RESEARCH ARTICLE

LaeA-Regulated Fungal Traits Mediate Bacterial Community

Assembly
Joanna Tannous,a,b Casey M. Cosetta,c Milton T. Drott,d,e Tomás A. Rush,b Paul E. Abraham,b Richard J. Giannone,b
Nancy P. Keller,a,d Benjamin E. Wolfec

a Department of Medical Microbiology and Immunology, University of Wisconsin—Madison, Madison, Wisconsin, USA
b Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
c Department of Biology, Tufts University, Medford, Massachusetts, USA
d Department of Plant Pathology, University of Wisconsin—Madison, Madison, Wisconsin, USA
e USDA-ARS Cereal Disease Laboratory, St. Paul, Minnesota, USA

ABSTRACT Potent antimicrobial metabolites are produced by filamentous fungi in

pure culture, but their ecological functions in nature are often unknown. Using an anti-
bacterial Penicillium isolate and a cheese rind microbial community, we demonstrate
that a fungal specialized metabolite can regulate the diversity of bacterial communities.
Inactivation of the global regulator, LaeA, resulted in the loss of antibacterial activity in
the Penicillium isolate. Cheese rind bacterial communities assembled with the laeA dele-
tion strain had significantly higher bacterial abundances than the wild-type strain. RNA-
sequencing and metabolite profiling demonstrated a striking reduction in the expression
and production of the natural product pseurotin in the laeA deletion strain. Inactivation
of a core gene in the pseurotin biosynthetic cluster restored bacterial community com-
position, confirming the role of pseurotins in mediating bacterial community assembly.
Our discovery demonstrates how global regulators of fungal transcription can control
the assembly of bacterial communities and highlights an ecological role for a wide-
spread class of fungal specialized metabolites.
IMPORTANCE Cheese rinds are economically important microbial communities where

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fungi can impact food quality and aesthetics. The specific mechanisms by which fungi
can regulate bacterial community assembly in cheeses, other fermented foods, and micro-
biomes in general are largely unknown. Our study highlights how specialized metabolites
secreted by a Penicillium species can mediate cheese rind development via differential in-
hibition of bacterial community members. Because LaeA regulates specialized metabolites
and other ecologically relevant traits in a wide range of filamentous fungi, this global reg-
ulator may have similar impacts in other fungus-dominated microbiomes.

KEYWORDS Penicillium, fungal metabolites, microbiome

F ungal specialized (or secondary) metabolites (SMs) gained attention in the 1900s fol-
lowing the discovery of the world’s first antibiotic, penicillin, produced by a Penicillium
isolate (1). Since then, fungal SMs have come to play pivotal roles in medicine, agriculture,
Editor John W. Taylor, University of California
—Berkeley
and biotechnology. Fungal SMs have been well-characterized in axenic cultures, including This is a work of the U.S. Government and is
not subject to copyright protection in the
genetic (2–5), biochemical (6), and physiological aspects of their regulation and production United States. Foreign copyrights may apply.
(7–9). In cocultures, fungal SMs have been suggested to play roles as signaling molecules Address correspondence to Nancy P. Keller,
that mediate the communication of the fungus with its surroundings (10–13), virulence [email protected], or Benjamin E. Wolfe,
[email protected].
factors to support pathogenic lifestyles (14, 15), microbial inhibitors that shape the compe-
The authors declare no conflict of interest.
tition with other microorganisms for finite resources (16–18), or defenses against fungi-
Received 28 March 2023
vores (19, 20). Accepted 3 April 2023
While these studies are critical first steps in understanding the potential functions Published 10 May 2023
of fungal SMs, the ecological roles of these compounds in multispecies communities

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are largely unknown. The activities of fungal SMs identified under laboratory condi-
tions may not translate to natural communities if concentrations used in vitro are not
reflective of those found in nature or if other community members are able to inacti-
vate or alter these compounds. Purified and concentrated fungal SMs can inhibit the
growth of some bacteria and fungi in large quantities in simplified lab environments
(21, 22), but how naturally secreted fungal SMs operate in microbial communities is
unknown. Fungal SMs could structure multispecies communities by mediating ecologi-
cal interactions that favor certain species over others, resulting in a shift in community
composition. We are unaware of studies that have demonstrated this scenario.
One approach for identifying fungal SMs that mediate bacterial communities is by
altering the activity of global regulators of fungal metabolites. In the fungal phylum
Ascomycota, the production of many SMs is under the control of the global regulatory
protein of the trimeric velvet complex, LaeA (23, 24). A growing body of research has
emphasized the role of LaeA in regulating SM production in monocultures of Aspergillus,
Fusarium, Penicillium, and other fungal genera (25–28). LaeA also regulates many biosyn-
thetic gene clusters (BGCs), including the aflatoxin and cyclopiazonic acid gene clusters in
Aspergillus flavus (25, 29), and the patulin gene cluster in Penicillium expansum (28).
Besides its impact on the metabolome, LaeA was also reported to regulate other traits in
filamentous fungi, such as conidiation (30), conidial morphogenesis (31), and sclerotia for-
mation (32). LaeA-regulated fungal traits may play important ecological roles in multispe-
cies microbial communities, but most studies of LaeA biology have been conducted with
fungal monocultures. Several studies have explored how LaeA can mediate fungal strain
competition and host-microbe interactions (8, 33), but the ecological roles of LaeA in the
assembly of polymicrobial communities has not been characterized.
Cheese rinds are microbial ecosystems where LaeA-regulated SMs could have signifi-
cant ecological impacts. These ecosystems are composed of bacteria, yeasts, and filamen-
tous fungi and form on the surface of many styles of cheese, including bloomy, washed,
and natural rind cheeses (34). Penicillium spp. are frequently encountered in cheese rinds
where they can be inoculated as industrial starter cultures (e.g., P. camemberti in
Camembert or Brie) or can colonize cheese from natural populations of fungi (e.g., P.
biforme, P. solitum, and P. nalgiovense in tomme style cheeses and clothbound cheddars
[34, 35]). Species within this genus are prolific producers of SMs, including polyketides
(e.g., patulin), nonribosomal peptides (e.g., roquefortine), and terpenes (e.g., expansolide)

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(36). While many Penicillium metabolites are valued as pharmaceuticals, such as the anti-
biotic penicillin (1) and the cholesterol-lowering drug lovastatin (37), others are consid-
ered mycotoxins, including the carcinogenic ochratoxin A (38), cyclopiazonic acid (39),
and patulin (40). Penicillium species isolated from cheese rinds produce an extensive
range of SMs, including mycotoxins (41, 42), and have been shown to impact the growth
of neighboring bacteria (43–45), suggesting a potential of fungi to control bacterial com-
munity diversity through antibiotic production.
In the present study, we determined the ecological significance of fungal SMs in the
cheese rind model system by inactivating LaeA in Penicillium sp. strain MB. The exact
species identity of this fungus is currently unknown due to limited availability of whole-
genome sequences of isolates from this section of the Penicillium phylogeny; a previous
whole-genome sequencing analysis of this strain placed it close to Penicillium polonicum
(42). When this fungus was originally isolated from a natural-rind cheese, it was the dom-
inant filamentous fungus growing on the cheese and was preventing typical growth of
the normal fungal and bacterial communities found in natural-rind cheeses. Given its
negative impacts on the cheese rind, we predicted that this strain produced metabolites
that could alter microbial community assembly. When we deleted laeA in this strain, an
in vitro cheese rind bacterial community increased in total abundance and shifted in
composition to resemble bacterial communities grown without the fungus. Both tran-
scriptomic analysis and metabolite profiling pointed to pseurotins as putative LaeA-regu-
lated antibacterial compounds. Inactivation of pseurotin production in the WT strain
through the disruption of the gene encoding the hybrid PKS-NRPS enzyme required for

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pseurotin synthesis eliminated much of the antibacterial activity and caused a shift in
bacterial community composition that was similar to the DlaeA strain. This study demon-
strates the ecological relevance of LaeA-regulated fungal SMs, their roles in shaping the
assembly of multispecies bacterial communities, and their possible influence on the de-
velopment of human food commodities.

