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Editor's Pick Research Article | Applied and Environmental Science

Rapid Phenotypic and Metabolomic Domestication of Wild Penicillium Molds on Cheese

Ina Bodinaku, Jason Shaffer, Allison B. Connors, Jacob L. Steenwyk, Megan N. Biango-Daniels, Erik K. Kastman, Antonis Rokas, Albert Robbat, Benjamin E. Wolfe
John W. Taylor, Editor
Ina Bodinaku
aTufts University, Department of Biology, Medford, Massachusetts, USA
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Jason Shaffer
aTufts University, Department of Biology, Medford, Massachusetts, USA
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Allison B. Connors
bTufts University, Department of Chemistry, Medford, Massachusetts, USA
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Jacob L. Steenwyk
cVanderbilt University, Department of Biological Sciences, Nashville, Tennessee, USA
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Megan N. Biango-Daniels
aTufts University, Department of Biology, Medford, Massachusetts, USA
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Erik K. Kastman
aTufts University, Department of Biology, Medford, Massachusetts, USA
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Antonis Rokas
cVanderbilt University, Department of Biological Sciences, Nashville, Tennessee, USA
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Albert Robbat
bTufts University, Department of Chemistry, Medford, Massachusetts, USA
dTufts University Sensory and Science Center, Medford, Massachusetts, USA
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Benjamin E. Wolfe
aTufts University, Department of Biology, Medford, Massachusetts, USA
dTufts University Sensory and Science Center, Medford, Massachusetts, USA
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John W. Taylor
University of California, Berkeley
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DOI: 10.1128/mBio.02445-19
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  • FIG 1
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    FIG 1

    Penicillium molds in the cheese environment. (A) The white mold known as Penicillium camemberti (show in pure culture in the Petri dish) is used to make Camembert (shown), Brie, and other bloomy rind cheeses. (Photo by Adam DeTour and used with permission.) (B) Wild Penicillium molds (also known as non-starter molds) can contaminate cheeses during production. (C) Some natural rind cheeses are intentionally colonized by wild Penicillium molds. Shown here is Penicillium sp. strain 12, a strain used in the experiments in this paper, colonizing wheels of a blue cheese in a cave in the United States. (D) A phylogenomic tree of Penicillium. Strains used in this work are highlighted in bold. Penicillium sp. MB was also isolated from a natural rind cheese and sequenced as part of this work but was not used in the experiments described.

  • FIG 2
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    FIG 2

    Experimental evolution of Penicillium on cheese. (A) Evolution of Penicillium commune strain 162_3FA on cheese curd alone (“Penicillium alone”) and in the presence of three competing cheese rind microbes (“Penicillium + community”; Staphylococcus xylosus, Brachybacterium alimentarium, and Debaryomyces hansenii). Lines connect points representing mean domesticated phenotype frequencies of four replicate populations, and error bars represent 1 standard deviation of the mean. “Penicillium + community” had a significantly lower domesticated phenotype frequency (repeated-measures ANOVA; see text for statistics). (B) Experimental evolution of P. commune strain 162_3FA in different cheese nutrient environments. “Normal cheese” = 10% cheese curd in agar medium. “Low cheese” = 1% cheese curd in agar medium. “Alternating normal/low” = alternating 10% and 1% cheese curd at each transfer. Both “Low cheese” and “Alternating normal/low” had significantly lower domesticated phenotype frequencies (repeated-measures ANOVA with Tukey’s HSD post hoc tests; see text for statistics). Lines connect points representing mean domesticated phenotype frequencies of four replicate populations, and error bars represent 1 standard deviation of the mean. The “low cheese” line is difficult to see because it is at 0%. (C) Morphology of four representative colony types. The ancestor phenotype was dark blue-green and dusty; domesticated phenotype 1 was white and flat; domesticated phenotype 2 was white and fuzzy/dusty; domesticated phenotype 3 was blue-green but had less intense coloration than the ancestor and a less fuzzy appearance. (D) Reproductive output and cyclopiazonic acid production of a range of strains isolated across the experimental evolution populations. Points are mean values, and error bars represent 1 standard deviation of the mean. Strains M5, M6, M7, and M8 had reduced reproductive compared to the ancestor (Dunnett’s test, P < 0.05). All domesticated phenotypes had significantly reduced CPA production compared to the ancestor (Dunnett’s test, P < 0.05). (E) Competition between ancestral P. commune strain 162_3FA and domesticated strain M9. Strain M9 outcompeted the ancestor after 10 days of growth on cheese curd agar. Points represent individual replicate competition communities, and the horizontal line indicates mean values. Error bars represent 1 standard deviation.

