Arc$c Microbes: Popula$on Changes Due to Warming TemperaturesGarre% Taylor1, Daphne Mueller1, Samantha Wade1, Ana Stringer2, Jaime Patkotak2, Olive Kanayurak2, Linda Nicholas-Figueroa2, Joanna Green1, Rebekah Hare1

1University of Alaska, Fairbanks, 2Ilisagvik College

Abstract

Permafrost and Microbes

Experimental Design

Results

MinION sequencing analysis (culture independent) yieldedmostly unclassified reads. One read yielded:

- Pseudomonas brassicacearum is a likely idenVficaVon of oneof the isolated cultures.- P. brassicacearum is a soil bacteria that has been shown tobe a plant pathogen and/or a growth promoter1,2.

Illumina MiSeq BaseSpace 16S analysis; two idenVficaVons each at approximately 2% of total reads:

- Mycobacterium simulans, potenVally pathogenic3

- Chthoniobacter flavus, carbon cycle4 and impacts on climate change.5

Acknowledgments

Sources

Work reported in this publicaVon was supported by the NaVonal InsVtute of General Medical Sciences of the NaVonal InsVtutes of Health under three linked awards numberRL5GM118990, TL4 GM 118992 and 1UL1GM118991. The work is solely the responsibility of the authors and does not necessarily represent the official view of the NaVonal InsVtutes ofHealth. This material is based upon work supported by the NaVonal Science FoundaVon under Grant No. 1622418.

1. Ortet, P., Barakat, M., Lalaouna, D., Fochesato, S., Barbe, V., Vacherie, B., & Achouak, W. (2011). “Complete Genome Sequence of a Beneficial Plant Root-Associated Bacterium, Pseudomonas brassicacearum.” Journal of Bacteriology, 193(12): 3146. 2. TianVan, Z., Chen, D., Li, C., Sun, Q., Li, L., Liu, F., Shen, Q., & Shen, B. (2012) “IsolaVon and characterizaVon of Pseudomonas brassicacearum J12 as an antagonist against Ralstonia solanacearum and idenVficaVon of its anVmicrobial components.” Microbiological Research, 167(7): 388-394.3. Tortoli E, Rogasi PG, Fantoni E, Beltrami C, De Francisci A, & Mariorni, A. (2010). “InfecVon due to a novel mycobacterium, mimicking mulVdrug-resistant Mycobacterium tuberculosis.” Clinical Microbiology & InfecCon, 14:1130–1134.4. Sangwan, P., Chen, X., Hugenholtz, P., & Janssen, P.H. “ChthoniobacterFlavus Gen. Nov., Sp. Nov., the First Pure-Culture RepresentaVve of Subdivision Two, Spartobacteria Classis Nov., of the Phylum Verrucomicrobia.” (2004). Applied & Environmental Microbiology, 70(10 ): 5875–81.5. Victoria, R., Banwart, S.A., Black, H., Ingram, J., Joosten, H., Milne, E., & Noellemeyer, E. (2012). Benefits of soil carbon. Foresight Chapter in UNEP

Yearbook 2012. United NaCons Environment Programme: 19–33.

Figure 1. Coastal erosion in Shishmareff, Alaska, (pubs.usgs.org)

Figure 3.Bacteria in ancient permafrost, (geocryology.com)

Figure 7. Streak plate of a bacterial culture

Figure 5. MinIONflow cell

Figure 2. Ice cellar from Barrow, Alaska, photo by KaVe Orlinsky

Figure 6. Nanopore Sequencing

Figure 4. Soil Samples, photo by Ana Stringer

Culture Independent

Culture Dependent

Soil

Culture Independent

Isolate Colonies

DNA ExtracVons

Illumina MiSeq

MinION

Culture Dependent

DNA Extractions

Methods

- Soil samples from organic layer (2 cm depth), topsoil (10 cm), and permafrost (uppermost horizon) were taken from Barrow Environmental Observatory, Utqiaġvik, AK and stored at -80∘ C. - Culture Independent: DNA samples were extracted directly from the soil using Mo Bio PowerMax or Qiagen Soil IsolaVon DNA Kits. - Culture Dependent: Bacteria were cultured directly from the soil using media types (EMB, BHI, TSA, SDA, CZD, NA), streaked to obtain isolated colonies and DNA obtained using Mo bio or Qiagen Microbial DNA extracVon kits. ConcentraVon and purity were measured using a Nanodrop OneCspectrophotometer. - DNA was sequenced and analyzed with MinION NGS and EPI2ME real Vme analyVcal workflow or sent to the University of Alaska Fairbanks CORE Lab for Illumina MiSeq NGS and BaseSpace 16S analysis.

The effects of global warming are most profound in the ArcVc,ranging from the rate of sea-ice decline, melVng permafrost, andmigraVon changes in plants and animals. In addiVon, an increase isexpected in exisVng and invasive microorganisms, which can haveadverse effects on the local food chain. We have hypothesized thatArcVc warming will conVnue to affect arcVc microorganisms. It isspeculated that changes within the arcVc microbial composiVonwill be followed by changes in arcVc vegetaVon, which willulVmately affect arcVc plants, animals, and the subsistence lifestyleof Alaska NaVves in rural villages. The proposed hypothesis istested via measurements of microbial community composiVon inArcVc soils.

ConclusionThese results reinforce the need for further research into microbial dynamics associated with climate changes in the ArcVc.