RESULTS
The deletion of laeA impairs several physiological traits in Penicillium sp. strain
MB. The deletion and complementation of laeA in Penicillium sp. strain MB resulted in
four strains: MB_WT, MB_DlaeA, MB_HygR (to test for effects of inserting the hygromycin
selectable marker into the genome), and MB_laeAc (complementation of the DlaeA strain)
(see Fig. S1a to d in the supplemental material). The MB_DlaeA strain had altered pigmen-
tation and reduced spore production relative to MB_WT, and complementation of the
DlaeA strain (MB_laeAc) restored the WT phenotype (Fig. 1a and b). Differences in pig-
mentation were most striking on cheese curd agar (CCA), a medium that mimics cheese
surface environments in cheese aging facilities (43, 46). No differences in pigmentation or
sporulation were observed between the MB_WT, the MB_HygR, and the MB_laeAc strains.
The deletion of laeA resulted in an accelerated germination of spores, reaching 100%
germination 13 h post-inoculation, while none of the control strains were able to achieve
complete spore germination (Fig. 1c). Many fungi produce self-inhibitors that reduce
germination rates, especially at high inoculum levels; we speculate that the deletion of
laeA may results in the diminishment of these self-inhibitors (47). The deletion of laeA
also resulted in reduced growth (as measured by colony diameter) regardless of the
media (see Fig. S2). Collectively, these growth and development data demonstrate that
LaeA regulates fungal traits that could have consequences for neighboring microbes.
Penicillium traits regulated by LaeA mediate cheese rind bacterial community
assembly and microbial interactions. To test how LaeA’s regulation of physiological
or metabolic traits alters the development of cheese rind microbiomes, we grew all
four Penicillium strains (MB_WT, MB_DlaeA, MB_HygR, and MB_laeAc) with a four-mem-
ber bacterial community that represents the dominant taxa found in typical natural
rind cheeses. We compared total bacterial community abundance (as total CFU) and
bacterial community composition (relative abundance of each community member) at 3,
10, and 21 days post-inoculation across five different treatments: (i) bacteria alone, (ii)
bacteria 1 Penicillium WT strain, (iii) bacteria 1 Penicillium DlaeA strain (MB_DlaeA), (iv)

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bacteria 1 Penicillium hygromycin resistance control strain (MB_HygR), and (v) bacteria
1 Penicillium laeA complement strain (MB_laeAc).
After 3 days of incubation, community composition was similar across all treatments
(Fig. 2a and b). However, the presence of MB_WT, MB_HygR, and MB_laeAc strains caused
a significant restructuring of the bacterial community through their strong inhibitory
effects at day 10 post-inoculation (Fig. 2c; see the Fig. 2 legend for PERMANOVA [permu-
tational multivariate analysis of variance] statistics). The bacteria alone treatments were
dominated by Actinobacteria (Brevibacterium [30%] and Brachybacterium [38%]), whereas
the MB_WT, MB_HygR, and MB_laeAc treatments were Staphylococcus-dominated (.82%
of the population) (Fig. 2c). In the presence of MB_WT, the absolute growth for all bacte-
ria was significantly reduced, with Brevibacterium and Brachybacterium having nearly a 4-
fold decrease in absolute growth, and Psychrobacter populations not reaching detectable
levels (Fig. 2d). Comparable results were observed with the Penicillium MB_HygR and
MB_laeAc strains. Strikingly, the abundance and structure of the bacterial community in
the presence of MB_DlaeA was similar to the community grown in the absence of the
fungus (a range of 57 to 68% Actinobacteria) (Fig. 2c). The shifts in community composi-
tion and dominance of Actinobacteria in the bacterium-alone and MB_DlaeA communities
persisted through day 21 (Fig. 2e and f).
Penicillium species often co-occur with yeasts in cheese rinds (34, 43), and the pres-
ence of another fungus may dampen the inhibitory effects of Penicillium sp. strain MB on
the bacterial communities. To test this, we repeated all community experiments with the
addition of the common cheese rind yeast, Debaryomyces hansenii. We observed nearly

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FIG 1 Deletion of laeA affects both morphological and physiological characteristics of Penicillium sp. MB strains (a) Colony aspect of Penicillium
sp. MB strains grown on different media for 5 days at 25°C. GMM = glucose minimal medium, YES = yeast extract medium, CYA = Czapek Yeast
(Continued on next page)

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FIG 2 Inactivation of LaeA increases bacterial diversity and abundance in cheese rind communities. The stacked barcharts show the shifts in the relative
abundances of a typical rind bacterial community in the presence of four strains of Penicillium sp. strain MB after (a) 3, (c) 10, and (e) 21 days of
incubation. Data are mean relative abundance from two independent experiments with five biological replicates each. The compositions of the Bacteria

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Alone and MB_DlaeA communities were not significantly different from one another, but were different from MB_WT, MB_HygR, MB_laeAc at 10 and 21
days (Day 3 PERMANOVA F = 0.9958, p = 0.425; Day 10 PERMANOVA F = 23.12, P , 0.0001; Day 21 PERMANOVA F = 11.14, P , 0.0001). The bar graphs
show absolute abundances of individual bacterial community members from the same experiments as in (a), (c), and (e) in the presence of Penicillium sp.
strain WT and mutants (MB_DlaeA, MB_HygR, and MB_laeAc) after (b) 3 (d), 10 and (f) 21 days of incubation. Each bar represents the mean with standard
errors and each dot represents a biological replicate. One-way analysis of variance (ANOVA) was performed for each bacterium. Dunnett’s multiple
comparison test was used and compared to the WT strain. (****) indicates P , 0.0001, (***) indicates P , 0.001, (**) indicates P , 0.01, (*) indicates P ,
0.05, and no asterisk indicates not significant (ns). For exact P-values for each treatment, see Data Set S1.

identical patterns of inhibition of bacteria by MB_WT and loss of inhibition in MB_DlaeA

communities in the presence of D. hansenii (see Fig. S3). This demonstrates that even
with a more realistic two species fungal community, laeA-regulated traits of Penicillium
sp. MB can control bacterial community diversity.
To determine the role of LaeA in mediating pairwise interactions between Penicillium
sp. MB and each of the individual bacterial species, we cocultured each bacterium sepa-
rately with the four Penicillium sp. MB strains on cheese curd agar. After 21 days, strains
that had a functional LaeA (MB_WT, HygR, and MB_laeAc) inhibited the growth of all four

FIG 1 Legend (Continued)

Autolysate Agar, PDA = potato dextrose agar, CCA = cheese curd agar. Top and bottom views of the agar plates are shown in the top and bottom
panels, respectively. (b) Spore counts for each strain over four days growth on GMM agar medium. The number of spores were assessed per
standard area that was sampled by plugging the fungal colony using the shaft-attaching end of a p5000 pipette tip. (c) Percentage of germinated
spores over 13 hours growth in GMM broth. Counting of germinated spores started 3 hours post-inoculation. There were no germinated spores
between hours 3 to 6. In the bar graphs, the error bars represent one standard error of the mean and each dot represents a biological replicate
(n = 3). One-way analysis of variance (ANOVA) was performed for each day for the sporulation data and each hour for the germination data.
Dunnett’s multiple comparison test was used and compared to the MB_WT strain. (****) indicates P , 0.0001, (***) indicates P , 0.001, (**)
indicates P , 0.01, (*) indicates P , 0.05, and no asterisk indicates not significant. For exact P-values for each treatment, see Data Set S1.

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FIG 3 Pairwise interaction assays showing inhibition of bacterial strains grown individually in the presence of each of the four strains of
Penicillium sp. strain MB. Data for (a) Staphylococcus, (b) Brevibacterium, (c) Brachybacterium, and (d) Psychrobacter are shown. Points indicate
means and error bars indicate standard errors from two independent experiments with five biological replicates each. In experiment 2, replication
5 was removed due to no growth of the bacteria in the control. Two-way analysis of variance (ANOVA) was performed for each bacterial
community. Dunnett’s multiple comparison test was used and compared to the WT strain. (****) indicates P , 0.0001, (***) indicates P , 0.001,
(**) indicates P , 0.01, (*) indicates P , 0.05, and no asterisk indicates not significant (ns). For exact P-values for each treatment, see Data Set S1.

bacteria (Fig. 3) with an inhibition hierarchy (log10% decrease alone versus MB_WT) of
Psychrobacter (100% inhibition) . Brevibacterium (73% inhibition) . Staphylococcus (24%
inhibition) . Brachybacterium (21% inhibition). These data demonstrate that Penicillium
sp. MB strongly and directly inhibits the growth of individual cheese rind bacteria, and
this inhibitory effect is mediated by LaeA.
Collectively, these community and pairwise data demonstrate that LaeA regulates some
aspect of Penicillium sp. strain MB physiology or metabolism that results in differential inhibi-

