Available online at www.sciencedirect.
com
Coprophilous fungi: antibiotic discovery and functions in an
underexplored arena of microbial defensive mutualism
Gerald F Bills1, James B Gloer2 and Zhiqiang An1
Microbial antibiotics can mediate mutualisms and
interorganism communications. Herbivorous animal dung
offers opportunities for discovery of new antibiotics from
microbial communities that compete for a nutrient-rich,
ephemeral resource. Distinct lineages form a specialized
community of coprophilous (dung-colonizing) fungi. Bacteria,
protists, invertebrates, the mammalian digestive system, and
other fungi can pose challenges to their fitness in the dung
environment. The well-characterized diversity of dung fungi
offers accessible systems for dissecting the function of
antibiotics and for exploring fungal genomes for new
antibiotics. Their potential for antibiotic discovery is evidenced
by a high frequency of antifungal antibiotics and bioactive
secondary metabolites from limited prior efforts and from
mapping biosynthetic pathways in the genomes of the
coprophilous fungi Podospora anserina and Sordaria
macrospora.
Addresses
1
Texas Therapeutics Institute, The Brown Foundation Institute of
Molecular Medicine, University of Texas Health Science Center, 1825
Pressler Street, Houston, TX 77030, USA
2
Department of Chemistry, University of Iowa, E331 Chemistry Building,
Iowa City, IA 52242, USA
Corresponding author: Bills, Gerald F (gerald.f.bills@uth.tmc.edu,
billsge@vt.edu)
Current Opinion in Microbiology 2013, 16:549–565
This review comes from a themed issue on Antimicrobials
Edited by Robert EW Hancock and Hans-Georg Sahl
For a complete overview see the Issue and the Editorial
Available online 23rd August 2013
1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.mib.2013.08.001
Introduction
Antibiotic-seekers have favored certain biological com-
munities for exploration of chemically mediated
microbial interactions, including soils, marine invert-
ebrates, and plant–microorganism symbioses. In this
review, we present reasons why animal dung, particularly
from herbivorous mammals, offers exceptional opportu-
nities for discovery of new antibiotics from microbial
communities that compete within a nutrient rich, yet
physically patchy and ephemeral, resource. Three groups
of antibiotic-producing microorganisms, the myxobac-
teria, the streptomycetes, and the fungi, are conspicuous
in the dung community. The fitness of these organisms in
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the dung environment is challenged by niche overlap
with other bacteria, protists, invertebrates, the mamma-
lian digestive system, and other fungi. Although myxo-
bacteria and streptomycetes continue to be of great
interest as antibiotic producers, this review will focus
on results and developments in mycology, chemistry,
and genomics and argue strongly that coprophilous fungi,
especially coprophilous ascomycetes, will be a productive
reservoir of antibiotics and other bioactive secondary
metabolites.
The fungi are at the forefront of eukaryote comparative
genomics. Annotated fungal genomes are yielding sub-
stantial evidence for a richness of secondary metabolite
pathways among the major kinds of fungi well beyond
that imagined to date, and the number of sequenced
genomes will grow exponentially in the near future
[1,2,3]. Next-generation sequencing and automated
gene prediction can preview the biosynthetic repertoire
of a fungus and can anticipate the pathways for known
and unknown antibiotics. Filamentous ascomycete gen-
omes encode a broad spectrum of enzymes that synthes-
ize secondary metabolites, including nonribosomal
peptide synthetases (NRPSs), polyketide synthases
(PKSs), and terpene synthases (TPSs). In contrast, the
genomes of basally divergent ascomycetes (yeasts and
allies) generally lack secondary metabolites. The case of
fungi in the Pezizales, another early divergent group of
ascomycetes, may be intermediate. Their genomes
appear to have only a limited array of secondary metab-
olite pathways, as evidenced by the preliminary release of
the genome of the coprophilous ascomycete Ascobolous
immersus (http://genome.jgi.doe.gov/programs/fungi/
index.jsf). In contrast, in the basidiomycetes, terpenoid
biosynthesis predominates as the major modality of sec-
ondary metabolite production, with PKSs and NRPSs
occurring to a lesser extent [4,5,6]. Secondary metab-
olism in the Fungi is almost exclusively associated with
the filamentous fungi of the dikaryomycota — the asco-
mycetes and basidiomycetes — of which species in the
Eurotiales, Onygenales, Microascales, Sordariales, Pleos-
porales, Hypocreales, and Xylariales are amply
represented in the coprophilous community (Table 1).
How can the lessons learned from coprophilous fungi
contribute to understanding the natural roles of antibiotic
metabolites? The genetic and cellular basis for how fungi
assemble, store, activate, transport, and allocate second-
ary metabolites is better understood than ever [7,8].
Understanding the rules by which organisms interact and
Current Opinion in Microbiology 2013, 16:549–565
Current Opinion in Microbiology 2013, 16:549–565

550 Antimicrobials
Table 1

The known orders, families, and genera of coprophilous Ascomycota, and species reported to have produced secondary metabolites

Order Family Genus Species Compounds and Biosynthetic family Biological activity
references
Pleosporales Phaeotrichaceae Trichodelitschia – – – –
Sporormiaceae Delitschia confertaspora Flutimide [100] Peptide? Viral endonuclease inhibitor
unidentified sp. Talaroderxines [101] Polyketide
Preussia aurantiaca Auranticins [102] Polyketide
fleischhakii Diphenyl ethers [103]
isomera Preussomerins [104,105] Polyketide Ras-farnesyl-protein
transferase inhibitors, cyotoxity
Semidelitschia
Sporormia
Sporormiella australis Australifungin [106] Polyketide? Antifungal, ceramide synthase inhibitor
intermedia Zaragozic acid B [107] Polyketide-TCA- Squalene synthase inhibitor, antifungal
amino acid
minimoides Sporminarins [108] Polyketide Antifungal
Unnamed [109] Peptide-polyketide Antifungal
similis Similins [110] Polyketide Antifungal
teretispora Terezines [111] Peptide
vexans Sporovexins [112] Mixed Both antifungal, antibacterial
Preussomerins [112] Polyketide
Onygenales Ajellomycetaceae Polytolypa hystricis Polytolypin [113] Terpenoid Antifungal
Gymnoascaceae Arachniotus punctatus Gymnastatin N [114] Amino acid-polyketide Kinase inhibitors
Aranorosinol
Arachnomyces
Ctenomyces
Gymnoascus reessii Gymnoconjugatin A [115,116] Polyketide Cytoprotective
Rumbrin
Neogymnomyces Gymnoascolides [117] Shikimate
Pseudoarachniotus roseus Aranorosins [118,119,120] Amino acid-polyketide Antibacterial
Eurotiales Trichocomaceae Penicillium
Uncertain Myxotrichaceae Myxotrichum
Pezizales Ascobolaceae Ascobolus
Saccobolus
Thecotheus
Ascodesmidaceae Ascodesmis sphaerospora Arugosin F [121] Polyketide Antifungal
Pyronemataceae Cheilymenia
Fimaria
Trichophaea
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Pseudoombrophila
Thelebolaceae Ascozonus
Thelebolus
Tricobolus
Uncertain Coprotus
Lasiobolidium
Lasiobolus
Orbicula
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Hypocreales Ceratostomataceae Melanospora

