
GH5 w/ CBM1
(middle ring), and P. chrysosporium (outer ring). Proteins not found in P.
placenta are underlined. Comparisons with additional species are listed in
ily 5 modules associated with family 1 carbohydrate binding modules; GT,
glycosyl transferases; CE, carbohydrate esterases; PL, polysaccharide lyases;
EXPN, expansin-related proteins.
Distribution of various CAZymes in P. placenta (inner ring), T. reesei
Martinez et al. PNAS ?
February 10, 2009 ?
vol. 106 ?
no. 6 ?
endo-?-mannanases, and ?-mannosidases). It is unclear whether
any of these enzymes could directly attack crystalline cellulose.
Extracellular H2O2Generation. Gene models potentially supporting
Fenton chemistry through the generation of extracellular H2O2
include copper radical oxidases and GMC oxidoreductases (Table
S3). Results summarized here focus on those genes with expression
patterns that are consistent with a role in cellulose depolymeriza-
accession no. GSE12540 si_table_6.xls.
On the basis of overall sequence similarity to P. chrysosporium
glyoxal oxidase (GLOX) and conservation of catalytic residues
(CROs). GLOX is 1 of 7 CROs in P. chrysosporium and physio-
particular relevance to potential Fenton systems, CRO genes
encoding proteins Ppl56703 and Ppl130305 are up-regulated in
microcrystalline cellulose, and Ppl56703 peptides were detected in
not closely related, and they do not have orthologs in P. placenta,
which suggests that there have been 2 independent losses of these
CRO lineages in Postia (Fig. 2). Ppl56703 is orthologous to the
cro3-4-5 lineage in P. chrysosporium, which therefore represents a
Phanerochaete-specific expansion of the gene family. As in the case
of the GHs, evolution of brown-rot is associated with a reduced
diversity of CROs.
Catalytically distinct from CROs, GMC oxidoreductases (Inter-
the former, P. placenta protein model Ppl118723 is similar to G.
trabeum methanol oxidase (GenBank DQ835989) (? 85% amino
acid identity over full length). Recent immunolocalization studies
strongly implicate the G. trabeum alcohol oxidase as a source of
P. placenta, microarray analysis revealed high transcript levels and
a sharp increase in transcription of the gene encoding Ppl118723 in
cellulose-grown cultures relative to noncellulolytic cultures. Com-
paratively high transcript levels in cellulose- and glucose-grown
cultures were also observed for genes encoding Ppl128830 and
Ppl108489, models tentatively identified as glucose-1-oxidases
based on conserved key residues (12). Peptides corresponding to
heuristic searches, 1000 bootstrap replicates), maximum likelihood (RAxML; 1000 bootstrap replicates, with models suggested by ProtTest), and Bayesian (MrBayes
v3.1.2; 2 runs of 4 chains, 10 million generations each, with mixed protein models) support values are indicated in the order MP/PP/ML. Topologies shown are from
Bayesian phylogenetic analyses. An alternative topology from parsimony analysis is shown for part of the GH10 phylogeny. Inferred gene losses, duplication events
(paralogy), and speciation events (orthology) are indicated within Postia and Phanerochaete only.
www.pnas.org?cgi?doi?10.1073?pnas.0809575106 Martinez et al.
these putative gox genes were detected in extracellular filtrates
(GEO accession no. GSE12540 si_table_6.xls and si_table_11.xls).
Aryl-alcohol oxidase, an extracellular GMC oxidoreductase coop-
erating with aryl-alcohol dehydrogenases for continuous peroxide
supply in some white-rot fungi (12) does not seem to be involved in
cellulose attack by P. placenta because the corresponding models
were not or only slightly up-regulated. Another extracellular GMC
oxidoreductase, pyranose-2-oxidase, has been implicated in ligno-
cellulose degradation in P. chrysosporium (13), but no orthologs
were detected in P. placenta.
