Discipline: Biological Sciences
Cristina Gonzalez - California State University, San Bernardino
Co-Author(s): Michelle Miranda, Lizett J. Gonzalez, Christian Sam, Senthil K. Murugapiran, Brian P. Hedlund, and Jeremy A. Dodsworth
Aigarchaeota is a deeply branching, thermophilic clade within the Domain Archaea that has no cultivated representatives. It is a member of the TACK superphylum, which may represent a sister lineage to the progenitor of eukaryotes. Based on 16S rRNA gene phylogenies, 9 genus level groups have been detected within Aigarchaeota so far. This work focuses on one of these groups, Group 4 Aigarchaeota (Aig G4), with the following goals: definition and analysis of an Aig G4 metagenome bin, and attempting to cultivate Aig G4 in the laboratory. Previous metagenomic work has created predictions on the biological function of Aigarchaeota group 4 (Aig G4), but like many uncultivated microbes, these predictions have yet to be confirmed. Aig G4 was found to be relatively abundant in in situ lignocellulose (corn stover) enrichments established at ~85°C in Great Boiling Spring (GBS), NV. A metagenome derived from the GBS in situ enrichments, yielded an Aig G4 metagenome bin ~1.48 Mb in size and an estimated completeness of 90%. Based on this metagenome, Aig G4 is predicted to be a fermenter of sugars and/or amino acids. Laboratory cultures were designed in an attempt to create an ideal environment to domesticate Aig G4. In total, 12 laboratory enrichment cultures were designed and inoculated in the field with in situ lignocellulose (corn stover) enrichments at GBS hot spring. In the lab, these cultures were incubated anaerobically at ~80°C and transferred every three weeks. Quantitative polymerase chain reaction (qPCR) was used to detect Aig G4 using sequence specific primers. Universal primers were also used to determine the relative abundance of Aig G4 compared to bacteria and other archaea present. Two of 12 original laboratory cultures had a relative abundance of 1-5% and 106 gene rRNA gene copies/mL culture according to quantitative PCR assays. These cultures were maintained at a temperature 80°C on semi-synthetic media containing GBS spring water and the major growth substrates: corn stover, keratin, and xyloglucan. These are, to our knowledge, the first established AigG4 enrichment cultures. Growth was not detected in media conditions that contained thiosulfate and nitrate (100 µM), suggesting that either substrate might be toxic to Aig G4. Successful cultures will be used in future experiments to determine what component(s) of GBS spring water is useful to Aig G4 and to test if thiosulfate or nitrate is toxic to Aig G4. Fluorescent in situ hybridization (FISH)-nanosims and isotope labeled substrates will be used to further examine Aig G4 metabolism.
Funder Acknowledgement(s): US NSF grant DEB-155704; NASA Exobiology grant EXONNX11AR78G; US-DOE grant DE-EE-0000716; Nevada Renewable Energy Consortium funded by the DOE; Joint Genome Institute (JGI) at the DOE (CSP-182); Partially supported by LSAMP NSF grant number HRD-1302873. NASA Exobiology grant EXONNX11AR78G US-DOE grant DE-EE-0000716 Nevada Renewable Energy Consortium funded by the DOE Joint Genome Institute (JGI) at the DOE (CSP-182) Partially supported by LSAMP NSF grant number HRD-1302873.
Faculty Advisor: Jeremy Dodsworth, JDodsworth@csusb.edu
Role: For this research project I worked on the cultivation and detection of Aig G4. These cultures were originally collected in the filed by Michelle Mirranda and our PI Professor Dodsworth at Great Boiling Spring, NV during March 2016. Since then, I have continued to help maintain these cultures. For this project, I also work alongside Professor Dodsworth to interpret our qPCR results and determine the relative abundance of Aig G4 in our cultures. I have begun analyzing the Aig G4 metagenome under the guidance of Professor Dodsworth. Identifying what genes are present will be useful in helping us determine the metabolism of Aig G4. In the future we will use isotope labels and FISH-nanosims to further understand Aig G4.