  • FIG 3
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    FIG 3

    Volatile organic compound (VOC) production of ancestral and domesticated Penicillium. Because total concentrations of VOCs are highly variable across different compounds, visualization was simplified by relativizing the relative peak areas from GC-MS chromatograms within each VOC to the highest concentration detected for that VOC. Only the VOCs that were detected across all replicates are shown. See Table S2 for all VOCs and their relative peak area values. The UPGMA tree is clustering the VOC profiles for each replicate based on Bray-Curtis dissimilarity. Asterisks indicate clusters with >70% bootstrap support. WT, ancestor phenotype. M2, M5, and M6 are all domesticated phenotypes. Numbers 1 to 4 after strain designations indicate biological replicates. Because of accidental sample loss during processing, only three biological replicates were collected from the WT. Descriptors on the right represent known aroma qualities of detected VOCs.

  • FIG 4
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    FIG 4

    Experimental domestication shifts global gene expression of Penicillium on cheese. (A) Differences in gene expression between ancestor and domesticated Penicillium commune 162_3FA. Each dot represents a transcript from across the genome. Yellow dots represent those transcripts that had higher expression, and blue dots represent those transcripts that had lower expression (5-fold change in expression; FDR-corrected P value < 0.05). (B) Pathway enrichment analysis showing the distribution of GO terms that were significantly enriched in genes with decreased expression in the domesticated phenotype (strain M5). (C) Representative mapping of reads to the ergot alkaloid synthesis (eas) gene cluster.

  • FIG 5
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    FIG 5

    Domesticated phenotypes of Penicillium are present in cheese caves. (A) A plate showing Penicillium sp. strain 12 isolated from a cheese cave in Vermont, USA. White colonies exist at low frequencies in this fungal population. The small smooth beige colonies represent the yeast Debaryomyces hansenii. (B) Wild-type phenotype of the mold isolated from the population and grown on cheese curd agar. The intensities of the green pigmentation differ between panel A and panel B because panel A shows a fungal population grown on plate count agar and panel B shows a fungal population grown on cheese. (C) A white domesticated strain isolated from the same population as the wild type.

Supplemental Material

  • Figures
  • FIG S1

    The genome-scale phylogeny of the genus Penicillium. (A) Concatenation phylogeny with section denominations. The two strains used in the experiments described in the text (indicated in bold) are placed within section Fasciculata. Furthermore, Penicillium commune strain 162_3FA is closely related to Penicillium biforme and Penicillium camemberti. The inset depicts phylogeny, with branch lengths representing substitutions per site. Penicillium sp. MB was isolated from a natural rind cheese at the same time as the other two strains and was sequenced as part of this work, but it was not used in the experiments described. (B) Comparison of concatenation-based (left) and coalescence-based (right) phylogenies reveals only one instance of incongruence. Specifically, whereas P. biforme is placed sister to P. commune 162_3FA in the concatenation analysis, coalescence supports P. camemberti as sister to P. commune 162_3FA. All internal branches received full support except the coalescence-inferred internal branch where Exilicaulis and Lanata-divaricata split, which received a local posterior probability value of 0.97. Branch lengths reflect substitutions/site for concatenation and coalescence units for the coalescence inferred phylogeny. Download FIG S1, DOCX file, 0.5 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Population size of Penicillium commune 162_3FA evolved alone and with a community of cheese microbes. Lines connect points representing mean CFUs from four replicate populations, and error bars represent 1 standard deviation of the mean. Total CFUs in the Penicillium-plus-community treatment were significantly different from those measured for the total measured for Penicillium alone (repeated-measures ANOVA F1,6 = 10.3, P = 0.02). Download FIG S2, DOCX file, 0.1 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    Experimental evolution of Penicillium sp. 12 alone and with a community of cheese rind microbes. (A) Population size of Penicillium sp. 12 evolved alone and with a community of cheese microbes. Lines connecting points represent mean domesticated phenotype frequencies of four replicate populations, and error bars represent 1 standard deviation of the mean. Total CFUs in the Penicillium-plus-community treatment were significantly different from those measured for Penicillium alone (repeated-measures ANOVA F1,6 = 16.8, P = 0.006). Lines connect points representing mean CFUs from four replicate populations, and error bars represent 1 standard deviation of the mean. (B) Domesticated phenotype frequency of Penicillium sp. 12 evolved alone and with a community of cheese microbes. Lines connect points representing mean domesticated phenotype frequencies of four replicate populations, and error bars represent 1 standard deviation of the mean. Domesticated phenotype frequencies in the Penicillium-plus-community treatment were significantly different from those measured for Penicillium alone (repeated-measures ANOVA F1,6 = 20.1, P < 0.005). Download FIG S3, DOCX file, 0.2 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S1

