Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

Abstract

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both fieldcollected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus-bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.

Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

“…Future transcriptomic analyses and additional genome sequencing will likely identify more enzymes used by this fungus for biomass degradation in leaf-cutter ant fungus gardens. Previous metagenomic characterizations of bacterial communities in Atta and Acromyrmex gardens recovered mainly oligosaccharide-degrading enzymes and few CAZy families associated with the degradation of recalcitrant polysaccharides (17), suggesting that bacteria are not playing a prominent role in biomass degradation in fungus gardens. Moreover, our metaproteomic results revealed that although Ͼ81,000 spectra could be confidently mapped to diverse L. gongylophorus lignocellulases, only a single spectrum could be mapped to a bacterial GH8, a CAZyme family that in bacteria is often associated with cell wall modification rather than plant biomass degradation (49).…”

“…S4). Peptide matches were filtered using Sequest scores (37), MS-GF software spectral probabilities (38), and false discovery rates, as previously described (17). Moreover, all peptides with a Ͼ10-ppm mass error were discarded.…”

“…DNA was extracted using a modified phenol-chloroform method. Primers used for the amplification of genes encoding one GH6 and two GH10 glycoside hydrolases were designed in this study from contigs present in previously reported community metagenomes of ant gardens (details are in Table S5), which contained a small amount of L. gongylophorus sequences in addition to the bacterial component (17). Amplification of partial-length GH7-encoding genes was performed using the primers fungcbhIF and fungcbhIR, designed in a previous study (43).…”

“…Culture-based studies have shown that a diversity of bacteria, yeasts, and microfungi can also be found in ant gardens (12)(13)(14), and recent culture-independent investigations have revealed that a distinct community of bacteria resides in the fungus gardens of leaf-cutter ants (15,16). Although the genetic potential of bacteria in Atta gardens has been shown to be consistent with the degradation of less-recalcitrant plant material (17), the relative contribution of these microbes to biomass degradation remains unknown.…”

Symbiotic adaptations in the fungal cultivar of leaf-cutting ants

“…This phenylalanine production is independent of the plant material that the ants offer as fungal substrate, because 3.86 mg free and 7.33 mg protein-bound phenylalanine could be extracted per gram dry weight of L. gongylophorus grown on Potato Dextrose Agar 50 . Genes encoding enzymes for part of the aromatic amino acid biosynthesis pathway have also been discovered among Klebsiella, Enterobacter, Pantoea and Pseudomonas bacteria in A. colombica fungus gardens 51 , suggesting that bacterial symbionts may contribute phenylalanine to the symbiosis as well.…”