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tion of bacterial species growth in the cheese rind community. The LaeA-regulated factor(s)
have the ability to completely transform the composition of the bacterial community.
RNA sequencing reveals global alterations in the expression of specialized
metabolite genes in the DlaeA strain. To identify SM biosynthetic gene clusters
(BGCs) and other genes regulated by laeA in Penicillium sp. strain MB that might be
driving bacterial-fungal interaction outcomes, we performed gene expression profiling
using RNA sequencing (RNA-seq). RNA-seq libraries prepared from mycelium of
MB_WT and MB_DlaeA harvested 48 h post-inoculation from CCA were examined for
differential gene expression. This time point was selected because physiological differ-
ences between both strains were visually apparent (differences in pigmentation and
spore production), and significant amounts of high-quality RNA could be obtained.
Using a log2 ratio cutoff of 2 (corrected P value of ,0.05), we identified 253 genes with
decreased expression and 57 genes with increased expression in the MB_DlaeA strain
(Fig. 4a; see also Data Set S2a). No transcripts for laeA were detected in the MB_DlaeA
strain confirming the successful deletion of this gene.
Using an enrichment analysis of differentially expressed genes’ GO terms, we identi-
fied depsipeptide, emericellamide, lactone, aspartic peptidase, indole alkaloid, fumagil-
lin, epoxide, and alkaloid biosynthesis as pathways with greater than 30% of genes in a
pathway being significantly downregulated (see Data Set S2b). All these biosynthetic
pathways play major roles in SM production of fungi. The GO terms glucose, hexose,
monosaccharide, and nucleobase transporters were enriched in the upregulated genes
of the MB_DlaeA strain (see Data Set 2b).

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FIG 4 Deletion of laeA impairs production of metabolites with associated antimicrobial properties. (a) RNA-sequencing results showed a significant
downregulation of biosynthetic genes in the pseurotin putative gene cluster when laeA was deleted. (b) Organization of the putative pseurotin
biosynthetic gene cluster in Penicillium sp. strain MB and log2 ratio of expression in MB_DlaeA. Gene names in bold are those known to be involved in
pseurotin production in Aspergillus species. (c) Visualization of zones of inhibition of crude extracts collected from all four Penicillium isolates on YES
medium against Brachybacterium alimentarium evaluated by the disk diffusion method. See Fig. S4 for additional zone of inhibition data for other bacterial
species. (d) Volcano plots representing the number of analytes significantly regulated in MB_DlaeA compared to the MB_WT strain on YES medium. The
blue dots indicate pseurotin A and the other putative pseurotins identified in this analysis. (e) Comparison of the peak area corrected values for pseurotin
A and the other putative pseurotins between strains. Each bar represents the means with 1/2 one standard error, and each dot represents a biological
replicate (n = 3). One-way analysis of variance (ANOVA) was performed for each Penicillium strain. Dunnett’s multiple comparison test was used and
compared to the MB_WT strain. (**) indicates P , 0.01 and no asterisk indicates not significant (ns). (f) Inactivation of psoA led to loss of antibacterial
activity and restored bacterial community composition. The compositions of MB_DpsoA-1 and MB_DpsoA-2 bacterial communities were not significantly
different from one another but were different from Bacteria Alone and MB_WT (PERMANOVA F = 49.97, P , 0.0001). (g) Histograms showing the inhibition
of community members grown in the presence of the four strains of Penicillium sp. strain MB at 10 days post-inoculation. Data are mean relative
abundance from three independent experiments with five biological replications each. In the box plots, the bar represents the standard errors of the
means and each dot represents a biological replication. One-way analysis of variance (ANOVA) was performed for each bacterial community. Dunnett’s
multiple comparison test was used and compared to the WT strain. (****) indicates P , 0.0001, (***) indicates P , 0.001, (**) indicates P , 0.01, (*)
indicates P , 0.05, and no asterisk indicates not significant (ns). For exact P-values for each treatment, see Data Set S1.

We used the list of downregulated genes and antiSMASH predictions to identify specific
BGCs that might be related to the observed inhibition of bacterial growth. BGCs were iden-
tified by locating groups of adjacent downregulated genes with annotations for hallmarks
of biosynthetic gene clusters, including polyketide synthases, nonribosomal peptide syn-
thetases, terpene cyclases, and prenyltransferases (48). The most downregulated BGC in
the MB_DlaeA strain contained genes homologous to the A. fumigatus fumagillin/pseurotin
supercluster (49) (Fig. 4a). Pseurotins are a family of fungal alkaloids that have been

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reported as having antibacterial and insecticidal activities (50). A detailed analysis of the
pseurotin BGC describing its genetic organization was reported in A. fumigatus (49), but
pseurotin biosynthetic pathways and modes of antibacterial action are not fully known
due to the presence of many intermediate compounds (51). The Penicillium sp. MB ge-
nome contains a 16-gene cluster with a complete set of predicted genes for pseurotin bio-
synthesis, including psoA, psoB, psoC, psoD, psoE, psoF, and psoG (Fig. 4b). Some genes
essential for fumagillin biosynthesis, including the terpene cyclase gene (fmaA) are missing,
suggesting the strain could synthesize pseurotin but not fumagillin (Fig. 4b). All these
genes were highly downregulated (24.7 to 27 log2-fold) and were among the most
downregulated genes in the MB_DlaeA strain (Fig. 4a). Based on antiSMASH predictions
(see Data Set S2c), the Penicillium sp. strain MB genome contains 42 regions that could
encode putative specialized metabolites. Some of these other BGCs that were downregu-
lated in the MB_DlaeA strain included a putative aspterric acid and quinolone BGC not
known to have antibacterial activity, as well as other BGCs encoding putative metabolites
(see Data Set S2a). None of these BGCs were downregulated to the same extent as the
pseurotin BGC.
Loss of laeA alters the production of all members of the 1-oxa-7-aza-spiro[4,4]
non-2-ene-4,6-dione class of antibacterial natural products. Our findings high-
lighted a significant role for laeA in the assembly of microbial communities that may
be partly due to a fitness cost associated with physiological traits of this mutant
(Fig. 1). The downregulation of several BGCs in the MB_DlaeA strain suggested that dif-
ferences in community structure between treatments could be due to antibacterial
activities of laeA-regulated metabolites. To explore this hypothesis, we assessed
metabolomic changes resulting from laeA deletion after 14 days of growth on four dif-
ferent media (CYA, PDA, YES, and CCA). Crude extracts collected from all four
Penicillium strains were later screened for antibacterial activity using the disk diffusion
method against the same bacterial species used in the community and pairwise inter-
action assays.
Crude extracts collected from MB_WT growing on YES medium showed the highest
antibacterial effects, with a clear circular zone of inhibition on all tested bacteria (Fig. 3c;
see also Fig. S4a). Interestingly, crude extracts from MB_DlaeA cultures generated signifi-
cantly smaller zones of inhibition than extracts from control strains regardless of the bac-
terial strain tested (see Fig. S4a). Crude extracts collected from cultures of the MB_DlaeA
and the control strains did not show significant differences in their ability to inhibit bac-

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terial growth, except against Psychrobacter (see Fig. S4b to d). Extracts collected from
CCA cultures showed the lowest inhibitory effects on all tested bacteria, likely due to the
high amount of fat in this medium resulting in low recovery of metabolites from the or-
ganic phase (see Fig. S4d). Similar problems with metabolite recovery have previously
been attributed to the high fat content in milk (52). Therefore, we focused on exploring
the metabolomic changes on YES medium to pinpoint the laeA-regulated metabolite(s)
responsible for the shift in bacterial community composition.
Analysis of metabolomic data on YES medium showed that the deletion of laeA led to
a 2-fold decrease in the production of 62% of the 2,703 significantly differentially produced
analytes and 2-fold increase in the production of 38% of all significantly differentially pro-
duced analytes in negative ionization mode (Fig. 4d). Pseurotins have a 1-oxa-7-aza-spiro
[4,4]non-2-ene-4,6-dione core and multiple forms exist (A through F) with slight modifica-
tions to this core (53). Using a list of chemical formulas assigned to known metabolites
produced by this Penicillium species and closely related species, we were able to identify
peaks that correspond to putative pseurotins. Strikingly, the antibacterial metabolite pseur-
otin A showed a 140-fold decrease in the DlaeA mutant compared to the WT strain
(P = 0.025) (Fig. 4e). The identification of pseurotin A (C22H25NO8) was confirmed by high-
resolution mass spectrometry (HRMS) [M-H]– (m/z 430.1503, calculated for C22H24NO8
430.1507) and by HRMS/MS fragmentation. The mass spectrum showed the two fragmen-
tation ions (m/z 270.0768 and 308.1137) consistent with the mass spectrum ion previously
reported for pseurotin A (49). We could not perform experimental confirmation of other
putative pseurotins due to the lack of commercially available standards or fragmentation

