Actinobacteria, one of the largest bacterial phyla, are ubiquitous in many of Earth’s ecosystems and often act as defensive symbionts with animal hosts. Members of the phylum have repeatedly been isolated from basidiomycete-cultivating fungus-farming termites that maintain a monoculture fungus crop on macerated dead plant substrate. The proclivity for antimicrobial and enzyme production of Actinobacteria make them likely contributors to plant decomposition and defense in the symbiosis. To test this, we analyzed the prophylactic (biosynthetic gene cluster [BGC]) and metabolic (carbohydrate-active enzyme [CAZy]) potential in 16 (10 existing and six new genomes) termite-associated Actinobacteria and compared these to the soil-dwelling close relatives. Using antiSMASH, we identified 435 BGCs, of which 329 (65 unique) were similar to known compound gene clusters, while 106 were putatively novel, suggesting ample prospects for novel compound discovery. BGCs were identified among all major compound categories, including 26 encoding the production of known antimicrobial compounds, which ranged in activity (antibacterial being most prevalent) and modes of action that might suggest broad defensive potential. Peptide pattern recognition analysis revealed 823 (43 unique) CAZymes coding for enzymes that target key plant and fungal cell wall components (predominantly chitin, cellulose, and hemicellulose), confirming a substantial degradative potential of these bacteria. Comparison of termite-associated and soil-dwelling bacteria indicated no significant difference in either BGC or CAZy potential, suggesting that the farming termite hosts may have coopted these soil-dwelling bacteria due to their metabolic potential but that they have not been subject to genome change associated with symbiosis.
IMPORTANCE : Actinobacteria have repeatedly been isolated in fungus-farming termites, and our genome analyses provide insights into the potential roles they may serve in defense and for plant biomass breakdown. These insights, combined with their relatively higher abundances in fungus combs than in termite gut, suggest that they are more likely to play roles in fungus combs than in termite guts. Up to 25% of the BGCs we identify have no similarity to known clusters, indicating a large potential for novel chemistry to be discovered. Similarities in metabolic potential of soil-dwelling and termite-associated bacteria suggest that they have environmental origins, but their consistent presence with the termite system suggests their importance for the symbiosis.
DATA AVAILABILITY : All genomes are publicly available, either from previous publications, submission to the NCBI database under accession number JAEKDS000000000.1 for RB110, or Zenodo at https://doi.org/10.5281/zenodo.4302144 (RB22, RB24, RB33, M32, and RB108). All other accession numbers are provided in Table S1.
TABLE S1 : Termite-associated Actinobacteria genome metadata and statistics. N50, number of contigs making 50% of the assembly; L50, length lower limit of contigs making up 50% of the assembly.
TABLE S2 : (Sheet 1) Predicted BGCs identified by antiSMASH with a similarity to a reported BGC in the MIBiG database of 50% or higher. The class of BGC, the similar compound identified in the MIBiG database, and whether it has documented antimicrobial activity are detailed. The number relating a sample to a BGC indicates the occurrences of this BGC being found in that sample. (Sheet 2) All BGCs identified by antiSMASH, both novel and predicted (regardless of similarity).
TABLE S3 : Overview results of HotPep-identified CAZymes. CAZy prediction summary indicating the EC number and corresponding enzyme name of CAZymes identified by HotPep, along with the enzyme substrate, category of enzyme (hydrolase, oxidoreductases, etc.), and whether the enzyme is catabolic or anabolic in nature. The number relating a sample to a specific enzyme is the frequency count of that enzyme in that sample.
TABLE S4 : All essential genes identified with ARTS and their frequency of marker occurrence (duplication, proximity to a BGC, horizontal gene transfer, and whether they are a known target). Essential genes are ordered into categories. First, “Most likely resistance factor or target” indicating that in an individual genome for this gene, three or more markers were positive, one being proximity to a BGC. Second, “Likely resistance target or factor,” requiring the same as the previous category, just without proximity to a BGC being one of the markers. Lastly, “Unlikely resistance target or factor,” where fewer than three markers were identified in association with this essential gene in any singular genome.
TABLE S5 : Overview of all BGCs previously identified from Actinobacteria isolated from the fungus-growing termite symbiosis.
FIGURE S1 : Frequency of essential gene markers produced by ARTS to indicate said gene being a resistance factor or resistant target. Each point is an identified essential gene, with three or more markers, observed in at least one termite-associated bacterial genome. The y axis is the frequency of how often that marker is positive in that specific essential gene across all genomes in which the essential gene was identified. Both axes have been jittered to improve visualization. “FGT” denotes that strains are fungus-growing termite-associated, and “free” indicates that strains are closely related soil-dwelling free-living bacteria.
FIGURE S2 : Length (bp) of BGCs with a similarity score of 50% or greater to a known BGC in the MIBiG database (the same BGCs as in Fig. 3).