Bionectriaceae Nigrosabulum globosum Pseudodestruxins [122] Peptide Antibacterial
Ascochlorin Terpenoid/polyketide Antifungal
Hypocreales Uncertain Selinia
Stilbella erythrocephala Antiamoebins [47] Peptide Antifungal
Myrocin B Terpenoid
aciculosa Fusidic acid [123] Sterol Antibacterial, anti-HIV
Microascales Microascaceae Doratomyces
Kernia
Microascus tardifaciens Tardioxopiperazines [124] Peptide/terpenoid Immunosuppressant
Petriella sordida Petriellins [125] Peptide Antifungal
Scopulariopsis Restricticins [126] Polyketide Antifungal
Antibiotic 1233A Polyketide
s Uncertain Sphaeronaemella
Sordariales Chaetomiaceae Chaetomidium
Chaetomium
Gilmaniella
Thielavia
Coniochaeta ellipsoidea Coniosetin [127] Polyketide-amino acid Antibacterial, antifungal
saccardoi Coniochaetones [128] Polyketide Antifungal
tetraspora Coniochaetones [129] Polyketide Monoamine oxidase inhibitor
Sordariales Lasiosphaeriaceae Apiosordaria
Arnium
Bombardioidea anartia Bombardolides [130] Modified polyketide Antifungal, antibacterial
Cercophora areolata Cercophorins [131] Polyketide Antifungal
Roridin F Terpenoid-polyketide

Antibiotics from coprophilous fungi Bills, Gloer and An 551

sordarioides Arthrinones [132] Polyketide Antifungal
Cerdarin Polyketide Antifungal
Podospora anserina Anserinones [133] Polyketide Antifungal, antibacterial
araneosa Sordarins [134,135] Terpenoid glycoside Antifungal, antitumor
appendiculata Appenolides [136] Polyketide Antifungal
Current Opinion in Microbiology 2013, 16:549–565

communis Communiols [137,138] Polyketide Antibacterial

curvicolla Curvicollides [139] Polyketide Antifungal
decipiens Decipenin A [140] Polyketide Antifungal
decipenolides
Terpenoid Antibacterial
pleiospora Sordarin [49] Terpenoid glycoside Fungal elongation factor-2 inhibitor
Schizothecium
Strattonia
Tripterosporella
Zopfiella
Zygopleurage
Zygospermella
Sordariales Sordariaceae Asordaria
Sordaria gondaensis Fumitremorgens [141] Peptide-terpenoid Immunosuppressive
macrospora Sordariols [142,143] Polyketide
Uncertain Pseudeurotiaceae Pleuroascus
Pseudeurotium ovalis Pseurotins [144,145] Polyketide + phenylalanine Antibacterial, chitin synthase inhibitors
552 Antimicrobials

defend themselves chemically, finding the triggers for

Inhibitor of eukaryotic DNA polymerase A,D

and in some viruses, induces apoptosis
pathway transcription, and then isolating and identifying
chemical products associated with microbial defense and
Type 2 methionine aminopeptidase

survival could lead to novel approaches in the develop-

inhibitor, angiogenesis inhibitor,

ment of chemical therapies. Dung fungi have already

contributed several candidate metabolites as model sys-
tems for elucidating ecological functions of antibiotics in

inducer in HeLa cells

Antifungal, cytotoxic
immunosuppressive

the dung microcosm (Table 1). Post-genomic methods

Biological activity

now enable experimental manipulation of this convenient

model community in ways previously unimaginable
Antifungal

[9,10,11,12]. Furthermore, as sequenced fungal gen-

omes continue emerging, the ideas that unknown biodi-
versity offers ample discovery space and that creatively
stimulating production of secondary metabolites from
underexplored fungi are supported as viable strategies
in the search for new bioactive natural products. Such
Polyketide-amino acid
Polyketide + C3 unit

emerging technologies offer multiple entry points to

Biosynthetic family

rationally dig beneath the fungal chemical phenotype

and unravel how and why antibiotics are made. We will
Diterpenoid
Terpenoid

Terpenoid

outline experiences from our own laboratories and explain

Terpene

how they can be merged with advances in genome

mining, analytical chemistry, and screening techniques
to improve the chances of finding new antibiotics.
Punctatins/punctaporonins

The coprophilous fungi — phylogenetic and

life style diversity
Apiosporamide [153]

Several fungal lineages have co-evolved with herbivores

Isoepoxydon [152]
[148,149,150,151]
Compounds and

Aphidicolin [154]

to disperse and compete in complex, antagonistic, tran-

Tulasnein [147]
Ovalicin [146]

Podospirone

sient, and resource-limited dung microcosms. Their life

references

cycles share the objectives of targeting and colonizing

specific nutrient microhabitats in dung, growing suffi-
ciently fast to reach reproductive maturity before the
habitat collapses, dispersing their spores to forage plants
of herbivores, moving their spores through the digestive
tract, and inoculating their spores in new dung [13].
coprophila
montagnei
punctata
Species

tulasnei

Mycologists have intensively studied coprophilous fungi

because of their ubiquity and ease of study [13,14]. Dung
fungi can be collected in the field, but more commonly,
dung samples are returned to the lab where they are
incubated in moist chambers to simulate naturally humid
conditions that encourage growth and sporulation
Onychophora
Podosordaria
Hypocopra