Iron Reduction and Homeostasis. Protein model Ppl124517 was
fungus G. trabeum, a QRD may drive extracellular Fenton systems
via redox cycling of secreted fungal quinones (6). Transcription of
medium (GEO accession no. GSE12540 si_table_3.xls), which is
consistent with a role for cellulolytic Fenton chemistry involving
quinone redox-cycling. In this connection, up-regulation of the
genes encoding phenylalanine ammonia lyase (Ppl112824) and a
putative quinate transporter (Ppl44553) may also be relevant by
virtue of their respective roles in the biosynthesis and transport of
In addition to hydroquinone-based iron reduction systems, low
molecular weight glycoproteins (GLPs) that can act as iron reduc-
tases have been hypothesized as components of extracellular Fen-
ton systems in G. trabeum and P. chrysosporium (14). Four P.
placenta models show significant similarity (?48% amino acid
identity) to P. chrysosporium glp1 and glp2, and the gene encoding
medium (GEO accession no. GSE12540 si_table_7.xls). The se-
quence corresponding to another fungal protein implicated in Fe3?
reduction, CDH (6), appears to be absent in P. placenta.
iron homeostasis must play a central role in modulating a func-
tioning Fenton system. The P. placenta genome features numerous
genes potentially involved in iron transport and redox state (GEO
accession no. GSE12540 si_table_7.xls). In addition to 7 ferric
reductases, 2 iron permease genes were identified one of which lies
immediately downstream from a canonical yeast ferroxidase or-
tholog (Fet3). Transcripts of these adjacent genes were among the
most highly up-regulated in cellulose medium (GEO accession no.
Modification of Lignin and Other Aromatic Compounds. Genes en-
coding the class II secretory peroxidases lignin peroxidase (LiP),
manganese peroxidase (MnP) and versatile peroxidase (VP) were
not detected in the P. placenta genome (Table S3). Peroxidase
model Ppl44056 lacks residues involved in Mn2?binding and
oxidation of aromatic compounds (15), and superimposition of
protein models strongly suggests that Ppl44056 is a low redox
potential peroxidase (Fig. S1). Consistent with this structural
evidence, phylogenetic analyses of class II peroxidase genes from
Postia, Phanerochaete, and other fungal genomes suggest that
Ppl44056 is not closely related to LiP and MnP, but is part of an
assemblage of ‘‘basal peroxidases’’ that includes the novel peroxi-
dase (NoP) of P. chrysosporium and peroxidases from Coprinopsis
cinerea and L. bicolor (Fig. 2) (16). The backbone of the class II
peroxidase phylogeny is not strongly supported, but analyses of
broadly sampled datasets (16) suggest that the LiP and MnP gene
lineages of P. chrysosporium were independently derived from the
basal peroxidases before the divergence of Postia and Phanero-
chaete. If so, then the absence of LiP and MnP in P. placenta may
reflect additional instances of gene loss.
by white-rot fungi but have not previously been demonstrated in
brown-rot fungi. The precise role of these enzymes remains uncer-
tain, but numerous studies have demonstrated laccase-catalyzed
oxidation of phenolic and nonphenolic lignin model substrates,
cellulose as sole carbon sources. In B, a cluster of 24 of highly expressed genes is expanded and the color scale recalibrated to illustrate differences in transcript
CAZYs with statistical analyses of expression data are presented in SI Appendix and GEO accession GSE12540 si_table_1.xls.
Regulation of CAZY-encoding genes. (A) Expression profile of 144 glycoside hydrolase-encoding genes in media containing glucose versus microcrystalline
Martinez et al. PNAS ?
February 10, 2009 ?
vol. 106 ?
no. 6 ?
results from P. placenta belie the usual picture of brown-rot in that
(Fig. 2). Transcript levels of the genes encoding Ppl89382 and
Ppl111314 appear differentially regulated by decreasing slightly
(?1.08-fold) and increasing (?2.29-fold), respectively, on cellulose
si_table_7.xls). These enzymes could contribute to hydroxyl radical
generation by oxidizing hydroquinones as described in ref. 18.
Interestingly, laccase genes are absent from the genome of P.
chrysosporium (19), suggesting that laccase (sensu stricto) is not a
core component of fungal wood decay mechanisms and is certainly
not essential for white-rot.
Other up-regulated genes potentially involved in quinone
redox-cycling, and oxidation of lignin derived products include
those encoding ‘‘polyphenol oxidase’’ (Ppl114245), i.e., tyrosi-
nase or catechol oxidase related to typical laccases and various
oxidoreductases of uncertain function (Ppl107061, Ppl28683,
Ppl34850, Ppl61437, Ppl24981) (GEO accesssion no.
Oxalate Metabolism. In addition to pH effects on a wide range of
enzymes, extracellular accumulation of oxalate by P. placenta may
affect ferric iron availability and thereby impact hydroxyl radical
formation (20; reviewed in ref. 6). A metabolic shunt between the
citric acid and glyoxylate cycles is central to oxalic-acid accumula-
tion by the brown-rot fungus Fomitopsis palustris (21). Analysis of
the P. placenta genome demonstrates a functional glyoxylate shunt
and substantially extends our understanding of the number, struc-
ture, and transcription of key genes (Fig. S2; SI Appendix; GEO
accession no. GSE12540 si_table_8.xls).
