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Editor's Pick Research Article | Novel Systems Biology Techniques

Spatial Molecular Architecture of the Microbial Community of a Peltigera Lichen

Neha Garg, Yi Zeng, Anna Edlund, Alexey V. Melnik, Laura M. Sanchez, Hosein Mohimani, Alexey Gurevich, Vivian Miao, Stefan Schiffler, Yan Wei Lim, Tal Luzzatto-Knaan, Shengxin Cai, Forest Rohwer, Pavel A. Pevzner, Robert H. Cichewicz, Theodore Alexandrov, Pieter C. Dorrestein
Janet K. Jansson, Editor
Neha Garg
aSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California, USA
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Yi Zeng
bDepartment of Chemistry and Biochemistry, University of California, San Diego, California, USA
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Anna Edlund
cGenomic Medicine, J. Craig Venter Institute, La Jolla, California, USA
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Alexey V. Melnik
aSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California, USA
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Laura M. Sanchez
aSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California, USA
dDepartment of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA
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Hosein Mohimani
kDepartment of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA
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Alexey Gurevich
lCenter for Algorithmic Biotechnology, Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg, Russia
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Vivian Miao
eDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
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Stefan Schiffler
fSCiLS GmbH, Bremen, Germany
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Yan Wei Lim
gDepartment of Biology, San Diego State University, San Diego, California, USA
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Tal Luzzatto-Knaan
aSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California, USA
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Shengxin Cai
hNatural Products Discovery Group, Department of Chemistry and Biochemistry, Institute for Natural Products Applications and Research Technologies, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, Oklahoma, USA
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Forest Rohwer
gDepartment of Biology, San Diego State University, San Diego, California, USA
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Pavel A. Pevzner
kDepartment of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA
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Robert H. Cichewicz
hNatural Products Discovery Group, Department of Chemistry and Biochemistry, Institute for Natural Products Applications and Research Technologies, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, Oklahoma, USA
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Theodore Alexandrov
aSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California, USA
fSCiLS GmbH, Bremen, Germany
iEuropean Molecular Biology Laboratory (EMBL), Heidelberg, Germany
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Pieter C. Dorrestein
aSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, California, USA
bDepartment of Chemistry and Biochemistry, University of California, San Diego, California, USA
jCenter for Computational Mass Spectrometry and Department of Computer Science and Engineering, University of California San Diego, La Jolla, California, USA
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Janet K. Jansson
Pacific Northwest National Laboratory
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DOI: 10.1128/mSystems.00139-16
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  • FIG 1
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    FIG 1

    (A) A needle was used to scrape off small amounts of material from 110 locations from the original piece of lichen. UHPLC-MS/MS data were acquired on these materials, and data analysis was performed using the online analysis infrastructure GNPS. (B) A 2.5-mm by 1.8-mm by 3-mm piece of lichen was sectioned from the original lichen piece (1.8 cm by 3 cm). This section was embedded in gelatin, and MALDI IMS data were acquired on three layers, the sun-exposed layer, the middle layer, and the bottom layer, to reveal metabolite distributions in false color. (C) The Venn diagram on the left shows the percentage of molecules detected in lichen that are of either fungal or bacterial origin. This Venn diagram is scaled to demonstrate the number of features detected. The origin was assigned by identifying common molecules in the MS/MS data acquired on the lichen and microbes cultured from this lichen. The Venn diagram on the right shows percentages of common molecules among lichen, cultured microbial isolates, and public data sets on soil fungi as well as freshwater cyanobacteria.

  • FIG 2
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    FIG 2

    The molecular family belonging to fungal pyridone alkaloid PF1140 is shown. (A) The representative molecule PF1140 was identified in both lichen and fungal isolate (pink node) as well as in the MALDI IMS data. (B) The cultured microbes also produced a previously known analogue, deoxy-PF1140 (purple node). (C) The isolated microbe also showed production of an unknown molecule at m/z 354.207 (purple node). The shift in the parent mass for this unknown molecule by 92.03 Da from the mass of deoxy-PF1140 suggested addition of a phenol group to deoxy-PF1140. The MS/MS fragments also showed a shift of 92.03 Da in mass (shown in dashed lines). A structure search in SciFinder suggested the molecule to be a new analogue of trichodin A with one additional methyl group. The putative structures and the tandem MS spectra of other molecules in this cluster are shown in Fig. S5 in the supplemental material.

  • FIG 3
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    FIG 3

    The molecular network of the fungal molecule asperphenamate (m/z 507.229) and its distribution are shown. The pink node represents a common molecule produced by both the lichen and the isolated microbe. The purple nodes are unique to the cultured microbe, and the orange node is unique to lichen. The underlying MS/MS spectra for asperphenamate in the orange node were recorded at lower intensity and also have a contaminating MS/MS spectrum from a molecule with similar mass. Hence, this node is not merged with the pink node corresponding to asperphenamate. The MS/MS spectra of asperphenamate (m/z 507.229) and the analogues (m/z 532.222 and 564.248) are annotated in the MS/MS spectra shown, and annotations are described in Text S1 in the supplemental material.