(Figure 1). Several excellent regional identification

Apiospora
Poronia

guides and monographs have been produced

Genus

[15,16,17,18]. As a result, their identification at the

generic, and often at the species level, is reasonably
straightforward, although unusual new species continue
to be discovered [19,20]. Their higher phylogenetic
Apiosporaceae

relationships have been substantially clarified in the last

Xylariaceae

decade [21,22,23,24], resulting in the recognition that

Uncertain

many historically important genera were artificially cir-

Family

cumscribed. Efforts to develop useful sets of marker

Table 1 (Continued )

genes for molecular identification of species have been

hampered by a lack of authentic strains tied to good
specimens [25,26]. Some of the most chemically inter-
Uncertain
Xylariales

esting genera based on the limited chemical studies

Order

published to date are very species-rich, for example,

Podospora and Sporormiella (Table 1), each having at least

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 Antibiotics from coprophilous fungi Bills, Gloer and An 553

Figure 1

(a) (b) (c) (d)

(e) (f) (g) (h)

Current Opinion in Microbiology

Examples of coprophilous fungi and their ascospores. (a) Podospora communis ascoma; (b) P. communis appendaged ascospore; (c) Poronia erici
ascoma; (d) P. erici ascus with ascospores and iodine-stained ascus apex. (e) Sporormiella (Preussia) intermedia ascomata protruding from surface of
white-tailed deer dung. (f) S. intermedia ascospores and part ascospore. (g) Delitschia confertaspora ascomata on V8-juice agar. (h) D. confertaspora
ascospores. Photos (a–d) taken by Jacques Fournier. Scale bars: (a, c, e, g) = 5 mm; (b, d, f, h) = 10 mm.

80 named species. If we extrapolate from what has already
been found about fungal species-level differences in
secondary metabolite diversity [5,27,28], then the
potential for discovery is enormous. In any event, the
existing taxonomic framework is adequate to guide anti-
biotic exploration toward the least-explored and the most
pathway-novel species.
Their trophic capabilities are complex and are partitioned
successionally to exploit the nutrient-rich environment
[13,29]. The typical coprophilous fungi, for example,
Podospora and Sordaria species, pre-position their myce-
lia in the dung cycle to capture energy from plant cell wall
deconstruction [12,30,31,32,33]. Some, like Chaetomium
species, are likely opportunists that invade the dung
environment from soil and plant debris using their potent
chemical arsenal to compete for lignocellulose. Other
species are mycoparasites of coprophiles [34,35], parasites
of coprophilous invertebrates [36], or degraders of hair
and skin mixed in from the diet or from animal burrows
[37,38]. Undoubtedly, the assimilation and concentration
of nutrients in the hyphae and spores makes fungi attrac-
tive targets for predation and parasitism, and thus has
been a factor in evolution of constitutive and induced
secondary metabolite pathways [39,40].
In the past, the trophic requirements of these fungi were
inferred by observation or tested experimentally with
cultured mycelia. Genomic annotation pipelines for
microbial genomes developed for carbohydrate-active
enzymes and general metabolism (CAZymes, www.ca-
zy.org; Kyoto Encyclopedia of Genes and Genomes,
www.genome.jp/kegg/) now can predict pathways for poly-
mer degradation, and hence can be used experimentally to
test gene function. The effects of the substratum in pro-
moting development of life-cycle stages associated with
secondary metabolism and pathway expression can be
tested by monitoring specific transcription events by
RT-PCR, microarray analysis, or high-throughput RNA-
Seq to obtain complete transcriptomes [9,28,41,42,
43,44]. In the case of keratinophilic fungi on dung, their
antagonistic behavior may be as much related to polymer
degradation as to secondary metabolite biosynthesis. Ker-
atinophilic fungi hydrolyze the cysteine disulfide bonds of
keratin, releasing high levels of cysteine. Sulfite is excreted
as a reducing agent, and in the presence of sulfite, disulfide
bonds of keratin are cleaved to cysteine and S-sulfocys-
teine; thus, reduced proteins are available for digestion by
fungal endoproteases and exoproteases [45]. These fungi
employ a cysteine dioxygenase (Cdo1) to oxidize the
L-cysteine to produce sulfite. Both cysteine and sulfite

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554 Antimicrobials

Figure 2

OH OH OH OH OH O
HO OMe O
N O
O Me H O OH
O O Me OH HO
O N O HOOC O HO
O O O OH HOOC
CHMe2 O
H COOH HO COOH O
OH Flutimide CHO
OH OH O
Zaragozic Acid B
Sordarin
Talaroderxine A
O
Preussomerin A CH3CO-Phe-Aib-Aib-Aib-Iva-Gly-Leu-Aib-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phol
H O
H Antiamoebin I
O N O
O N O
O OH O
N
N H N
OH O O H H3C O
O N O
CH3 O CH3 CH3
CH3 HO OH H3C
O O N CH3
OH O H3C O H
CH3
O O CH3 O OH
N N N OH Coniochaetone Communiol D Curvicollide A
N N H
O CH3 O CH3

Petriellin A
CH3
O O OH OH
HO N CH3 CH3 CH3 CH3 CH3 CH3 CH3
HO O CH3 OH
O
O N OCH3 CH3 CH3
H OH OH OH OH O
O
O OH
Terezine A Sporminarin A
Bombardolide
Podosporin A
Current Opinion in Microbiology

Selected chemical structures of biologically active metabolites from coprophilous fungi. See Table 1 for references to original discoveries and
bioactivities.