Cytochrome P450 Monooxygenases. P450s have various roles in
secondary metabolism and are thought to be involved in biodeg-
radation of lignin and various xenobiotic compounds. The P.
placenta genome features an impressive set of 236 P450 genes
(SI Appendix, GEO acccesion no. GSE12540 si_figure_3.jpg), com-
pared with 149 in P. chrysosporium, and expansions of certain
families (CYP64, CYP503, CYP5031 and CYP617) were observed.
Genes encoding Ppl110015 (CYP53) and Ppl128850 (CYP503)
were significantly up-regulated in cellulose medium (GEO acces-
sion no. GSE12540 si_table_3.xls). The former is highly conserved
in fungi and thought to catalyze benzoate hydroxylation.
Other. The genome was systematically examined for genes involved
in oxidative phosphorylation, stress-related genes, signal transduc-
glycoside hydrolase expression and mating type (complete listings
and analysis in SI Appendix and Figs. S3–S5).
Analysis of the P. placenta genome elucidated a repertoire of genes
and expression patterns distinct from those of other known cellu-
lose-degrading microbes. The overall number of CAZY-encoding
genes in P. placenta, 242, is not particularly low, and among these,
the number of glycosyl transferases, 75, is fairly typical. However,
the genome completely lacks cellulose-binding domains and the
number of GHs is relatively low owing in part to the paucity of
cellulases. No exocellobiohydrolases and only 2 potential ?-1,4
is expressed at relatively high levels.
Comparisons with genomes of other cellulolytic microbes reveal
a strikingly distinct set of glycoside hydrolase genes in P. placenta.
Among aerobes, only the cellulolytic gliding bacterium Cytophaga
hutchinsonii lacks exocellobiohydrolases and endoglucanases fused
to cellulose-binding domains (22). The precise mechanism used by
C. hutchinsonii is somewhat mysterious, but it has been suggested
that cellulose chains are peeled away from the polymer and
transported into the periplasm (23). There, nonprocessive endo-
glucanases might readily degrade the cellulose. Such a mechanism
seems unlikely in P. placenta because all evidence suggests that
cellulose depolymerization by brown-rot fungi occurs at a distance
from the advancing hyphae. In contrast, C. hutchinsonii is in direct
contact with cellulose.
clearly expressed in cellulose-containing media (Fig. 3), may pos-
sess processive activity that enables it to liberate the cellobiose that
?-glucosidases then hydrolyze to assimilable glucose. Indeed, the
accumulation of putative ?-glucosidase transcripts and the corre-
sponding proteins that we observed are consistent with the avail-
ability of cellobiose in our cultures. Precedents for crystalline
cellulose hydrolysis by ?-1,4-endoglucanases within GH family 5
have been reported (24, 25), but it seems unlikely that the
Ppl115648 endoglucanase alone can account for the efficient cel-
lulose depolymerization by P. placenta. Other GHs and/or hypo-
thetical proteins, perhaps some of those expressed in microcrystal-
line cellulose cultures (Fig. 3; GEO accession no. GSE12540
si_table_1.xls), may be necessary for the complete breakdown of
cellulose. Heterologous expression of P. placenta GH-encoding
genes followed by biochemical characterization of the purified
proteins may resolve this question.
implicated the participation of low molecular weight oxidants,
particularly Fenton-generated hydroxyl radicals. As recently re-
viewed (6), 3 somewhat overlapping mechanisms of oxidative
degradation have been advanced. One view emphasizes the impor-
tance of CDH. In the case of P. placenta, CDH is absent. Another
view invokes the role of low molecular weight glycopeptides that
sporium (14), potential orthologs of these glycopeptide-encoding
genes were identified in P. placenta, and in 1 case, increased
transcript levels were observed in cellulose medium. Accordingly,
a role for these glycoproteins in a P. placenta Fenton system is
cycling (26). Evidence supporting this system includes cellulose
induction of genes encoding QRD, quinate transporter, phenylal-
anine ammonia lyase, and laccase. However, the importance of
hydroquinone-driven Fenton chemistry in P. placenta remains
unclear because this fungus secretes high levels of oxalate (27), and
Fe3?-oxalate chelates are poorly reducible by hydroquinones (28).