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databases. The published chemical formulas of pseurotins B, C, D, and E (54) were applied
to MAVEN, which identified a significant reduction of production of these putative metab-
olites in the absence of laeA (Fig. 4e).
The significant decrease in the synthesis of pseurotin A and the other pathway deriv-
atives on YES medium matched the transcriptomic data showing a downregulation of
the pseurotin gene cluster in the MB_DlaeA strain (Fig. 4a). Combined with previous
reports of antibacterial properties of pseurotin (50), our concurring datasets suggested
that pseurotins could explain the striking antibacterial activity of this Penicillium isolate.
Inactivation of pseurotin production leads to a loss of antibacterial activity and
restores bacterial community composition. To test whether pseurotins were the fun-
gal metabolites regulating bacterial community structure, we disrupted the hybrid
PKS-NRPS synthase gene psoA required for pseurotin production in Penicillium sp.
strain MB using a CRISPR-Cas9 system (see Fig. S1e and f). The disruption of this gene
in two independent mutants (MB_DpsoA-1 and MB_DpsoA-2) and the absence of Cas9
off-target cleavage events were confirmed using whole-genome resequencing (see
Fig. S1g). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of
MB_DpsoA-1 and MB_DpsoA-2 confirmed a lack of pseurotin A production (see Fig. S5).
In contrast, MB_WT produced on average 1.36 mg of pseurotin A (see Data Set S1).
When we repeated the bacterial community assembly assays described above with
the DpsoA mutants, we saw two striking patterns (Fig. 4f and g). First, the two mutants
did not reduce total bacterial growth as much as the MB_WT strain; while MB_WT had a
34.7% reduction in total CFU across all four bacterial species, the average total bacterial
reduction across the two pseurotin mutants was only 13.9% (Fig. 4g). As we observed ear-
lier (Fig. 2), Brevibacterium, Brachybacterium, and Psychrobacter were most inhibited by
the MB_WT strain. Similar to coculture with MB_DlaeA, the DpsoA mutants had minor in-
hibitory impacts on these bacterial genera (Fig. 4g). Second, we observed that bacterial
community composition was significantly different between the MB_WT and the
MB_DpsoA-1 and MB_DpsoA-2 mutant communities. As with the DlaeA knockout above,
the bacterial community composition shifted from dominance of Staphylococcus in the
MB_WT community to a mix of all bacterial species in the MB_DpsoA-1 and MB_DpsoA-2
communities (Fig. 4f). These data combined with our transcriptomic and metabolomic
data above demonstrate that the pseurotin BGC is regulated by LaeA in this Penicillium
species and that the loss of pseurotin production eliminates much of the strong inhibi-
tory effect of this fungus on bacterial community assembly.

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Components of the pseurotin biosynthetic gene cluster are widely distributed
across the Ascomycota. Considering the strong ecological impacts of the pseurotin BGC,
we used comparative genomics to identify other fungi whose genomes encode this cluster
or closely related clusters with similar antimicrobial properties. Previous studies have dem-
onstrated that the pseurotin/fumagillin supercluster was subject to rearrangements and
gene loss in A. clavatus, Neosartorya fischeri, and Metarhizium anisopliae (49). Scanning
across a broad sample of fungal genomes, we found that pseurotin genes are only present
in the Ascomycota (Fig. 5; see also Fig. S6). Only a small subset of fungi are predicted to
have all eight pseurotin genes, including species in the genera Aspergillus, Penicillium,
Metarhizium, Tolypocladium, and Venustampulla (see Data Set S3). Of the 77 species of
Aspergillus in this data set, only 11 have all 8 genes. Of the 24 species of Penicillium, only 2
(in addition to Penicillium sp. strain MB) have all 8 genes (Fig. 5a). Another closely related
Penicillium strain that we isolated from a separate cheese production facility in the United
States (Penicillium sp. strain 261) also has the full pseurotin BGC (Fig. 5b).
Given the taxonomic breadth of fungi containing the pseurotin cluster, we sought to
understand the evolutionary origins of this cluster within and outside the Ascomycota,
including whether horizontal or vertical transmission explains patterns of pseurotin gene
distribution. The overall history of this BGC appears consistent with vertical transmission
and gene loss in some lineages; the topology of a phylogeny constructed from 290
benchmarking single copy orthologs (BUSCOs) was compatible with another phylogeny
constructed using PsoA, PsoB, and PsoC sequences (Fig. 5b to d). We cannot entirely rule
out the possibility of horizontal gene transfer, but such transfers would have to be

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FIG 5 Components of the pseurotin biosynthetic gene cluster are widespread across Ascomycota, but the full cluster is mainly found in Aspergillus and
Penicillium species. (a) The subclade of Ascomycota identified as having higher prevalence of pseurotin cluster genes (Fig. S6) presented with a single leaf
per species. Phylogenetic relationships were determined from the consensus of 290 maximum-likelihood trees constructed for benchmarking single copy
orthologs (BUSCOs). The outer ring indicates the taxonomic order that species pertain to. Terminal branch lengths are not calculated. The number of
pseurotin genes was determined using reciprocal best-hit BLAST analysis using the Aspergillus fumigatus pseurotin genes psoA, psoB, psoC, psoD, psoE, psoF,
psoG, and fapR is indicated as the intensity of the tip color. (b) A phylogeny constructed in the same way as (a) representing a subset of species identified
as having seven or eight pseurotin genes that were manually selected to represent taxonomic breadth. (c) An alignment of pseurotin gene clusters. Genes
in the pseurotin cluster are labeled in blue while constituents of the intertwined fumagillin cluster are indicated in green. Regulatory genes of these
clusters are labeled in orange. Coloration of genes is based on homology searches and visualizations performed by clinker. The weight of lines between
homologous genes indicates the percent identity; genes sharing identity below 30% are not indicated. (d) A maximum likelihood phylogeny constructed
from the concatenation of PsoA, PsoB, and PsoC sequences. Note that phylogenetic relationships between (d) and (b) are compatible, suggesting vertical
transmission of the pseurotin gene cluster. The strain information of the species used in this analysis are provided in Data Set S3.

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ancient to allow for BGC and species histories to align. Patterns of vertical transmission
are also apparent in the synteny of the BGC. For example, an inversion of several genes
at one end of the cluster (fmaD-fapI) that is present in most Penicillium spp. but not in
Aspergillus spp. is missing in the early-divergent Penicillium arizonense (Fig. 5c), suggest-
ing that this mutation occurred sometime after the diversification of this genus.
To explore the possibility of an ancient horizontal gene transfer event from outside
the fungi, we BLASTed the PsoA protein against all NCBI databases, excluding fungi.
The highest-scoring hit from this search (94% of the query cover, 32.38% identity) was
an uncharacterized hypothetical protein that is annotated as PKS in the plant Carpinus
fangiana (accession KAB8336832.1). However, the reciprocal BLAST of this protein
against fungi found better hits (100% coverage and ;60% identity) against other
uncharacterized PKSes and putative lovastatin nonaketide synthases. While our results
demonstrate that the pseurotin BGC is widespread in a subclade of Ascomycota, fur-
ther analyses exploring the evolution of this gene cluster are needed.