are toxic to other organisms above threshold concentrations (=S. erythrocephala) produced peptaibol antibiotics (anti-
[46]. Their cells protect themselves by a sulfite efflux amoebins, Figure 2) in rabbit dung pellets at concen-
pump (Ssu) that secretes sulfite. Thus, by-products of trations well above concentrations needed for inhibiting
substrate cleavage appear to provide a protective benefit. most competing coprophilous fungi and other fungi and
bacteria. The fungus also coproduced the diterpene
The dung environment as a model for natural myrocin B, but the levels measured in colonized dung
antibiotic function were below the predicted antifungal thresholds. Stilbella
Many secondary metabolites have been proven exper- fimetaria was self-resistant to its own metabolites. As a
imentally to mediate interactions between microorgan- result, it was hypothesized that exclusive colonization of
isms, plants, and animals, indicating that they are individual rabbit pellets by S. fimetaria observed in nature
functional in nature. Most of our understanding of fungal or inoculated dung samples was caused by antiamoebin-
natural–product function has been learned from exper- based antibiosis [47]. Sordarin, a diterpene glycoside,
imentation in plant pathogen–host plant and mycotoxin– and its aglycone, sordaricin (Figure 2), were first isolated
commodity systems. However, coprophilous fungi offer a from Podospora araneosa (=Sordaria araneosa) as antifun-
tractable system for developing chemical ecological hy- gal compounds lacking antibacterial activity. The dis-
potheses about how secondary metabolites enhance covery and effects of sordarins on fungal protein
overall fitness in populations that inhabit an ecologically biosynthesis via interaction of elongation factor 2 at
unpredictable landscape, either by observational infer- the ribosomal P-protein have been reviewed extensively
ences or by artificially manipulating the dung microcosm [48]. Experimentation with a sordarin-producing copro-
and its associated organisms [29]. Two examples philous fungus, Podospora pleiospora, demonstrated that
show the potential for experimentation. Stilbella fimetaria sordarin, its aglycone sordaricin, and sordarin analogs

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were produced in autoclaved rabbit dung [49]. Consist-
ent with its observed in vitro activity, sordarins produced
in simulated dung habitats exhibited antibiosis toward
the yeasts Nematospora coryli and Sporobolomyces roseus,
but not against the filamentous fungus Penicillium clavi-
forme. Sordarin production by P. pleiospora was unaf-
fected by the presence of other fungi when grown in
liquid medium co-cultures.
The detection and manipulation of biosynthetic pathway
products in complex habitats is challenging. The above
examples demonstrate that rabbit dung can be easily
inoculated, either by inoculating sterilized forage or by
inoculating sterilized dung pellets, to produce single-
species or multiple-species colonies that simulate a mini-
mal natural system [29]. Assessing secondary metabolite
expression as the antibiotic-producing fungus switches
among growth forms as it explores, captures, or defends
the dung pellets is technically feasible. This can be
accomplished either by metabolite extraction followed
by direct MS analysis or by RT-PCR monitoring tran-
scription of key secondary metabolite pathway genes, as
has been done with Podospora anserina [12] and Sordaria
macrospora [9] in agar. High-throughput RNA-Seq could
also map and quantify transcriptomes while surmounting
the limitations of hybridization-type approaches because
sequencing not only detects transcripts corresponding to
known genomic sequences, but also unknown genes from
any genome-sequenced organism. Thus, the method
would be suitable for evaluating fungus-fungus and fun-
gus-bacterium interactions and gene expressions, even
when dealing with newly genome-sequenced microor-
ganisms.
Targeted disruption of regulating genes, pathway promo-
ters, or key early pathway enzymes can suppress metab-
olite production and alter the outcome of interorganism
interactions [50,51]. Therefore, experimental manipula-
tion of antagonistic antibiotic-producing fungi in the dung
microcosm in parallel with their pathway-silenced
mutants could enable testing of hypotheses about inter-
organism interactions. Once a system is validated, possi-
bilities for additional targeted gene disruptions or
pathway overexpression can open multiple paths to crea-
tive chemo-ecological experiments to dissect antibiotic
function.
Antibiotics and other bioactive secondary
metabolites from prior studies of
coprophilous fungi
Reports of novel bioactive secondary metabolites from
coprophilous fungi have been limited to date, and come
mainly from only a few laboratories, as coprophilous fungi
apparently have not been incorporated in screening pro-
grams at levels competitive with fungi from other sub-
strata and niche groups. Even so, compounds from all
major biosynthetic classes are represented (Table 1),
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Antibiotics from coprophilous fungi Bills, Gloer and An 555
indicating that these fungi have robust secondary meta-
bolic capabilities, and this observation is consistent with
genomic annotations (see below). Secondary metabolites
described in the literature from coprophilous fungi in-
clude metabolites reported from other fungi, as well as a
variety of novel products. Analogs of some of these novel
compound-types were later reported from other fungi,
while others remain distinctive, with no close analogs
having been described from other sources. Noteworthy
examples of bioactive secondary metabolites reported
from coprophilous fungi (Figure 2) include preussome-
rins, flutimide, australifungin, sordarins, zaragozic acid B,
antiamoebins, talaroderxines, and petriellins. Other
reported compounds have perhaps less notable bioactiv-
ities, but display biosynthetically interesting or distinc-
tive structural features, and include coniochaetones,
communiols, curvicollides, podosporins, bombardolides,
terezines, and sporminarins. While details of these chemi-
cal studies are not reviewed in depth here, such reports
offer considerable support for the concept that the types
of coprophilous fungi highlighted in this review are well-
armed with secondary metabolite-producing capabilities,
and that some of the secondary metabolites they produce
have potent biological effects and/or distinctive chemical
structures. The most commonly reported biological
activity associated with these metabolites so far has been
antifungal activity. Only in a few cases has the mechanism
of antifungal activity been elucidated (Table 1). We have
firsthand knowledge of the screening history of a large
percentage of these compounds, and the bias toward
antifungal activity reflects a dependence on fungal phe-
notypic assays in the initial detection systems. These
compounds have not been extensively evaluated in bac-
terial or human targets. Therefore, a major goal of future
work with coprophilous fungal secondary metabolites, as
well as for other high quality natural product collections,
should be to ensure that new extract collections and
purified metabolites make their way into chemical collec-
tions of both commercially and publically funded drug
discovery centers so that they are circulated to and
evaluated in many more targets including biochemical,
human-cell-based, and antibiotic assays.
Predictive framework for previewing
secondary metabolite pathways
Genomic sequencing is establishing a predictive frame-
work for natural product biosynthesis, regulation, and
resistance, and has left no doubts about the importance
of functional natural products as pigments, siderophores,
virulence factors, growth regulators, and yet unknown
determinants in microbial life cycles [7]. Microorgan-
isms regulate their metabolite synthesis, presumably to
ensure that energy and precursors used in such processes
are expended only where and when the metabolites are
advantageous. In some cases, synthesis may be regulated
globally or tightly orchestrated with growth and devel-
opmental stages. It is also evident from the filamentous
Current Opinion in Microbiology 2013, 16:549–565
556 Antimicrobials

