Mapping microhabitats of lignocellulose decomposition by a microbial consortium

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

more conservative taxonomic assignment.In addition, we keep the taxonomic assignment at a higher rank (e.g., fungi, bacteria, insect) to avoid potential overlaps between different taxonomic groups.Functional assignment: In addition to JGI metagenome workflow (Functional assignment, Method 1), the protein (with >= 2 quality-filtered peptides) was assigned with the functional annotation that the top hits with the same highest bitscore agreed on, otherwise remained unclassified by NCBI clustered NR database (Functional assignment, Method 2).

Annotation of 4-Carboxymethylenebut-2-en-4-olide is supported by the identification of the enzyme (Carboxymethylenebutenolidase) and product (2-Maleylacetate) within the same microscale regions (LM1, LM2, LM3).
i) LC-MS/MS -Identified with confidence level 4 by accurate mass and isotope information.Supplementary Table 6.Metabolites confirmed by annotated LC-IMS-MS features from the fungal garden sample.A list of targets with accurate masses for precursor and fragment ions was generated using predicted spectra by CFM-ID.Signals were extracted using Skyline from the LC-IMS-MS/MS data acquired in DIA mode from the fungal garden sample.
The column # of detected fragments indicates the count of fragments that had good peak shape similarity compared to the precursor.
Results are from the data acquired using the HILIC method from Bilbao et al.
i) LC-IMS-MS/MS -Identified with confidence level 1 by accurate mass, RT, CCS, and MS/MS match with the standard.ii) LC-MS/MS -Identified with confidence level 2 by identification by accurate mass, RT, and MS/MS match with the standard.STANDARD SAMPLE iii) LESA-MS/MS -Identified with confidence level 3 by accurate mass and MS/MS that makes sense for the ID.iv) GC-MS -Identified with confidence level 2. Retention index from sample 1314.4,retention index in standard: 1315.4.Fragments used to validate identification: 193, 223 and 267.The metabolite is co-eluting with other compounds that generate the adjacent ions observed in the MS.Scan 1360 (14.594 min): x_LCA_FunalGarden_Confirm_IDs_12_GC.D\data.ms(-1355) 4-hydroxybenzoic acid (p-salicylic acid) Head to Tail MF=539 C6H4O4 -H] -This molecular formula matches to candidate molecules 4-Carboxymethylenebut-2-en-4-olide and 5-Formyl-2-furoate in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Supplementary Figure 3 .
ii) LC-IMS-MS/MS -Identified with confidence level 5 by accurate mass.e) Benzene-1,2,4-triol i) LC-IMS-MS/MS -Identified with confidence level 3 by accurate mass, RT, CCS, and MS/MS match with predicted spectra using CFM-ID.ii) LC-MS/MS -Identified with confidence level 3 by accurate mass and MS/MS with external databases.f) 2-Maleylacetate i) LC-IMS-MS/MS -Identified with confidence level 3 by accurate mass, RT, CCS, and MS/MS match with predicted spectra using CFM-ID.ii) LC-MS/MS -Identified with confidence level 3 by accurate mass and MS/MS that makes sense for the ID.g) Homogentisate i) LC-IMS-MS/MS -Identified with confidence level 1 by accurate mass, RT, CCS, and MS/MS match with the standard.i) LC-MS/MS -Identified with confidence level 4 by accurate mass and isotope information.ii) LC-IMS-MS/MS -Identified with confidence level 3 by accurate mass, RT, CCS, and MS/MS match with predicted spectra using CFM-ID.i) Fuconate i) LC-IMS-MS/MS -Identified with confidence level 5 by accurate mass.ii) LC-MS/MS -Identified with confidence level 3 by accurate mass and MS/MS that makes sense for the ID.j) 2-Dehydro-3-deoxy-fuconate i) LC-IMS-MS/MS -Identified with confidence level 5 by accurate mass.ii) LC-MS/MS -Identified with confidence level 3 by accurate mass and MS/MS that makes sense for the ID.iii) LESA-MS/MS -Identified with confidence level 3 by accurate mass and MS/MS that makes sense for the ID.k) Glucose i) LC-IMS-MS/MS -Identified with confidence level 5 by accurate mass.Caveat: Due to the low intensity of this metabolite, we used GC-MS (see below) to confidently identify glucose in the garden sample.ii) GC-MS -Identified with confidence level 1. MS from in-house library.Retention index from sample 1607.4,retention index in standard: 1608.3.Fragments used to validate identification: 160, 205 and 319.GC-MS -Identified with confidence level 3. MS from in-house library.Retention index from sample 2077.5 retention index in standard: 2049.0.Fragments used to validate identification: 299, 357 and 387.Caveat: Due to the low intensity of this metabolite, we used LC-MS/MS (see below) to confidently identify glucose-6-phosphate in the garden sample.ii) LC-IMS-MS/MS -Identified with confidence level 1 by accurate mass, RT, CCS, and MS/MS match with the standard.STANDARD SAMPLE Scan 2542 (21.988 min): x_LCA_FunalGarden_Confirm_IDs_15_GC.D\data.ms(-2540) d-Glucose, 2,3,4,5-tetrakis-O-(trimethylsilyl)-, o-methyloxime, 6-[bis(trimethylsilyl) phospha Head to Tail MF=390 Spatial multi-omics integration unravels complex catabolic pathways in the leaf-cutter ant fungal garden ecosystem.Metabolite MALDI-FTICR-MSI images visualize the relative abundance of each metabolite across Section 9 (yellow, highest intensity).These pathways include a) Conversion of 4-