DISCUSSION
The chemistry, genetics, and physiology of fungal specialized metabolism are widely
studied and recognized, but their ecological consequences in microbiomes are largely
unknown. Previous genetic and -omics approaches identified key metabolites and path-
ways regulated by LaeA (28, 29, 55–57), but these data have not been integrated into a
comprehensive model for studying microbial interactions that influence community as-
sembly. By toggling LaeA between on and off, we were able to identify one class of
metabolites produced by a strongly inhibitory fungus that allows it to dramatically
remodel the bacterial communities of cheese. Our integration of physiological assays, tran-
scriptomics, and metabolomics more broadly demonstrates how LaeA can be used to
identify fungal metabolites that control the assembly of microbiomes.
The intertwined pseurotin and fumagillin supercluster was initially identified in
Aspergillus fumigatus and reported to be under the control of LaeA (49). Our study
shows that this BGC exists in the cheese-isolated Penicillium sp. strain MB and is also pos-
itively regulated by LaeA (Fig. 4a and b). This metabolite was previously found to exhibit
a range of bioactivities at moderate to high concentrations (up to 50 m g/mL) with poten-
tial therapeutic applications due to its immunosuppressive (58), antibacterial (50, 59),
nematocidal (60), insecticidal (61), antiparasitic, and anticancer activities (62). Previous

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studies of antibacterial properties have focused on purified versions of the compound
and have not used natural production from a host for assays. In our analysis, the produc-
tion of pseurotin A by the MB_WT strain was roughly 2.7 times higher than the concen-
tration used in the aforementioned studies (see Data Set S1) and may explain why this
fungus has such strong inhibitory effects on bacterial communities. Characterization of
how other fungi that produce pseurotins impact bacterial community assembly will fur-
ther clarify how this class of metabolites plays roles in multispecies microbiomes. In addi-
tion, why pseurotins have antibacterial activity is not currently known, and additional
work characterizing the mode of action is needed.
In addition to its regulation and role in the cheese Penicillium isolate, we have found
that components of the pseurotin BGC can be found intermittently in the Ascomycota, but
not in any other fungal phyla. Pseurotin A has been reported in several different filamen-
tous fungi, including species of Aspergillus, Penicillium, and more recently the distantly
related taxon Metarhizium (51, 61, 63, 64). Our finding of all eight genes of this BGC in sev-
eral other genera (Tolypocladium and Venustampulla) suggests that fungal pseurotins may
be widely distributed across a range of fungal niches, from food production and indoor
environments to forest soils. Additional studies of these fungi and neighboring bacteria are
needed to fully characterize the ecological significance of pseurotins across ecosystems.
Our data strongly suggest that pseurotins are key LaeA-regulated metabolites that
can mediate bacterial community assembly, but we acknowledge that other fungal
metabolites and traits could be playing roles in this system. Other BGCs, including
some putative clusters with unknown functions, were downregulated in the

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transcriptome of the MB_DlaeA strain (see Data Set S2), suggesting that they are regu-
lated by LaeA. These may also contribute to the antibacterial effects of Penicillium sp.
strain MB. Future work using untargeted metabolomics and other approaches may
identify these additional LaeA-regulated metabolites in this and other filamentous
fungi that affect bacterial community assembly.
Every time a consumer eats a piece of a naturally aged cheese, they ingest the many
metabolites secreted into the cheese by rind microbes. Surprisingly little is known about
the diversity and functions of these microbial metabolites (41, 65–68). Our work demon-
strates that fungal SMs produced in cheese environments can serve as mediators of
microbiome formation at concentrations relevant to naturally aged cheeses. The interac-
tions described here occurred in a highly controlled laboratory environment and their
relevance in full-scale cheese production remains to be determined. The Penicillium sp.
strain MB used in this study was causing cheese production problems by inhibiting nor-
mal rind formation, suggesting that the disrupted community assembly observed in the
lab could also happen in cheese caves. Many other filamentous fungi found in cheese
rinds possess uncharacterized metabolites that might be responsible for similar negative
outcomes. Systematic exploration of LaeA-regulated metabolites and their ecological
roles will not only discover microbial mechanisms underlying traditional cheese-making
but will also illuminate how fungal SMs mediate microbiome composition in all environ-
mental niches where fungi reside.

MATERIALS AND METHODS

Microbial strain isolation and culture conditions. The Penicillium sp. strain MB used in this study
was isolated from a natural rind cheese made in the United States. Its genome has been deposited in
NCBI with accession number GCA_008931935.1. The exact species identification of this strain is
unknown at this time because genomes of type strains of closely related species are not currently avail-
able. However, previous comparative genomic analysis suggests it is near P. polonicum in section
Fasiculata of the genus Penicillium (69). The beta-tubulin marker gene (benA) of Penicillium sp. strain MB
has a high similarity with two Penicillium species: 98.6% pairwise identity with a reference strain of P.
cyclopium (strain CBS 14445) and 96.1% pairwise identity with a reference strain of P. polonicum (strain
CBS 22228). Preliminary observations demonstrated that this Penicillium strain had potent antibacterial
activity and was therefore an interesting strain to explore microbial interactions and secondary metabo-
lite regulation through the inactivation of the global regulator of fungal secondary metabolism, LaeA.
To prepare a fresh spore suspension prior to experiments, the Penicillium sp. strain MB was activated
on glucose minimal media (GMM) (70) for 7 days at 25°C, and spores were harvested in 0.01% Tween 80
and counted using a hemocytometer. Bacterial strains were also maintained as glycerol stocks and

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streaked on brain heart infusion agar prior to experiments. The CYA, YES, PDA, and GMM agars were pre-
pared as previously described (70), and the CCA medium was also prepared as previously described (43).
Four bacterial strains (Staphylococcus equorum strain BC9, Brevibacterium aurantiacum strain JB5,
Brachybacterium alimentarium strain JB7, and Psychrobacter sp. strain JB193) were used in bacterial commu-
nity experiments. These species span three bacterial phyla (Firmicutes, Actinobacteria, and Proteobacteria) (43)
that are most abundant in cheese rinds. They have been used as a model community in previous work from
our lab (44, 45, 71) and have been demonstrated to have various responses to the presence of Penicillium (43,
45). This model community also has a well-defined community succession with Staphylococcus and
Psychrobacter dominating early in succession (days 0 to 10) and the Actinobacteria Brevibacterium and
Brachybacterium dominating later in succession (days 10 to 21). We did not use bacteria isolated from the
same cheese where the Penicillium sp. strain MB was originally isolated because we were interested in identi-
fying the basic ecological impacts of LaeA on bacterial communities, not the specific way in which this mold
was interacting with bacteria on the cheese surface. By using previously well-characterized responding bacte-
ria, we can compare our community assembly assays and interactions in this work to many previous experi-
ments. We acknowledge that the bacteria that co-occurred with the Penicillium sp. strain MB may have had a
different response compared to our model bacterial community.
Construction of gene deletion cassettes. To knockout laeA in the Penicillium sp. strain MB, the isolate
was first subjected to antimicrobial susceptibility testing toward hygromycin and phleomycin, two antibiot-
ics commonly used by our group as selectable markers in fungal transformations. The isolate showed a con-
firmed sensitivity to both antibiotics. A three round PCR deletion strategy was used to replace the laeA
open reading frame (ORF) in the Penicillium sp. strain MB with the hph gene, whose expression confers
selection on hygromycin. The schematic representation of the laeA gene replacement with the hph gene is
depicted in Fig. S1a. Each deletion cassette (59flank-hph-39flank) was constructed using three sequential
PCRs. In the first PCR round, about 1 kb of genomic sequence that flanks either the 59 or the 39 end of the
laeA ORF was amplified from strain MB using, respectively, the primer set PMB_KOlaeA_59 or 39F/R. The hph
gene was amplified from plasmid pUCH2-8 using primers hph_F and hph_R. A second PCR was performed
to assemble by homologous recombination the three individual fragments from the first-round PCR. The
deletion cassettes were finally amplified using nested primer sets (PMB_KOlaeA_NestedF/R).