Figure 3

72 Sordaria macrospora SMAC 03130

0.1 Chaetomium globosum pks1
Hypoxylon pulicicidum NSPKS1
78 61
Colletotrichum lagenarium CLPKS1
89 PODANSg7836 Non-reducing PKSs
Ceratocystis resinifera PKS1 (melanins)
Glarea lozoyensis GLPKS1
Aspergillus parasiticus WA1
Wangiella dermatitidis WdPKS1
100 Elsinoe fawcettii efpks1
87 Alternaria alternata ALM1
Ascochyta rabiei ArPKS1
Penicillium aethiopicum GsfA
Aspergillus fumigatus encA Non-reducing PKSs
Aspergillus terreus ACAS (griseofulvin,
Aspergillus nidulans mdpG viridicatumtoxin)
58 Penicillium aethiopicum vrtA
Cercospora nicotianae CTB1
PODANSg8570 Non-reducing PKSs
Dothistroma septosporum pksA
100
(sterigmatocystin)
Aspergillus nidulans stcA
Aspergillus parasiticus pksL1
Gibberella fujikuroi fsr1
60 Gibberella fujikuroi GFPKS4
57
Aspergillus oryzae aoiG
Gibberella zeae PKS12
70
Talaromyces marneffei alb1
60 65 Pencillium griseofulvum WA Non-reducing PKSs
54 Aspergillus nidulans ANWA (conidial pigments)
Aspergillus niger albA
Aspergillus fumigatus AFALB1
PODANSg8506
95 86 Pochonia chlamydosporia RDC1
Non-reducing PKS
100 Chaetomium chiversii RADS2
Gibberella zeae PKS13 (radicicol, zearalenone)
69 Hypomyces subiculosus Hpm3
93 Aspergillus nidulans orsA Non-reducing PKS
Talaromyces marneffei pks12 (orsellinic acid)
PODANSg600
99 Aspergillus nidulans ausA
71 72 Aspegillus terreus trt4
90 Penicillium brevicompactum MpaC Non-reducing PKSs
52
PODANSg3829 (citrinin)
72 PODANSg2680
Chaetomium globosum cazM
99
Monascus purpureus PksCT
Sarocladium strictum MOS
53
Aspergillus niger azaA
53 Talaromyces stipitatus tropA
Talaromyces marneffei pks11
PODANSg2301
Aspergillus westerdijkiae aomsas
100
Penicillium griseofulvum PKS2 Partially reducing PKSs
Glarea lozoyensis GLPKS2 (6-methyl-salicylic acid)
Penicillium patulum PPMSAS
Penicillium nordicum otapksPN
Aspergillus terreus ATATX
Aspergillus parasiticus pksL2
PODANSg1941
Cochliobolus heterostrophus CHPKS1
PODANSg1131
Aspergillus nidulans EasB
Aspergillus westerdijkiae aoks1
Alternaria solani PKSN
Gibberella moniliformis Fub1
Phoma sp.C2932 phPKS1
55 Chaetomium globosum cazF
50 Fungal reducing PKSs
PODANSg5999
86
Aspergillus niger azaB
Aspergillus terreus LovF
Hypomyces subiculosus Hpm8
Cladonia metacorallifera CmPKS1
PODANSg1178
Gibberella zeae FSL1
PODANSg1570
Botryotinia fuckeliana BcBOA6
68 PODANSg9775
Fungal PKS-NRPS hydrids
61 PODANSg2214
Fusarium heterosporum EqiS
Aspergillus terreus LovB
99 Gibberella fujikuroi fusA
Metarhizium robertsii NGS1
Aspergillus fumigatus PsoA
73 Penicillium expansum CheA
Beauveria bassiana TenS
Aspergillus flavus CpaS
Xylaria sp. BCC1067 pks3
Aspergillus nidulans ApdA
99 Alternaria solani Sol1
PODANSg2799
Botryotinia fuckeliana BcBOA9
34
98 Gibberella fujikuroi GFFUM5
82 Glarea lozoyensis GLPKS4 Fungal reducing PKSs
54 PODANSg7686
PODANSg1326
PODANSg1601
PODANSg5890
Rattus norvegicus FAS