  • FIG 4
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    FIG 4

    The molecular network of a family of sesquiterpene lactones and the distribution of a representative molecule with an MS/MS spectrum match to alantolactone are shown. All the labeled MS/MS peaks for alantolactone matched the MS/MS spectra available on the METLIN metabolite database. The known analogues at m/z 249.149 and 235.169 are annotated as hydroxyalantolactone and dihydroisoalantolactone based on the MS/MS data available on METLIN. The molecule at m/z 265.143 is annotated as dihydroxyalantolactone due to an increase in the parent mass of 15.99 Da from the mass of hydroxyalantolactone. The corresponding fragments with a 15.99-Da shift are labeled in the MS/MS spectra in blue. Orange represents spectra found in lichen samples only, pink represents spectra found in both lichen and cultured isolates, purple represents spectra detected only in cultured isolates, and grey nodes represent other combinations.

  • FIG 5
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    FIG 5

    The MS/MS spectra and MALDI IMS distributions of mannitol and the corresponding polysaccharide containing mannitol are shown. The polysaccharide at m/z 345.138 contains an additional hexose residue, and the polysaccharide at m/z 689.272 contains two additional hexose sugar residues. The molecular network corresponding to the polysaccharide family is shown in Fig. S6B in the supplemental material.

  • FIG 6
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    FIG 6

    The chlorophyll a pigments pheophytin A and pheophorbide A were identified in both UHPLC-MS/MS data and MALDI-IMS data. The two major fragments at m/z 593.276 and m/z 533.255 are annotated in the structure of pheophytin A.

  • FIG 7
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    FIG 7

    The mass spectrum of heterocyst glycolipid and the corresponding structures are shown on the right. The cyanobacterial heterocyst glycolipid colocalizes with the cyanobacterial chlorophyll (Fig. 5 and 8B). The heterocyst biosynthetic gene cluster was identified by running antiSMASH on the metaSPAde assembly of the short-read data set. The gene cluster identified by using antiSMASH is shown as the query sequence, and the gene cluster previously deposited in antiSMASH corresponds to the gene cluster named BGC0000869_c1.

  • FIG 8
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    FIG 8

    The distribution of fungal molecules PF1140, asperphenamate, and alantolactone(A) and cyanobacterial molecules (chlorophyll and heterocyst glycolipid) (B) and a representative member of the molecular family of compounds with spectral similarity to lupeol is shown. The complete overlap of cyanobacterial chlorophyll pigment (green) and heterocyst glycolipid (red) results in cyanobacteria appearing yellow.

Tables

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  • TABLE 1

    Eukaryotic species and bacterial phyla associated with the lichen under studya

    OrganismbAbsolute gene countRelative gene count (%)
    Eukaryota (species level)
     Ajellomyces capsulatus (A)5,49411.9
     Talaromyces stipitatus (A)4,3619.9
     Arthroderma gypseum (A)1,9594.2
     Botryotinia fuckeliana (A)1,6873.6
     Cryptococcus neoformans (B)1,1322.4
     Geomyces destructans (A)7971.7
     Sclerotinia sclerotiorum (A)7461.7
     Rhizopus oryzae (O)6981.5
    Unresolved fungus4,4749.7
    Bacteria (phylum level)
     Proteobacteria 102,58251.4
     Bacteroidetes/Chlorobi35,46517.7
     Actinobacteria 20,17610.1
     Cyanobacteria 10,4055.2
     Fibrobacteres/Acidobacteria10,3115.2
     Chlamydiae/Verrucomicrobia6,2203.1
     Planctomyces 6,2133.1
     Firmicutes 2,8131.4
    • ↵a Taxonomic predictions were derived from assembled metagenome annotations and relative gene counts by using the JCVI-supported METAREP analysis pipeline. Taxonomic units contributing >1% to the total community abundance are presented.

    • ↵b A, Ascomycetes; B, Basidiomycetes; O, other fungus.

Supplemental Material

  • Figures
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  • Figure S1

    Pie charts representing taxonomic diversity of bacterial (A) and fungal (B) genes associated with the lichen under study. Bacterial taxa are presented at the phylum level, and fungal taxa are presented at the species level. Taxonomic annotation of genes was performed using the METAREP Web tool. Download Figure S1, EPS file, 0.8 MB.

    Copyright © 2016 Garg et al.