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To test whether pseurotin was involved in the antimicrobial activity and shifts in bacterial commu-
nity composition observed with this Penicillium strain, we knocked out the psoA gene that encodes for
the hybrid polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS) of the putative pseurotin
biosynthetic gene cluster to disrupt the production and accumulation of all pseurotin. The same strategy
described earlier was adopted for the construction of the psoA deletion cassette. The primer sets
PMB_KOpsoA_59/R and 39F/R were used to amplify the 1-kb homology arms flanking the psoA gene on
the 59 and 39 ends, respectively. The hph gene under the control of the Tef1 promoter and terminator
was amplified from the plasmid pCS01 using the primer set Hyg-tef1F/R. The plasmid map is given and
annotated in Fig. S1e. A second PCR was performed to assemble by homologous recombination the
three individual fragments from the first round PCR. The deletion cassette was finally amplified using
the nested primer set (PMB_psoA_nestedF/R). The sequences of the primer sets used for the construc-
tion of the deletion cassettes are shown in Table S1.
PEG-mediated protoplast transformation. To generate the deletion strains, a protoplast-mediated
transformation protocol routinely used by our group was employed and optimized to achieve a success-
ful protoplasting of Penicillium sp. strain MB. Briefly, 109 fresh spores from each strain are cultured in
500 mL of GMM broth supplemented with 1 g/L yeast extract for 12 h under 25°C and 280 rpm. Newly
born hyphae were harvested by centrifugation at 8,000 rpm for 15 min and hydrolyzed in a mixture of
30 mg of lysing enzyme from Trichoderma harzianum (Sigma-Aldrich) and 20 mg of Yatalase (Fisher
Scientific) in 10 mL of osmotic medium (1.2 M MgSO4 and 10 mM sodium phosphate buffer). The quality
of protoplast was monitored under the microscope after 4 h of shaking at 28°C and 80 rpm. The proto-
plast mixture was later overlaid gently with 10 mL of chilled trapping buffer (0.6 M sorbitol and 0.1 M
Tris-HCl [pH 7.0]) and centrifuged for 15 min under 4°C and 5,000 rpm. Protoplasts were collected from
the interface, overlaid with an equal volume of chilled STC (1 M sorbitol, 10 mM Tris-HCl, and 10 mM
CaCl2) and decanted by centrifugation at 6,000 rpm for 10 min. The protoplast pellet was resuspended
in 500 m L of STC and used for transformation. For protoplast transfection, 100 m L of freshly isolated pro-
toplasts and 5 m g of linear DNA containing the deletion cassette were mixed to a final volume of 200 m L
of STC buffer. The contents were mixed by gently inverting the tubes. After 50 min incubation on ice,
1.2 mL of 60% (wt/vol) PEG solution (60 g of PEG 3350, 50 mM CaCl2, and 50 mM Tris-HCl [pH 7.5]) was
added to the mixture, followed by incubation for an additional 20 min at room temperature. The mixture
was supplemented with 5 mL of STC and mixed into 50 mL of SMM top agar (GMM supplemented with
1.2 M sorbitol) containing hygromycin at a final concentration of 150 m g/mL. The mixture was inverted
several times, and each 5-mL portion was poured onto a selective SMM bottom agar plate. The transfor-
mation plates were incubated at 25°C for 5 to 7 days.
(i) CRISPR/Cas9-mediated knockout of psoA gene. The large size of the psoA gene (;12 kb) made
the standard transformation method described for laeA deletion ineffective. Therefore, we switched to a ri-
bonucleoprotein-CRISPR-Cas9 (RNP-CRISPR-Cas9) system. The schematic representation of the CRISPR cas9
system used for engineering the psoA knockout strain is depicted in Fig. S1f. The fungal transformation
steps followed were the same as described above, the only difference being the codelivery of the Cas9-
gRNA RNP complex along with the linear donor DNA (psoA deletion cassette) to the protoplasts. The
CRISPR RNA (crRNA) was designed on the PKS-NRPS conserved domain in the psoA sequence using the
CRISPOR webtool (http://crispor.tefor.net/) that offers off-target and efficiency predictions. The selected
crRNA (59-GGAUCGAUCUUGAACAGCAG-39) showed a predicted targeting efficiency of 100% and 0 pre-

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dicted off targets. This crRNA was subjected to an additional confirmation using the RNAfold web server
(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) that allows to determine the gRNA secondary
structure. The interpretation of the results and validation of the gRNA were done following the guidance
by Hassan et al. (72). The crRNA and tracrRNA (IDT, catalog no. 1072534) were synthesized using the Alt-R
CRISPR-Cas9 system from Integrated DNA Technologies (IDT; San Diego, CA). The crRNA and tracrRNA
were mixed at a concentration of 200 nM each in IDT duplex buffer in a final volume of 20 m L. The gRNA
complex was formed by incubation at 95°C for 5 min, followed by slowly cooling down for 20 min at room
temperature, and then stored at 220°C. To form the RNP complex, 8.3 m L of HiFi Cas9 Nuclease (IDT, cata-
log no. 1081060), and 6 m L of the previously made gRNA was mixed in nuclease-free water to a 25-m L final
volume. The mixture was incubated for 15 min at room temperature prior to use.
(ii) Confirmation of gene deletion strains. After 5 to 7 days of incubation at 25°C, colonies grown
on SMM plates supplemented with hygromycin (150 m g/mL) were subjected to a second round of selec-
tion on hygromycin. Single-spored transformants were later tested for proper homologous recombina-
tion at the ORF locus by PCR.
To confirm deletion of laeA, 25 hygromycin-resistant transformants were isolated after a rapid selection
procedure on SMM supplemented with hygromycin. The correct replacement of the laeA with the hph
gene was first verified by PCR analysis of genomic DNA derived from the transformant strains using pri-
mers that amplify the laeA ORF. One ORF-specific confirmation primer set (PMB_laeA_F/R) was chosen for
the strain. About 40% (10/25) of the monoconidial lines generated from primary transformants of strain
MB were PCR-positive for the absence of the laeA ORF (data not shown). The positive deletion strains iden-
tified by PCR were further checked for a single insertion of the deletion cassette by Southern blot analysis.
Probes corresponding to the 59 and 39 flanks of the laeA gene in each strain were labeled using [a-32P]
dCTP (Perkin-Elmer, USA) according to the manufacturer’s instructions. A single-site integration of the dele-
tion cassette was revealed in a single transformant of the strain MB (see Fig. S1b). A HygR control strain,
which has the hygromycin cassette inserted into the genome but not at the target locus, was also included
in the study as a control for absence of selectable marker gene effects. To confirm deletion of psoA in
Penicillium strain MB_WT using Cas9/sgRNA RNP complex, four hygromycin-resistant transformants were
isolated after a rapid selection procedure on SMM supplemented with hygromycin. Two out of four of the

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monoconidial lines generated were PCR positive for the absence of the psoA ORF using the primer set
PMB_psoA_F/R (data not shown). Later, identification of exact gene deletion locations and assessment of
off-target effects of the cas9 were analyzed by whole-genome resequencing of the two PCR- positive
knockout strains. DNA was extracted from each DpsoA strain using a Qiagen DNeasy PowerSoil extraction
kit. DNA was sent to the Microbial Genome Sequencing Center for library preparation and sequencing on
an Illumina NextSeq 2000. To determine the specific location of CRISPR deletion, reads were mapped to
the Penicillium sp. MB_WT genome using Bowtie. To assess whether any off-target mutations were caused
by the CRISPR deletion of psoA, FreeBayes was used to identify variants in the mappings of both DpsoA
strains. Overall, only two high-confidence single nucleotide polymorphisms (SNPs) were detected in both
CRISPR knockout strains that could have resulted from off-target effects: one SNP (G!A with predicted
amino acid substitution of D!N) in a gene annotated as “chromatin structure-remodeling complex subu-
nit rsc1” and another SNP (DNA G!A with predicted amino acid substitution of P!L) in a predicted pro-
tein with an unknown function (see Fig. S1g).
Construction and confirmation of complement strains. To confirm that the phenotype exhibited
by the MB DlaeA strain is caused by the deletion of this specific gene, the MB DlaeA strain was comple-
mented with a wild-type (WT) copy of the laeA gene using phleomycin as a selectable marker.
Restriction sites for NotI were introduced at the predicted native promoter and terminator of the laeA
gene using primers PMB_laeAcomp_F and PMB_laeAcomp_R. The laeA gene was cloned into the pBC-
phleo plasmid at the multiple cloning site located within the lacZ gene (see Fig. S1c). The ligation of the
digested insert into the recipient plasmid was performed using T4 DNA ligase (New England Biolabs) fol-
lowing the manufacturer’s instructions. The ligation reaction was later transformed into Escherichia coli
DH5a competent cells according to the manufacturer’s directions (Thermo Fisher Scientific). Five white
bacterial colonies were randomly selected from the blue-white screening lysogeny broth (LB) agar plate
and screened for successful ligations by conducting a diagnostic restriction digest with the NotI restric-
tion enzyme. The E. coli strain carrying the correct plasmid (labeled PJT3) was then grown in 50 mL of LB
supplemented with chloramphenicol (35 m g/mL), and the plasmid DNA was isolated using a Quantum
Prep-Plasmid Midiprep kit (Bio-Rad) according to the manufacturer’s instructions. Next, 10 m g of plasmid
DNA was used for the transformation of the MB_DlaeA strain following the same protocol described
above. Prior to transformation, the plasmid was linearized using the NotI restriction enzyme.
Southern blotting was performed to confirm the single integration of laeA into the MB deletion
strain using the laeA ORF sequence (amplified using the primer set laeA_ORF_F/R) for making the probe.
Genomic DNAs from both laeA complement and deletion strains were digested with the same enzyme
used for cloning. A positive control corresponding to the PJT3 plasmid was incorporated in the
Southern blot analysis, and the MB_DlaeA strain was used as a negative control. 10 phleomycin-resistant
transformants were isolated and subjected to Southern blot to confirm the single insertion of the laeA
ORF. One strain out of 10 showed a single band of 2.8 Kb that matches the band obtained with the posi-
tive control (plasmid PJT3 generated after subcloning the laeA fragment into plasmid pBC-phleo). As
expected, the MB_DlaeA mutant strain used as a negative control did not show any band (see Fig. S1d).
Morphophysiological analysis. The impact of laeA deletion on the morphophysiological traits of the
cheese Penicillium sp. strain MB was evaluated by monitoring the growth, sporulation, and germination of
the MB_WT strain in comparison to the MB_DlaeA, MB_HygR, and MB_laeAc control strains. The pheno-
typic appearance and vegetative growth were evaluated on five different media: GMM, CYA, YES, and CCA.

