Fungus-growing ants (tribe Attini) form symbioses with a cultivar fungus (Leucoagaricus) that they grow in underground fungus gardens as their essential food source (1).
Other bacteria also inhabit these fungus gardens and provide nutrients to the cultivar fungus, at least in some cases (2)(3)(4)(5). These bacteria have the genetic potential to produce secondary metabolites that may mediate interspecific interactions in fungus gardens, although this remains poorly understood (6).
Trachymyrmex septentrionalis is the northernmost fungus-growing ant, occurring throughout the Eastern USA (7). Its colonies are relatively small (~1000 ants/colony) and subsist largely on caterpillar frass, oak catkins, and some fresh plant material (8). The T. septentrionalis fungus garden microbiome remains poorly characterized (9). We therefore isolated several bacteria from T. septentrionalis fungus gardens and sequenced their genomes to better understand their potential functions within this symbiotic niche.
T. septentrionalis fungus gardens were collected in Florida, New Jersey, and North Carolina following established protocols (10). Fungus garden fragments were resuspended in phosphate-buffered saline (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4) and bacteria were isolated on tryptic soy agar (Difco; adjusted to pH 6) using the spread plate technique. Genomic DNA was extracted from each isolate and their 16S rRNA gene was PCR amplified as described previously (11). PCR amplicons were Sanger sequenced at the University of Connecticut DNA Biotechnology Center and the resulting sequences were compared to the NCBI nr database (12) to identify each strain.
Genomes from eight T. septentrionalis fungus garden bacteria were sequenced at the Department of Energy Joint Genome Institute (JGI). Pacific Biosciences (PacBio) SMRTbell libraries were constructed following the manufacture's protocols and sequenced using a PacBio RS instrument. The resulting reads were assembled using the HGAP pipeline (v2.3.0_p5). Genes were predicted using Prodigal (13) and GenePRIMP (14), and annotated using the IMG nr (15), UniProt (16), TIGRFam (17), Pfam (18), KEGG (19), COG (20), and InterPro (21) databases.
Non-coding RNAs were annotated using tRNAScanSE (22), INFERNAL (23), and the IMG's ribosomal RNA gene models (15). Additional gene prediction and annotation was performed using the JGI's IMG ER platform (24).
The sequenced bacteria belong to the genera Bacillus, Burkholderia, Micrococcus, Pantoea, and Serratia, and a poorly resolved taxon within the Enterobacteriaceae (Table 1). The genome of Serratia sp. JKS000199 was assembled into a single contig and is therefore complete.
All other genomes were assembled into 2-6 contigs and are therefore high-quality drafts. These bacteria likely include both persistent and transient colonists of T. septentrionalis fungus gardens. Their genomes will inform future studies of these strains' ecology in the T. septentrionalis symbiosis and how secondary metabolites might mediate interspecific interactions within this niche.