The ‘Great Plate Anomaly’ described by Staley and Konopka has presented two significant obstacles in the field of microbiology, (1) to cultivate as well as characterize all of the microbial diversity within the natural environment as individual species, the uncultivated organisms being ‘microbial dark matter’, and (2) as a community, such as the microbiome. Importantly, the characterization of bacterial organisms involved in human health and disease is of particular importance. To further illuminate these implicated ‘microbial dark matter’, a Schaalia odontolytica (formerly Actinomyces odontolyticus subsp. actinosynbacter) strain, XH001, the bacterial basibiont, was recently co-cultivated from the human oral cavity along with its epibiont, a Nanosynbacter lyticus type strain TM7x (HMT_952), which thrives on surface of XH001, for characterization. Surprisingly, TM7x, is a member of the Saccharibacteria phylum (formerly TM7 phylum) that is part of a new bacterial lineage denoted as the Candidate Phyla Radiation (CPR), which may comprise of more than 25 percent of all bacterial diversity.
To determine genetic mediators in their relationship, RNA-seq was performed and found the highest upregulated gene in TM7x-asssociated XH001 versus XH001 to be lsrB, the receptor for the Auto-Inducer-2 molecule. We confirmed the lsrB upregulation via quantitative real time PCR from the transcriptomic analysis and developed a genetic modification system to create deletion mutants to elucidate the genetic determinants involved in the establishment of XH001 and the association with its parasitic epibiont, TM7x. We targeted the lsrB and luxS (encodes AI-2 synthase) homologues in XH001 and successfully generated an XH001ΔlsrB and XH001ΔluxS single deletion mutants. Phenotypic analyses provided data suggesting that the association with TM7x enhances wildtype XH001’s biofilm formation capability in a lsrB and luxS dependent manner. Herein we also present an established genetic system, including a high-throughput mutagenesis platform (Tn-seq), that enabled a full conditionally essential gene study of XH001 and evaluation of the genetic mediators during the TM7x-associated parasitic event with XH001 in real time. This essential gene study revealed 10.5% of genome to be necessary for growth and elucidated several genes in XH001 directly involved in the epibiotic-parasitic relationship between TM7x and XH001.
Bridging the second knowledge-gap of microbial community dynamics stated above, the oral and gut microbiome of BALB/c mice as part of NASA’s Rodent Research 5 mission were assessed for longitudinal changes upon returning to earth for the first time in US/NASA history as well as the effects of long duration microgravity exposure. Intriguingly, overall microbial diversity was shown to be increased in microgravity conditions, as corroborated by recent human astronaut studies, and found to reduce to pre-flight levels, albeit permanently shifted away from baseline upon returning to earth as show in this study. Furthermore, Lactobacillus murinus and Dorea spp. were enriched, along with functionally assigned metabolic pathways involved with short-chain fatty acid production, in microgravity compared to terrestrial ground controls revealing a potential mechanism for mediating bone homeostasis.