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Spore production and germination were assessed on GMM agar and broth, respectively. For conidial
counts, fresh spores from each strain were diluted to 105 spores/mL in GMM top agar and overlaid onto
agar plates of the same medium. The plates were incubated at 25°C and agar plugs removed on the sec-
ond, third, and fourth day post-inoculation were homogenized in 3 mL of 0.01% Tween 80 using the
VWR 200 homogenizer. Total spore counts were made using a hemocytometer. Conidial germination
rates were evaluated over a 24-h growth period using a Nikon Ti inverted microscope. A spore suspen-
sion of 105 spores/mL of GMM broth was prepared for each strain, and about 1 mL was distributed into
three replicate wells of a 24-sterile well plate. Five pictures per well were taken an hour apart beginning
4 h postincubation. The number of germlings were counted for each strain and the percentage of germi-
nated spores was plotted against time to estimate the germination rates.
Community and pairwise interaction assays. To determine how the deletion of laeA impacted mi-
crobial community assembly, we reconstructed cheese rind bacterial communities on cheese curd agar
(CCA) with each of the Penicillium strains and measured total bacterial community abundance (as total
CFU) and bacterial community composition (relative abundance of each community member) at 3, 10, and
21 days of community assembly. Each member of a four-member bacterial community (Staphylococcus
equorum BC9, Brevibacterium auranticum JB5, Brachybacterium alimentarium JB7, and Psychrobacter sp.
strain JB193) was initially inoculated at 200 CFU per species in five treatments: (i) bacteria alone, (ii) bacte-
ria 1 Penicillium WT strain, (iii) bacteria 1 Penicillium DlaeA strain (MB_DlaeA), (iv) bacteria 1 Penicillium
hygromycin resistance control strain (MB_HygR), and (v) bacteria 1 Penicillium laeA complement strain
(MB_laeAc). Penicillium strains were also inoculated at 200 CFU from experimental glycerol stocks (46). For
each treatment, replicate communities were inoculated on the surface of 150 m L of cheese curd agar dis-
pensed into each well of a 96-well plate. To determine bacterial community composition, communities
were harvested from individual wells with a sterile toothpick, suspended in 500 m L of phosphate-buffered
saline (PBS) in a 1.5-mL microcentrifuge tube, homogenized with a sterile micropestle, and serially diluted
onto plate count agar with milk and salt (PCAMS) media (46). To selectively plate bacteria, 100 mg/L of cy-
cloheximide was added to PCAMS. To quantify Penicillium abundance, 50 mg/L of chloramphenicol was
added to PCAMS. Each of the four bacteria have very distinct colony morphologies, making it easy to
determine the abundance of each community member.

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To determine whether the presence of another fungus could modify the inhibitory effects of Penicillium
sp. strain MB, we repeated the community assays above with an isolate of the yeast Debaryomyces hansenii
(see Fig. S3). This is a very widespread yeast in cheese rinds, has neutral or sometimes positive effects on the
growth of cheese rind bacteria, and often co-occurs with Penicillium species (39). These experiments with the
additional yeast were repeated as described above except that 200 CFU of Debaryomyces hansenii strain
135B were added to all treatments. Bacterial and fungal abundances were quantified as described above.
Pairwise interactions between each individual bacterium and the four Penicillium strains (MB_WT,
MB_DlaeA, MB_HygR, and MB_laeAc) were assessed using the same experimental setup as the community
experiments. Each bacterium was inoculated on the surface of a well of a 96-well plate with PCAMS either
alone or with 200 CFU of each of the four Penicillium strains. Bacterial abundance was determined at 3, 10,
and 21 days by plating harvested cocultures on PCAMS supplement with 100 mg/L of cycloheximide.
To determine the role of pseurotin in shaping cheese microbial communities, these community assays
were conducted with the DpsoA-1 and DpsoA-2 strains. The experimental setup and data collection and analysis
were identical to the experiments with the MB_WT, MB_DlaeA, MB_HygR, and MB_laeAc strains noted above.
RNA sequencing analysis and antiSMASH BGC prediction. Transcriptome changes in Penicillium
sp. strains MB_WT and MB_DlaeA were investigated using RNA-sequencing analysis of cultures growing
on CCA medium. Inoculum of both strains were prepared from 1-week cultures on PCAMS medium. A
1-cm2 plug was taken from the leading edge of mycelium and homogenized in 500 m L of PBS. A 20 m L
inoculum was spotted onto a CCA plate at three evenly spaced positions. After 48 h of growth in the
dark at 24°C, the spots were about 1.5 cm in width. The MB_WT had produced blue colored spores
whereas the MB_DlaeA spores were lighter in color. The entire fungal growth from each spot was cutoff
from the CCA plates, placed in RNAlater (Qiagen), and stored at 280°C. Four biological replicates were
sampled for each strain.
RNA was extracted from one of the three spots from each replicate plate using the Qiagen RNeasy
Plant minikit after grinding the sample in liquid nitrogen. Approximately 100 mg of ground fungal biomass
was mixed in 750 m L of Buffer RLT supplemented with 10 m L of b -mercaptoethanol. The manufacturer’s
recommended protocol was followed for RNA extraction, including an on-column DNase treatment. To iso-
late mRNA, the NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs) was used. This
mRNA was used to generate RNA-seq libraries using the NEBNext Ultra II RNA Library Prep kit for Illumina
according to the manufacturer’s recommended protocol. The RNA-seq libraries were sequenced using
125-bp paired-end Illumina sequencing on a HiSeq at the Harvard Bauer Core.
Duplicate reads were removed, and the total number of reads was subsampled to 3.8 million forward
reads that were used for read mapping and differential expression analysis. Reads were mapped to a draft
genome of Penicillium sp. strain MB. Read mapping was performed with TopHat v2.1.0 (73). Differentially
expressed genes were identified using DeSeq2 (74). Genes with a .5-fold change in expression and false
discovery rate (FDR)-corrected P values of ,0.05 were considered differentially expressed. To identify spe-
cific biological pathways that were enriched in the sets of downregulated or upregulated genes, we used
a KOBAS 2.0 (75) to conduct a hypergeometric test on functional assignments from the Gene Ontology
(GO) database (using the Aspergillus flavus genome as a reference for GO ID assignment) with Benjamini-
Hochberg FDR correction. To identify putative BGCs in the Penicillium sp. strain MB genome beyond the
pseurotin gene cluster, we used the fungal version of antiSMASH v 6.1.1 (76).
Metabolite profiling by UHPLC-MS analysis. To determine the effect of laeA deletion on the bio-

