Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential

Abstract

The attine ants of South and Central America are ancient farmers, having evolved a symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal partner against co-evolved fungal pathogens of the genus Escovopsis. Pseudonocardia mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring secondary metabolites of Pseudonocardia on a genome-wide scale. Here, we report a genomic analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 strains revealed that the phylotypes are distinct species and each encodes between 11 and 15 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. Strains show variations in the arrangement of these BGCs that resemble those seen in gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with Acromyrmex ants and supports the hypothesis that Pseudonocardia species could provide a valuable source of new antimicrobials.

The evolution of abdominal microbiomes in fungus‐growing ants

“…This could imply that ActAcro1 was initially a gut or abdominal‐organ symbiont, whose antibiotic functions were later co‐opted for external hygienic use in the evolutionarily derived higher attine ants. This hypothesis deserves further testing because it may shed novel light on the ongoing discussion of whether and to what extent Pseudonocardia strains co‐evolved with the attine ant symbiosis and its Escovopsis mycopathogens (Cafaro et al, ; de Man et al, ; Heine et al, ; Holmes et al, ; Sen et al, ).…”

“…These effects appear to be complex, to probably interact with the “A” transition, and to be mostly reflected in distributional shifts among rarer OTUs rather than the abundant ones (Figure ). Given the known production of antibacterials by the ActAcro1 cuticular Pseudonocardia symbiont (Holmes et al, ), these interactive effects do not come as a surprise, but they will require functional experiments to assess whether and to what extent specific bacterial OTUs benefit from the presence of cuticular actinobacterial antibiotics or are inhibited.…”

“…Cuticular actinobacterial biofilms have been maintained by many, but not all attine genera, so that attine ants are exceptional in often having both an external (cuticular) microbiome and an internal abdominal microbiome (Sapountzis et al, ). Considering that the cuticular actinomycetes produce a range of antibiotics and antifungals (Barke et al, ; Holmes et al, ; Oh, Poulsen, Currie, & Clardy, ; Seipke et al, ; Seipke, Grüschow, Goss, & Hutchings, ) that are applied to fungus gardens that the ants ingest, it is conceivable that cuticular Actinobacteria may affect the composition and diversity of microbiomes in the guts and associated organs, but this hypothesis has remained untested.…”

Cooperative Involvement of Glycosyltransferases in the Transfer of Amino Sugars during the Biosynthesis of the Macrolactam Sipanmycin by Streptomyces sp. Strain CS149

“…However, due to the low hit rate for novel compounds obtained using classical screening approaches and the frequent rediscovery of alreadyknown compounds (2), novel strategies must be developed. Some of these approaches involve the isolation of bioactive compound microbial producers from unexplored environments, paying special attention to microorganisms associated with marine or terrestrial macroorganisms, such as sponges, plants, ants, termites, and wasps (3)(4)(5)(6), but also to mammals (7), including human beings (8). Furthermore, the development of DNA sequencing technologies and the increasing number of genomes sequenced facilitate the use of other strategies, such as transcriptome analysis by RNA sequencing (9), mining of microbial genomes or metagenomes in searching specific gene homologs (10), or new biosynthetic gene clusters (BGCs) (11), and the activation of low-expressed or silent clusters (12).…”

Biogeography and Microscale Diversity Shape the Biosynthetic Potential of Fungus-growing Ant-associated Pseudonocardia

“…Although the environmental forces driving the distribution of fine-scale microbial diversity are poorly understood for most taxa, those that are associated with extreme environmental conditions (8) or with eukaryotic hosts (9) can be used to address biogeographical and population-scale ecological questions more readily. Lineages of bacteria from the actinobacterial genus Pseudonocardia that form a defensive mutualism with many fungus-growing ant species (10,11) provide a useful model system to do this. These filamentous spore forming bacteria grow on the external surface of the ants, where they produce natural products that inhibit the growth of Escovopsis, a co-evolved pathogen of the ant's fungus garden (12).…”