    (A) Description of domesticated phenotype classes and specific isolates identified in the evolution of Penicillium commune strain 162_3FA. (B) Distribution of domesticated phenotypes across replicate populations and transfers in the experimental evolution of Penicillium commune 162_3FA. Data at the bottom show the number of colonies of ancestor or domesticated phenotypes counted from 10−4 dilution plates of experimental populations. T1, T2, etc. = transfer numbers. (C) Description of specific domesticated phenotype isolates identified in the evolution of Penicillium sp. strain 12. (D) Distribution of domesticated phenotypes across replicate populations and transfers in the experimental evolution of Penicillium sp. 12. Data at the bottom show the number of colonies of ancestor or domesticated phenotypes counted from 10−4 dilution plates of experimental populations. T1, T2, etc. = transfer numbers. Download Table S1, XLSX file, 0.1 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S4

    Population size of Penicillium commune 162_3FA evolved in different cheese nutrient environments. “Normal cheese” = 10% cheese curd in agar medium. “Low cheese” = 1% cheese curd in agar medium. “Alternating normal/low” = alternating 10% and 1% cheese curd at each transfer. The “Low cheese” treatment suppressed population size (repeated-measures ANOVA F2,9= 105.1, P < 0.0001, with Tukey’s HSD post hoc tests). Lines connect points representing mean CFUs from four replicate populations, and error bars represent 1 standard deviation of the mean. Download FIG S4, DOCX file, 0.2 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S5

    Spore production of ancestor and domesticated strains of Penicillium commune 162_3FA. Spores were harvested from plugs taken from the center of fungal colonies and were quantified using a hemocytometer. Bars with the same letter are not significantly different from one another (ANOVA F3,16 = 105.6, P < 0.001 with Tukey’s HSD post hoc test). Error bars represent 1 standard deviation. n = 5. Download FIG S5, DOCX file, 0.1 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S6

    Stability of Penicillium commune 162_3FA domesticated phenotypes. Domesticated strains were transferred weekly to new cheese curd agar, and colony morphology was photographed. The white morphology was stable over time. Download FIG S6, DOCX file, 0.2 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S2

    Overview of all volatile organic compounds detected in the ancestor (ANC) strain and domesticated strains M2, M5, and M6 of Penicillium commune strain 162_3FA. Download Table S2, XLSX file, 0.1 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S3

    Overview of genes that were differentially expressed between the ancestor strain and strain M5 of Penicillium commune strain 162_3FA. Download Table S3, XLSX file, 0.3 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S4

    Single-nucleotide polymorphisms detected in the genomes of domesticated strains of Penicillium commune strain 162_3FA. Download Table S4, XLSX file, 0.01 MB.

    Copyright © 2019 Bodinaku et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Rapid Phenotypic and Metabolomic Domestication of Wild Penicillium Molds on Cheese
Ina Bodinaku, Jason Shaffer, Allison B. Connors, Jacob L. Steenwyk, Megan N. Biango-Daniels, Erik K. Kastman, Antonis Rokas, Albert Robbat, Benjamin E. Wolfe
mBio Oct 2019, 10 (5) e02445-19; DOI: 10.1128/mBio.02445-19

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Rapid Phenotypic and Metabolomic Domestication of Wild Penicillium Molds on Cheese
Ina Bodinaku, Jason Shaffer, Allison B. Connors, Jacob L. Steenwyk, Megan N. Biango-Daniels, Erik K. Kastman, Antonis Rokas, Albert Robbat, Benjamin E. Wolfe
mBio Oct 2019, 10 (5) e02445-19; DOI: 10.1128/mBio.02445-19
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KEYWORDS

Penicillium
cheese
evolution
mycotoxins
secondary metabolism
transcriptome
volatile organic compound

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