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

  • Text S1

    Supplemental materials and methods. Download Text S1, DOCX file, 0.04 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S2

    Hierarchical cluster analysis of taxonomic annotations of genes and their relative frequencies from metagenomes representing a diversity of environments (lichen annotations boxed in red). Metagenome annotations and relative abundance values are available via the J. Craig Venter Institute through the JCVI-supported METAREP (http://www.jcvi.org/hmp-metarep ). Colors represent relative percentages of total annotated genes belonging to orders of bacteria (B), fungi (F), and eukaryotes (other than fungi) (E). Light pink to red, >3 to 14%; white, 3%; light blue to blue, 0.1 to <3%. Pearson correlation distance is shown next to cluster topography. Download Figure S2, EPS file, 1.3 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S3

    Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-level annotations and relative counts of assembled genes representing the lichen metagenome. The JCVI-supported METAREP (http://www.jcvi.org/hmp-metarep ) tool was used for classification. Numbers of total gene counts that could be classified for each functional group are presented inside brackets. Download Figure S3, EPS file, 1.5 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S4

    The molecular network of MS/MS data collected on 110 regions from a lichen piece and cultured bacterial and fungal isolates. The nodes in orange are molecules detected only in lichen, the nodes in pink are molecules detected both in lichen and in microbial cultures, and the nodes in purple are molecules detected only in cultured microbes. Download Figure S4, TIF file, 2.2 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S5

    Putative structures and MS/MS spectra of two fungal molecules detected in the pyridone alkaloid PF1140 family. The molecule on the left has the identical parent m/z as deoxyakanthomycin, and the molecule on the right is 92.03 Da in mass higher than deoxyakanthomycin. This molecule likely represents a new analogue of deoxyakanthomycin with a phenol moiety on the pyridine ring. This phenol group is also present on other known pyridine alkaloids such as trichodin A. Download Figure S5, EPS file, 0.4 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S6

    (A) The delta m/z histogram obtained from GNPS analysis of the molecular network is shown (http://gnps.ucsd.edu/ProteoSAFe/result.jsp?task=675991870933480293c10f0bfcf69e20&view=pairs_histogram ). This analysis is obtained under the network summarizing graphs tab on the results page. Common delta m/z shifts of sugar residues between parent masses are labeled. The y axis represents the number of pairs in the network that have these shifts, and the x axis represents the delta m/z shift itself. (B) The molecular network of the polysaccharide family is shown and is accessible online at http://gnps.ucsd.edu/ProteoSAFe/result.jsp?view=network_displayer&componentindex=224&task=675991870933480293c10f0bfcf69e20#%7B%7D , where delta m/z between nodes can be highlighted for visualization purposes. The nodes in orange are unique to lichen, the nodes in purple are unique to microbial isolates, and the nodes in gray are polysaccharides that are also present in the culture medium. (C) The molecular network of the polyacetylated polysaccharide family is shown. The nodes differ by 42.01 Da and 84.02 Da (2 × 42.01) in mass, indicative of polyacetylated residues. Download Figure S6, EPS file, 2.2 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S7

    The distributions and MS/MS spectra of UDP-N-acetylglucosamine and the polyacetylated polysaccharide are shown. The polysaccharide contains a hexose sugar residue (162 Da) and glucuronic acid (176 Da) as well as polyacetylations (n × 42.01) on sugar residues. Download Figure S7, TIF file, 1.2 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S8

    (A to G) MALDI IMS images of adjacent slices that were sliced 10 µm apart. (H) Fluorescence microscopy image of Peltigera sp. lichen under study. The distribution of fluorescence matches the distribution of chlorophyll pigments visualized by MALDI IMS in Fig. 4. Download Figure S8, TIF file, 2.1 MB.

    Copyright © 2016 Garg et al.

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

  • Figure S9

    The molecular network of the family of a triterpene and a phytosterol, lupeol, is shown. All the labeled peaks in the spectra of lupeol acetate, lupeol, and the insource fragment at m/z 409.381 match the fragments of MS/MS spectra available in the METLIN metabolite database. Based upon the fragmentation pattern and spectral match with reference spectra in METLIN, this family is putatively assigned to triterpene-lupeol. Download Figure S9, EPS file, 1.1 MB.

    Copyright © 2016 Garg et al.

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

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Spatial Molecular Architecture of the Microbial Community of a Peltigera Lichen
Neha Garg, Yi Zeng, Anna Edlund, Alexey V. Melnik, Laura M. Sanchez, Hosein Mohimani, Alexey Gurevich, Vivian Miao, Stefan Schiffler, Yan Wei Lim, Tal Luzzatto-Knaan, Shengxin Cai, Forest Rohwer, Pavel A. Pevzner, Robert H. Cichewicz, Theodore Alexandrov, Pieter C. Dorrestein
mSystems Dec 2016, 1 (6) e00139-16; DOI: 10.1128/mSystems.00139-16

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Spatial Molecular Architecture of the Microbial Community of a Peltigera Lichen
Neha Garg, Yi Zeng, Anna Edlund, Alexey V. Melnik, Laura M. Sanchez, Hosein Mohimani, Alexey Gurevich, Vivian Miao, Stefan Schiffler, Yan Wei Lim, Tal Luzzatto-Knaan, Shengxin Cai, Forest Rohwer, Pavel A. Pevzner, Robert H. Cichewicz, Theodore Alexandrov, Pieter C. Dorrestein
mSystems Dec 2016, 1 (6) e00139-16; DOI: 10.1128/mSystems.00139-16
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KEYWORDS

lichen
mass spectrometry
microbial assemblages
natural products
metagenomics

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