Current Opinion in Microbiology

Current Opinion in Microbiology 2013, 16:549–565 www.sciencedirect.com


fungal genomes studied to date that they all have the
kinds of pathways necessary to produce small molecules
that can affect the behavior or growth of other organisms.
In every case, the number of presumed secondary metab-
olite gene clusters exceeds the number of metabolites
detectable in a given species in laboratory culture,
suggesting the presence of a significant number of unex-
pressed pathways. These unexpressed gene clusters,
commonly referred to as ‘silent’ or ‘orphan’, have been
found in all filamentous fungal genomes examined to
date. Some may encode for known secondary metabolite
biosynthetic pathways, including those not expressed by
the organism under previously studied laboratory con-
ditions. More importantly, however, they may correspond
to previously unknown biosynthetic pathways respon-
sible for novel secondary metabolites.
The common dung fungus S. macrospora has little pub-
lished history of producing secondary metabolites or
antibiotics (Table 1). However, bioinformatic analysis
[52,53] of its genome sequences uncovers the pre-
sence of approximately 14 core pathways involved in
secondary metabolite biosynthesis. As mentioned above,
secondary metabolism in the Sordiariaceae sensu strictu,
which includes the sister genus Neurospora, appears to be
especially streamlined. The genome of N. crassa itself
only has about 17 secondary metabolite pathways [54],
possibly a result of evolutionary contraction associated
with its adaptation to lack of competition in fire-scoured
habitats. In S. macrospora, melanin biosynthesis via the
dihydroxynaphthalene pathway [55], as well as eight
different type I PKSs and three NRPSs, were significantly
upregulated during the sexual cycle [9]. Disruption of
one of the pathway dehydrogenase genes ( fbm1) down-
stream of SMAC 527 impaired perithecium development
and ascospore ejection [56].
Likewise, little is known about secondary metabolite
expression or identification of secondary metabolites in
P. anserina (Table 1). However, bioinformatics analysis of
its genome reveals a significantly more complex second-
ary metabolome, with about 31 core biosynthetic genes
and pathways (Figure S1). Bioinformatic searches across
fungal genomes revealed an intact 24-gene, 57-kb cluster
in P. anserina with extraordinary conservation of gene
homology and microsynteny with the Aspergillus nidulans
sterigmatocystin cluster [57] (Figure S1). Its origin was
believed to be a result of horizontal gene transfer (HGT)
because of incongruencies between multigene phyloge-
nies of the Eurotiales and Sordariales and phylogenies
based on the gene-coding sequences of the cluster
enzymes. Searches of a collection of 51 862 expressed
( Figure 3 Legend ) Evolutionary relationships of type I PKSs from the coprophilous fungus Podospora anserina (red), and functionally characterized fungal
type I PKSs inferred by neighbor-joining analysis of the amino acid (aa) sequence of the ketosynthase domain. Some of the major PKS subtypes sharing a
common domain organization are indicated by shaded boxes. Branch length indicates number of inferred aa changes. The percentage of replicate trees in
which the associated taxa clustered together at least 50% of the bootstrap tests (1000 replicates) are indicated at the branches. Branch lengths are
proportional to the number of inferred aa changes. Rat fatty acid synthase is the outgroup. Fungal gene abbreviations can be found in Table S1.
www.sciencedirect.com
Antibiotics from coprophilous fungi Bills, Gloer and An 557
sequence tags (ESTs) generated from cDNA libraries
constructed at seven successive life-cycle stages of P.
anserina with sequences from the putative sterigmatocys-
tin cluster proteins identified transcripts for 14 of the 24
cluster genes (including the aflR regulatory gene), indi-
cating some level of pathway expression at five of the
seven life cycle stages. Independent observation of ster-
igmatocystin production in a Podospora species [58],
possibly conspecific with P. anserina, implicated this
horizontally acquired cluster as the pathway for sterigma-
tocystin in Podospora. The report as of yet could not offer
a plausible mechanism for this HGT event. However,
Aspergilli and Podospora species coexist in dung. Various
HGT mechanisms conceivably might operate [59] within
the confined environment of the dung microcosm where
microorganisms must coexist or die. Clearly, the dung
microcosm could provide an exceptional experimental
arena for simulating HGT events.
Supplementary material related to this article can be
found, in the online version, at http://dx.doi.org/
10.1016/j.mib.2013.08.001.
By analysis of the annotated genomic sequence of P.
anserina [12], one can begin to predict its potential to
make secondary metabolites (Figure S1). We can specifi-
cally anticipate some of the chemistry from the putative
PKS pathways of the fungus by comparison a segment of
the PKS ketosynthase domain which was previously
validated to be predictive of its encoding chemistry
to ketosynthase domains of functionally characterized
PKSs (Figure 3, Table S1). Its genome encodes the main
types of fungal PKSs; type I PKSs, including non-
reducing PKSs (NR-PKSs), highly reducing PKSs (HR-
PKSs), hybrid PKS-NRPSs, fully reducing PKSs, and
at least one type III PKS. Besides sterigmatocystin
(PODANs8570), we can anticipate biosynthesis of mel-
anin (PODANSg7836) and possibly another pigment
(PODANs8506), an equisetin-like tetramic acid
(PODANs2214), and three other pathways with products
of a polyketide-amino acid hybrid architecture
(PODANs1178, 1570, 9775), a solanopyrone-like metab-
olite produced by an HR-PKS (PODANSg2799), and a
product of a type III PKS (PODANSg2924, Figure S1)
which is an ortholog of the Neurospora crassa ORAS gene
responsible for biosynthesis of pentaketide resorcylic
acids [60]. Three PKS genes are found in clusters where
they are paired with NRPS genes (Figure S1,
PODANSg599–600, 1940–1941, 2680–2681) indicating
the biosynthesis of complex hybrid products reminiscent