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synthetic metabolome of the Penicillium sp. strain MB, all four strains (MB_WT, MB_DlaeA, MB_HygR, and
MB_laeAc) were cultivated by centrally inoculating 106 fresh spores on 60-mm petri dishes containing
10 mL of the agar media PDA, CYA, YES, and CCA. Three technical replicates per strain and condition
were prepared. The cultures were incubated at 25°C for 2 weeks. After the incubation period, all cultures
were freeze-dried (;3 g [dry weight]) and ground into 5 mL of sterile water. Soluble metabolites were
later extracted by solvent extraction procedure using 5 mL of ethyl acetate. An organic metabolite frac-
tion was generated by liquid-liquid partitioning and dried under vacuum. The crude extract was then
dissolved in 400 m L acetonitrile-water (80:20 [vol/vol]) at a concentration of 100 m g/m L. Samples were
later analyzed by UHPLC-MS as previously described (77). The total data set was first evaluated using the
software MAVEN and the XCMS open-source package. Differential masses found via XCMS were filtered by
having a maximum intensity greater than 4  104. Identified masses that had a maximum intensity lower
than 4  104 were considered as background. A volcano plot was later constructed to determine statistically
significant data points in crude extracts analyzed for both MB_WT and MB_DlaeA in negative ionization
mode. For volcano plot construction, metabolites were filtered based on a P value of ,0.05 and a fold
change higher than 2 and lower than 22.
To confirm the lack of pseurotin production by the two DpsoA mutants, crude extracts from 14-day cul-
tures on YES agar medium of both MB_DpsoA and MB_WT were obtained and assessed by high-resolution
parallel reaction monitoring (PRM) LC-MS/MS using a Vanquish uHPLC plumbed directly to a Q-Exactive Plus
mass spectrometer (Thermo Scientific) outfitted with a 75-m m ID nanospray emitter packed with 15-cm
Kinetex C18 resin (1.7-m m particle size; Phenomenex). Mobile phases included solvent A (95% H2O, 5% aceto-
nitrile, 0.1% formic acid) and solvent B (30% H2O, 70% acetonitrile, 0.1% formic acid). Nanoliter flow rates
were achieved by uHPLC split-flow and measured as 300 nL/min at the nanospray emitter. First, 5 m L of each
sample was autoinjected prior to the split, leading to the separation and analysis of 10 nL of extract over a
30-min chromatographic method: 0 to 100% B over 17 min; 100 to 0% B over 3 min; hold at 0% B for 10 min
to re-equilibrate the column. Pseurotin A was targeted for PRM analysis in positive-ion mode with a duty
cycle that included a selected ion monitoring scan (427 to 437 m/z range; resolution, 35,000; 2 microscan
spectrum averaging), followed by a PRM scan targeting the pseurotin A ion (m/z of 432.1653 [M1H]1; resolu-
tion 17,500; 2.0 m/z isolation window with a 0.5 m/z offset; normalized collision energy [NCE] in the HCD cell

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LaeA-Mediated Regulation of The Cheese Rind Microbiome mBio

at 35). Sample extracts were measured across three technical replicates of each strain (WT_MB, MB_DpsoA1,
and MB_DpsoA2), including 10 m M pseurotin A standard (Cayman Chemical). Standard addition experiments
were also performed using the above PRM method on samples extracted from YES grown MB_WT strain to
assess the pseurotin A concentration. All PRM data were analyzed using Skyline (78) and FreeStyle (Thermo
Scientific) software.
In vitro antimicrobial assay. To determine whether the findings observed with community and
pairwise interaction assays are due to secreted metabolite(s), the antimicrobial activities of all crude exu-
dates collected from cultures of MB_WT, MB_DlaeA, MB_HygR, and MB_laeAc strains on various media
were evaluated using the paper disk agar diffusion method. The antimicrobial properties were assessed
against the same bacterial strains used for community and pairwise interaction assays except the B. aur-
antiacum strain JB5 due to the inability of growing this strain for these in vitro experiments. Bacterial
strains were first cultured in 5 mL of LB broth under 280 rpm at room temperature for 24 to 48 h. The
optical density of the bacterial suspension was later adjusted to 1. One milliliter of the bacterial suspen-
sion was then added to 20 mL of LB top agar, and 5 mL was gently applied on agar dishes of the same
medium. In sequence, sterile disks impregnated with 10 m L of extracts (at a concentration of 100 m g/m L)
dissolved in acetonitrile-water (80:20 [vol/vol]) was placed over the bacterial culture plates. One disk
containing the solvent previously used for resuspension was used as negative control. For each bacte-
rium, one disk of ampicillin at a concentration of 100 m g/mL was applied as a positive control. All dishes
were incubated at room temperature, for 24 to 48 h. At the end of the incubation period, each dish was
examined, and inhibition halo diameters were measured.
Comparative genomic analysis of the pseurotin gene cluster. To contextualize the ecological impor-
tance of pseurotin in Penicillium spp. relative to other fungi, we used a data set of all annotated publicly avail-
able genomes originally downloaded from NCBI on 20 April 2020. This data set comprised 1,464 genomes rep-
resenting 808 species. We performed pairwise reciprocal-best hit analysis of all proteins in the Aspergillus
fumigatus genome against all 1,464 genomes using methods described previously (79). The results of this anal-
ysis were used to identify psoA, psoB, psoC, psoD, psoE, psoF, psoG, and fapR orthologs across fungal phyla.
We mapped the phylogenetic relationship of fungal genomes based on 290 benchmarking single
copy orthologs (BUSCOs) as identified with BUSCO (80). We aligned sequences of each BUSCO using
MAFFT and trimmed alignments using TrimAl (81) with the parameter -automated1. We constructed
phylogenies for each gene using IQtree (82) after testing for the best fit model. We then created a single
consensus tree using ASTRAL (83).
We selected a subset of genomes to represent taxonomic breadth based on visual inspection of phy-
logenetic relationships between species where seven or eight pseurotin genes were found. In addition,
we added a set of genomes that were not present on NCBI but were found to contain the pseurotin
gene cluster from our analysis of this cluster in the Penicillium genus (see above). Phylogenetic relation-
ships between these genomes were determined using the same methodology as described above.
Pseurotin gene clusters were determined from antiSMASH (84) by selecting cluster calls that contained
the psoA ortholog (as determined above). The resulting clusters were aligned and visualized using
Clinker (85). When BGC border calls made by antiSMASH extended beyond the pseurotin BGC, we
trimmed these calls to facilitate visualization of this gene cluster. Validation of gene calls in genome
annotations was beyond the scope of this study. To explore the evolutionary history of the pseurotin
gene cluster, we aligned PsoA, PsoB, and PsoC sequences of the selected species using MAFFT (86) using

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the same parameters described above. The resulting alignments were trimmed with TrimAl (81) and
then concatenated to form a single sequence for each species. A phylogeny was generated from this
alignment using IQtree (82). Phylogenies were visualized in R (87) using ggtree (88).
Availability of biological materials. All unique materials, including the Penicillium sp. strain MB_WT
isolated from cheese, the Penicillium sp. strain MB_laeA deletion mutant, and the bacterial strains used
in the interaction and antimicrobial assays, are readily available from the authors upon request.
Data availability. Sequence data that support the findings of this study have been deposited in the
NCBI SRA database with accession numbers PRJNA861320 for the whole-genome resequencing data
and PRJNA861316 for the RNA-seq data. The LC-MS raw data have also been deposited to MassIVE
(https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp), and data are available at ftp://massive.ucsd
.edu/MSV000090563/ (data set ID MSV000090563, password: cheese).
Source data used to create all figures are available in the supplemental material.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
DATA SET S1, XLSX file, 0.6 MB.
DATA SET S2, XLSX file, 0.2 MB.
DATA SET S3, XLSX file, 0.1 MB.
FIG S1, TIF file, 4.7 MB.
FIG S2, TIF file, 0.8 MB.
FIG S3, TIF file, 1.8 MB.
FIG S4, TIF file, 0.9 MB.
FIG S5, TIF file, 0.8 MB.
FIG S6, TIF file, 0.8 MB.
TABLE S1, DOCX file, 0.02 MB.

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LaeA-Mediated Regulation of The Cheese Rind Microbiome mBio

ACKNOWLEDGMENTS
This study was supported by a grant from the National Science Foundation (CAREER
1942063) to B.E.W., a Secure Ecosystem Engineering and Design project funded by the
Genomic Science Program of the U.S. Department of Energy, Office of Science, Office of
Biological and Environmental Research as part of the Secure Biosystems Design Science
Focus Area to P.E.A.
Ruby Ye provided feedback on the manuscript and experimental design.
Conceptualization was carried out by J.T., B.E.W., and N.P.K. Experiments were
performed by J.T., C.M.C., M.T.D., and B.E.W. The article was written by J.T., C.M.C., and
B.E.W. and revised with input from all authors. The figures and statistical analyses were
made by J.T., C.M.C., T.A.R., and B.E.W., except for Fig. 4 (M.T.D.); see also Fig. S5 (R.J.G.),
and Fig. S6 (M.T.D.). The RNA-seq experiments were conducted and analyzed by B.E.W.
The LC-MS curation of data and analysis were conducted by R.J.G., P.E.A., T.A.R., and J.T.
The study was supervised by N.P.K. and B.E.W.
This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-
00OR22725 with the U.S. Department of Energy (DOE).
We declare there are no competing interests.

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