Tungsten is Required for Lab Maintenance of Aigarchaeota Group 4

SMSU Event Center BC

Aigarchaeota, a major group of microorganisms that are related to the ancestors of Eukaryotes, is found in geothermal and hydrothermal systems worldwide and consists of 9 genus-level groups. One of them, Aigarchaeota Group 4 (AigG4), was previously found to be abundant in corn stover incubations in Great Boiling Spring (GBS). This incubation sample was introduced into culture media containing corn stover or other substrates under a variety of conditions. Initially, AigG4 was only observed in culture media containing GBS water. AigG4 reconstructed genomes from environmental and laboratory samples contained multiple genes coding for hypothesized tungsten-utilizing enzymes, and ~1 µM tungsten was previously detected in GBS spring water, leading to the hypothesis that tungsten may be a necessary nutrient for AigG4. To test this, cultures containing AigG4 were introduced into an entirely artificial culture medium with corn stover in the absence or presence of 0.1 µM or 2 µM sodium tungstate. After two transfers in these media, AigG4 abundance in artificial medium with added tungstate was 2.8±1.0 x 10^6 16S rRNA gene copies/mL as determined by qPCR, similar to values in spring water medium positive controls with or without added tungsten (4.3±2.5 x 10^6 16S rRNA gene copies/mL), but AigG4 was below the level of detection (10^4 16S rRNA gene copies/mL) without added tungsten. Furthermore, expression of several of the AigG4 tungsten-utilizing enzymes were detected in the laboratory cultures using reverse transcriptase PCR. These results strongly suggest that tungsten is required for laboratory cultivation of AigG4.