of the biosynthesis of pneumocandins [61]. In
PODANSg599-600 and PODANSg1940-1941, homology
Current Opinion in Microbiology 2013, 16:549–565
558 Antimicrobials
searches with either the PKS or NRPS genes revealed no
obvious orthologous genes, indicating potential novelty,
while in PODANSg2680-2681, the PKS gene shows some
homology to a PKS responsible for 5-methylorsellinic acid
biosynthesis in Penicillium brevicompactum. Among the
remaining NRPSs, PODANSg524, PODANSg1248,
and PODANSg1535 show relatively high similarity to
an NRPS in Neurospora spp., S. macrospora, Thielavia
terrestris, and Myceliophthora thermophile. PODANSg3383,
with a single adenlylation domain, shows relatively low
homology to other fungal NRPSs (most similar is 44%
with a NRPS from Arthroderma gypseum). PODANSg8626,
a bimodular NRPS, shows some similarity to a NRPS in C.
glosbosum (76% similarity). Probably the most intriguing
cluster is the tandem NRPS consisting of PODANSg3425
(5 adenylation domains) and PODANSg3444 (3 adenyla-
tion domains). One can conclude from this summary that,
at present, only a few of the secondary metabolite path-
ways in P. anserina can be related to known fungal
metabolites or pigments, and that at least half of the
secondary metabolite pathways in P. anserina show little
similarity to pathways in other genome-sequenced fungi.
Supplementary material related to this article can be
found, in the online version, at http://dx.doi.org/
10.1016/j.mib.2013.08.001.
Stimulation of fungal biosynthetic pathway
expression
Some experts have raised concerns that biodiversity-dri-
ven and fermentation-driven approaches to antibiotic
discovery have reached their useful limits because of
the severe decline in discovery rates for new structural
classes of antibiotics [62,63]. In our view, this perceived
unproductivity is not surprising, as resources dedicated to
such efforts have plummeted, while much existing effort
is directed toward re-inventing screening methods pre-
viously practiced in industry [64]. At the same time, most
experts agree that the probability of success in drug
discovery increases with the size and chemical diversity
of screening libraries. Unfortunately, the size metric is
more easily measured than chemical diversity, and this
bias often causes pressure to prepare larger sample col-
lections at the expense of quality and novelty. As a result,
(the significantly reduced) ongoing efforts to seek new
bioactive microbial metabolites have continued to focus
on historically productive microorganisms using well-
established fermentation methods, often with inadequate
attention to nutrition and signals for secondary metabolite
expression. If that scenario is combined with simple
empirical growth inhibition assays or single gene enzyme
assays, then expectations will be low [65]. Even so,
experts continue to argue for new discovery paradigms
that penetrate into more inaccessible regions of the
microbial metabolite reservoir [66]. Our experience is
that we have never suffered from a lack of chemical
Current Opinion in Microbiology 2013, 16:549–565
novelty from fungi, but rather a limitation in access to
evaluation of their chemistry in novel targets. Therefore,
more in-depth exploration of the coprophilous fungi for
antibiotic discovery is justified from the point of view of
untested biodiversity.
Once these organisms are brought into culture, the next
challenge is provoking production of their secondary
metabolites in the laboratory so that their effects on other
organisms can be evaluated. Effective generation of
screening samples for drug discovery would ideally strive
for activation of all secondary metabolite pathways in each
organism. Such a goal may be too ambitious. However, a
number of creative techniques can be combined to expand
metabolite expression from sets of strains. These methods
include growth in media charged with natural elicitors (e.g.
oat flour, tomato paste), nutritional arrays [67], the FER-
MEX (Fermentation Extract) method [68,69], addition of
ion exchange resins [70,71], late fermentation medium
replacements [72], directed biosynthesis of halogenated
derivatives [73,74], and more recently, addition of epige-
netic agents to modulate expression [69]. How best to
combine these methods to efficiently obtain maximum
chemical diversity remains an open question [68]. The
ability to assess transcription of secondary metabolite path-
way genes in model species and correlate those data with
metabolomic analyses should now be able to address these
questions, as was recently assessed in the model fungus A.
nidulans [75]. Growth of the fungus on a well-informed
selection of three media led to the detection of over 59
different secondary metabolites, of which 42 could be
identified through database searches. Even more revealing
was that transcriptome profiling of mycelia from the three
media detected mRNAs from 58 of the A. nidulans gen-
ome’s 70 predicted PKSs, NRPSs, dimethyallyl trans-
ferases, terpene synthases, and prenyl transferases.
Several of the same metabolites that were recently acti-
vated by genomic mining techniques [76,77,78] were
encountered in these simple solid medium extracts. These
positive results contradict recent discouraging generaliz-
ations such as ‘Most fungal biosynthetic pathways are
cryptic, however, producing no products under normal
laboratory growth conditions’ [79].
As an alternative to growth manipulation of pure cultures,
the idea that chemical interactions between microbes can
lead to a reaction followed by the production of new
metabolites which may not be observed in axenic culture
extracts has received renewed attention [76,80]. As dis-
cussed above, investigations of simplified pairings of
members of the coprophilous community can reveal
molecular networks that contribute to coprophilous com-
munity structure through modulation of signaling, hyphal
extension, sporulation, and motility. The idea to system-
atically apply these methods to dissect interactions among
dung fungi and bacteria has considerable merit because of
the close contact between fungi, actinomycetes, and
www.sciencedirect.com