The elevated expression in cellulose medium of Fet3 and Ftr1,
partially explained by such chelates. Whereas cellulose itself may
sequester Fe3?(29), the generation of Fe3?-oxalate and poten-
tially other redox active iron-chelates might also contribute to
lower the effective concentration of bioavailable iron that is
accessible to the organism. Thus, cellulolytic conditions might
turn on the high-affinity iron-uptake system to ensure proper
levels of intracellular iron.
Also compatible with Fenton mechanisms is the observed cel-
lulose-induced expression of structurally divergent oxidases (e.g.,
copper radical oxidases, glucose-1-oxidases, and methanol oxi-
dases) and putative iron reductases. Given the significant number
of secreted hemicellulases observed, wood decay by P. placenta
likely involves attack by oxidative and hydrolytic mechanisms.
Elevated hemicellulase expression may reflect increased substrate
exposure and availability, relative to cellulose and lignin, especially
early in the decay process. Products of the hydrolytic attack could
ilarly, methanol released via demethoxylation of lignin (3, 4) may
play an important role in H2O2 generation as a substrate for
methanol oxidase. Such a role is consistent with our observed
expression patterns and with previous investigations with G. tra-
beum (11). Of course hydroxyl radical may also play an important
role early in decay, and it has been demonstrated to preferentially
attack hemicellulose in wood (30). Interestingly, ?OH attack on
www.pnas.org?cgi?doi?10.1073?pnas.0809575106Martinez et al.
cellulose oxidizes chain ends (31) and the depolymerized mate- Download full-text
rial becomes less amenable to cellulase action (30), providing a
plausible explanation for the lack of exocellobiohydrolase genes
in this fungus.
Comparison of the P. placenta and P. chrysosporium genomes
the contraction or loss of multiple gene families that are thought to
be important in typical white-rot, such as cellulases, LiPs, MnPs,
CROs, CDH, and pyranose-2-oxidase. This general pattern of
simplification is consistent with the view (32) that brown-rot fungi,
having evolved novel mechanisms for initiating cellulose depoly-
merization, have cast off much of the energetically costly lignocel-
lulose-degrading apparatus that is retained in white-rot fungi, such
as P. chrysosporium.
Materials and Methods
approach was used to sequence P. placenta strain MAD-698-R (USDA, Forest
sequenced paired reads by using JAZZ assembler. By using an array of gene
evidence, annotations, and analyses are available through interactive visualiza-
tion and analysis tools from the JGI genome portal www.jgi.doe.gov/Postia.
Detail regarding the assembly, repetitive elements, ESTs and annotation, are
provided separately (SI Appendix).
Mass Spectrometry. Soluble extracellular protein was concentrated from shake
cultures containing ball-milled aspen, microcrystalline cellulose (Avicel), or de-
waxed cotton as previously reported (33). Sample preparation and LC-MS/MS
analysis were performed as described (www.biotech.wisc.edu/ServicesResearch/
MassSpec/ingel.htm). Peptides were identified by using a Mascot search engine
(Matrix Science) against protein sequences of 17,173 predicted gene models
described above. Complete listings of CAZYs and oxidative enzymes, including
Expression Microarrays. Roche NimbleGen arrays were designed to assess ex-
pression of 12,438 genes during growth on microcrystalline cellulose or on
glucose as sole carbon sources (Fig. S6). The corresponding set of coding regions
was manually annotated to include only the ‘best allelic model’ among CAZY-
encoding genes (GEO accession no. GSE12540 si_table_1.xls). Methods are de-
tailed in SI Appendix and all data deposited under GEO accession no. GSE12540.
aspen. This work was supported by the U.S. Department of Energy’s Office of
Science, Biological and Environmental Research Program, and University of Cal-
ifornia, Lawrence Berkeley National Laboratory Contract DE-AC02–05CH11231;
Lawrence Livermore National Laboratory Contract DE-AC52–07NA27344; Los
Alamos National Laboratory Contract DE-AC02–06NA25396; University of Wis-
of Agriculture, Cooperative State Research, Education, and Extension Services
Grant 2007–35504-18257; National Institutes of Health Grant GM060201 (to
University of New Mexico); Centro de Investigaciones Biolo ´gicas (Madrid) EU-
project NMP2–2006-026456; Ministry of Education Czech Republic Grant
Agenda (U.S. Department of Energy, Washington, DC), DOE/SC-0095 (DOE).
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no. 6 ?