Article Figures & Data
Figures
- 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
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
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
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
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
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
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
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
- TABLE 1
Eukaryotic species and bacterial phyla associated with the lichen under studya
Organismb Absolute gene count Relative gene count (%) Eukaryota (species level) Ajellomyces capsulatus (A) 5,494 11.9 Talaromyces stipitatus (A) 4,361 9.9 Arthroderma gypseum (A) 1,959 4.2 Botryotinia fuckeliana (A) 1,687 3.6 Cryptococcus neoformans (B) 1,132 2.4 Geomyces destructans (A) 797 1.7 Sclerotinia sclerotiorum (A) 746 1.7 Rhizopus oryzae (O) 698 1.5 Unresolved fungus 4,474 9.7 Bacteria (phylum level) Proteobacteria 102,582 51.4 Bacteroidetes/Chlorobi 35,465 17.7 Actinobacteria 20,176 10.1 Cyanobacteria 10,405 5.2 Fibrobacteres/Acidobacteria 10,311 5.2 Chlamydiae/Verrucomicrobia 6,220 3.1 Planctomyces 6,213 3.1 Firmicutes 2,813 1.4
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.
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Text S1
Supplemental materials and methods. Download Text S1, DOCX file, 0.04 MB.
Copyright © 2016 Garg et al.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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