myxobacteria. The design of screening experiments to
detect metabolites resulting from such experiments is
complex and outcomes will be highly method-dependent.
As pointed out by Seyedsayamdost et al. [81], and in
agreement with our own experience, reliance on antimi-
crobial phenotypes as a sensor of interactions would likely
miss a large proportion of the interactions. In two differ-
ent experimental designs with actinomycetes, the exper-
imental pairing system relied on phenotypic changes in
Streptomyces colonies (changes in sporulation, aerial
mycelium, pigmentation) relative to a control colony to
detect interspecies recognitions [81,82]. Carrying out
agar pairing experiments with fungi is substantially more
complicated than in actinomycetes because of differen-
tials in growth rates. The logistical complexity and data
processing needed to analyze these kinds of pairing
experiments is well-illustrated in a recent study of inter-
actions among 83 strains of Fusarium species, environ-
mental fungi, and fungal dermatophytes [83]. The
interaction zones of 600 co-cultures were analyzed
visually and classified by interaction types. As expected,
a large array of phenotypic changes and interactive beha-
viors were observed. Interestingly, long-distance antagon-
istic reactions indicative of antibiotic-mediated
recognition were infrequent (<5% of the interactions).
A subset (138 interactions) were analyzed by ultra-high-
pressure liquid chromatography coupled to electrospray
ionization time-of-flight mass spectrometry (UHPLC–
TOF-MS) specifically for appearance of new molecules
in the confrontation zone. Analysis of the restrictive
confrontation zones and recognition of differential pro-
duction of metabolites presented a significant technical
challenge. Evidence was observed for large numbers of
chemical species in interaction zones that were not
detected in axenic controls, and many of these were
not found by database searches based on m/z values
and molecular formula. How the frequency of unknown
ions compared to that of unknowns found in control
cultures was unclear, nor was it clear whether these
molecules were responsible for any of the interactions.
One striking antagonistic interaction between colonies of
Bionectria ochroleuca and Trichophyton rubrum resulted in the
isolation of a sulfonated form of PS-990, a tridepside
consisting of three substituted orsellinic acid monomers
(4-hydroxy-2-methoxy-3,5,6-trimethylbenzoic acid) [84]
which was previously reported as a metabolite of an Acre-
monium species. Production of PS-990 was attributed to the
B. ochroleuca half of the pair because it could be found only
in B. ochroleuca axenic cultures. The biogenic origin of the
sulfonation was unclear. One wonders what would have
been the outcome had this set of fungi had been grown on a
set of several carefully selected complex media, extracted,
and fractionated, with the fractions then tested against a
panel of pathogens and environmental organisms. We
speculate that in terms of work per new active antibiotic
molecule, the results would speak for themselves.
www.sciencedirect.com
Antibiotics from coprophilous fungi Bills, Gloer and An 559
The above-mentioned screening experiments with strep-
tomycetes are among the few applications of the co-
culture technique that have been directly applied to
bioactivity-guided antibiotic discovery. With fungi, co-
cultivation has been more commonly practiced as a
method to simply screen for new compounds (see refer-
ences cited in [83]), but its success relative to other
methods is difficult to judge. Co-cultures are generally
monitored by UV-detector and/or MS-detector for
changes in secondary metabolite profiles, and newly
recognized constituents are purified and subjected to
structural characterization. Bioactivities are evaluated
after components are purified. Thus far, most such reports
describe isolated successful cases, and the level of invest-
ment in screening paired strains required to detect the
corresponding new secondary metabolites is generally
unclear. To the best of our knowledge, none of these
studies has been accompanied by direct comparison with
results from conventional multiple media conditions to
know whether the induced metabolites might be formed
under alternative culture conditions.
Two interesting examples of a global approach that could
be implemented across many species of fungi is disrup-
tion of ubiquitin-mediated protein degradation within the
fungal cell [85], and disruption of SUMO (Small Ubiqui-
tin-like Modifier) [86], a small ubiquitin-like protein that
post-translationally modifies a number of proteins in the
cells, including proteins involved in the regulation of
transcription. Both protein targets are highly conserved
in eukaryotes, including fungi. Rendering the COP9
signalosome complex inactive by disruption of the fungal
Csn5 homolog CsnE prevents degradation of regulators,
including transcription activators, of secondary metab-
olites and reproductive development, resulting in fungal
mutant strains with altered expression of secondary
metabolite genes and sporulation [87]. Disruption of
SUMO in A. nidulans resulted in significant upregulation
of biosynthesis of the polyketide asperthecin, and a
decrease in the synthesis of austinol, dehydroaustinol,
and sterigmatocystin. Therefore, disruption of global
regulatory genes, for example, CsnE and SUMO, could
effectively activate multiple biosynthetic gene clusters in
a variety of organisms leading to simultaneous production
of their corresponding secondary metabolites. The over-
riding benefit of global regulator approaches are analo-
gous to those of chromatin modification approaches
because underlying knowledge of regulation in specific
gene clusters is not necessary, and the influence of
transcription factors in the cluster is largely irrelevant.
Improved technologies for secondary
metabolite isolation and structure elucidation
Of parallel importance, chemical isolation techniques and
instrumentation have vastly improved in recent years,
leading to an ability to isolate and identify minor metab-
olites that were inaccessible one or two decades ago.
Current Opinion in Microbiology 2013, 16:549–565
560 Antimicrobials
These improvements, together with assay miniaturization
technology, enable discovery and exploration of the
potential of new and/or trace secondary metabolites with
greater speed and at lower cost, partially alleviating con-
cerns about the pace and expense of screening for new
bioactive secondary metabolites. Even so, such efforts
require capital-intensive resources and custom-designed
informatics, so these advances alone may be insufficient
to reverse declining discovery rates in the absence of
significant investment. Emerging technologies described
above have the potential to eventually attain the goal of
identifying all secondary metabolites from all organisms
for which genomic information can be obtained, such as in
the case of A. fumigatus [88,89] and A. nidulans [7,75].
The ability to anticipate or propose structures expected to
arise as the end products of deciphered secondary metab-
olite gene clusters would be extremely useful in the
search for novel or unusual natural products. One could
envision in silico screening of putative products in an
effort to target those belonging to a structural class of
interest, for example, large peptides. Ultimately, how-
ever, such proposed secondary metabolites would need to
be produced in the laboratory, not only to independently
verify their structures, but also to enable biological testing
and, of course, to exploit any utility they may have.
The extensive array of separation methodologies avail-
able for isolation of secondary metabolites includes a
growing variety of stationary phases that can be employed
in high performance liquid chromatography (HPLC)
methods and instrumentation. HPLC in general remains
a flagship separation technique, and its prominence in this
regard is enhanced by the ability to readily interface it to
other relevant analytical tools such as MS and/or UV
spectroscopy to assist in rapid identification of previously
known compounds (dereplication) [90,91], as well as in
distinguishing those that may be new or unusual. Other
separation method improvements, for example, counter-
current chromatography [92,93], have continued, and
such methods are also valuable in certain situations.
Significant advances in techniques for rapid and detailed
structure analysis have also been made. Access to instru-
ments that provide high resolution MS (i.e. for elemental
composition) and MS–MS data have become consider-
ably more common. Benchtop HPLC–MS instruments
have become more routine and affordable. Nuclear mag-
netic resonance (NMR) spectrometers at increasingly
higher field (offering better signal resolution and sensi-
tivity) are commercially available, as are NMR probe
improvements providing additional, sometimes rather
dramatic sensitivity enhancements (microprobes, cryop-
robes) [94,95]. Such advances enable not only more
rapid data collection on routine samples, but also acqui-
sition of quality data on trace amounts of material. Com-
putational methods and commercial software are
emerging to help deconvolute the process of mixture
analysis, and to help automate the data interpretation/
Current Opinion in Microbiology 2013, 16:549–565
structure elucidation process to a certain extent [96,97].
The sophistication and applicability of computational
methods in stereochemical analysis is also increasing
[98,99]. Thus, a convergence of technologies in both
chemistry and biology should help foster a resurgence
of microbial natural product chemistry.
Final remarks
Coprophilous fungi are commonplace organisms because
of their ubiquity, yet they are exotic in other respects
because of a paucity of prior chemical studies. These
fungi possess a unique combination of features that point
to them as elite candidate organisms for continued dis-
covery of new secondary metabolites with antibiotic and
other effects, and for exploration of possible functions
that these molecules may have in nature. They have
succeeded in thriving in, if not dominating, a highly
competitive environment. Relatively limited efforts
undertaken to date have shown that they are chemically
rich and capable of producing novel bioactive secondary
metabolites. These limited chemical evaluations are con-
sistent with preliminary genomic annotations of the few
fully genome-sequenced coprophilous fungi. Some of
these, for example, P. anserina and S. macrospora, are
genetic model organisms and are closely related to
genetic model species of Neurospora, therefore, genetic
methods to manipulate the corresponding secondary
metabolite genes are at hand. These model fungi, and
their unstudied relatives, are easily manipulated in the lab
and could be genetically engineered to test hypotheses
about the natural roles of antibiosis and how antibiotic
expression is triggered in dung microcosms.
We believe that natural antibiotic sources and other
bioactive secondary metabolites are far from exhausted,
especially when it comes to fungi. Generally speaking, we
have never experienced a lack of interesting chemistry
from previously unexplored fungi that have been ade-
quately grown on a suitable set of complementary media.
We believe the greatest challenge for the future of
antibiotic discovery is to re-establish infrastructures that
can support the complex process of antibiotic discovery
and development, and to do so with recognition of the
importance of such work as a long-term investment. Such
infrastructures must foster the building of cohesive teams
of scientists with creative know-how in exploiting sources
of natural chemical novelty and biological functions,
together with those seeking new molecular entities effec-
tive against pathogenic microorganisms.
Acknowledgments
We thank Rodolfo Aramayo, Texas A&M University, for helpful discussions
on bioinformatics analyses and Jacques Fournier, Rimont, Ariège, France,
for generously contributing his photographs of dung fungi. Lynn Silver
made insightful suggestions on the manuscript. JBG’s prior work on the
chemistry of coprophilous fungi was supported by grants from the National
Institutes of Health.
www.sciencedirect.com

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