Phylogenetic Diversity of Microbial Isolates

Phylogenetic Diversity of Microbial Isolates Associated with the Phoenix Mission Luke Hecht, Ari Morgenstern, Parag Vaishampayan, James Benardini, Adriana Blachowicz, Wayne Schubert Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA

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P F.W7B-2.2 PF4H_1 PF3H-2 P H O E N IX _ X 5 D -2 PF_4F_5 PF6-4.5 PF3H.3 PF6-4.6 PF1-3.1 PHOENIX_PF4G-2 PF3A-1 PF1-3.2 Abstract PF6-4.3.1 P H O E N I X _ P F 4 A - 3 - 1 PF5-1.2 PF4A-1.2 PF7-1.2 PH01A PF2-12.5.1 PF3-1.1 Planetary protection is concerned with preventing the transfer of biological material between planets. Flight projects are allocated P H O E N I X _ P h - B 5 PF6-4.3.2 PF_3F_2F.5D-1 PHOENIX_PF4J-1-3 PF4H_2.1 PF3-1.4 F.3B-1-1 F.3B-1-2 limits on total bioburden and bioburden density. Compliance is currently determined by a culture-based method that selects for PH04B6_2 PF4J_2.1 PHOENIX_Ph-01B1-2 PH01B1_1 heat-tolerant, heterotrophic aerobes. Microbial contaminants of the Phoenix spacecraft and associated hardware were identified PF4G_3 F.9G-1 19A-1.1 PHOENIX_PF4D-412A-5 PF9-10.2.3 to species level based on 16S rDNA similarity. Most (73%) of the 211 identified isolates were spore-formers, belonging to the PHOENIX_PF4J-1-2 PF4K_1 12A-6.112A-2 PHOENIX_4B-1.1 PHOENIX_PF4D-2 12A-1 PF5-1.1 genera Bacillus (67%) or Paenibacillus (6%). Non-spore-formers were predominately Staphylococcus (20%). Several extreme- PF4-9.1.3 PF4F-3.1 PF3i-1.2 associated species were identified, including stratospheric Bacillus aryabhatti and halophilic facultative anaerobe, Sporosarcina PF3-1.3.3 F.W12B-4PH15A PF8-3.1 aquimarina. Five potentially novel Bacillus species were identified. F.W12B-1.3 PF2-12.4.1 F.W12B-2.1 PF2-12.4.2 PF3-1.3.1 F.W12B-2.2 PF4-9.1.1 PF3i-1.1 PF44A-1 PF3-1.3.2 12A-4 PF4-9.1.2 PF3D-1 PF5-1.6 F.41A-2 PH25A PF44B-1 PHOENIX_Ph-03A5-2-3b Methods PH04A6_1 PHOEXNIX_Ph-04B1-2-2 PF9-10.4.3 PHB9 PF7-1.3.1 PHOENIX_Ph-02-B5-1-2 PHOENIX_Ph-4A Sample preparation PF5-1.5 PF4i_3.2 PF5-1.4 PF9-10.2.1 The spacecraft components were regularly sampled with swabs and wipes during assembly, test and launch operations. PF2-12.5.2 PF5-1.3.2 PHOENIX_Ph-11B PF6-4.7 Cultivation followed the NASA Standard Assay method. Microbes were sonicated off of the swab into DI-water, heat-shocked at PF24A.2.1 PF9-10.1.1 PF9-10.1.2 o p PF24B-2 PF24B-1 PF4i_3.1 80 for 15 minutes, and incubated in Tryptic Soy agar at 32 for 3 days. Isolates were subcultured and stocks frozen in glycerol at - PF5-1.3.1 o o PF24A-2.3 F.W12B-3.1 80 C. Frozen isolates were cultured onto Tryptic Soy Agar plates and incubated at 32 C for at least 48 hours. PF4F-2.1 F.W12B-5.2 PF2-12.2.2 F.W12B-6.2 PF2-12.2.3 F.W12B-6.1 PF4F-2.2 F.W12B-1.2 DNA processing and sequencing PF4A-1.1 F.6F-1.2 PF3-1.5 F.6B-1.2 PHOENIX_X4G-1.2 DNA was extracted using the Maxwell 16 MDx system (Promega) with the Tissue LEV Total RNA Purification Kit. A portion of the PF2-12.2.1 19B-1.2 PH03A1_1.1 F.2B-6 16S ribosomal RNA gene sequence (primers: 8F/1525R) was amplified by polymerase chain reaction (PCR) and sequenced. F.29D-2 F.2B-1-1 18B-2.1 F.4B-2-2 F.2B-5 F.5C-1-2 PHOENIX_PF4i-1-1 F.3E-3-2 Data analysis F.41A-1 F.2G-1-2 PHOENIX_PF4C-1-1 F.29D-1.1 PHOENIX_41A-4.2 F.3E-1-2 To determine the species of a microbial isolate, its 16S rDNA sequence was compared, using BLAST, against homologous PF3L-1 F.4B-2-1 PHOENIX_W2A-1F.23A-1 F.5C-1-1 sequences from known species found in the NCBI GenBank and GreenGenes databases. Quality sequences were aligned and their F.40A-1 F.4B-1-2 F.41A-3 F.5C-2 18B-1.1 F.2B-4 F.5C-3 phylogenetic relationships determined using MEGA 6.0, according to the neighbor-joining method with the Kimura 2-parameter PF9-10.3 F.3E-2-1 F.3E-3-3 F.2B-1-2 substitution model. PF3H-1.2.1 F.3E-1-1 PF4J_2.2 F.3E-3-1 F.2B-2 12L-1.1 F.1A-1 F.W12B-1.1PH26B1 F.1A-2 12L-2 F.6F-1.1 PHOENIX_Ph-02A6-2 F.7G-4.2 F.7G-1.2 F.7G-1.1 10m-1.1 F.4G-1-1 10m-2.2 F.2G-1-1 19B-1.1 10m-2.1 PH20A1 F.7G-2.1 10m-1.2 F.36A-1.1 F.7G-3.1 PF24B-3 PHOENIX_PF4K-2-2 PF24A-3 P F 2 4 A -2 .2 .1

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PH6A1PF4G_4.1 Discussion PH02B5_2 P H O E N IX _P F 4F -1 PH04A6_2

PH02A6_1 PH04B1_3.1 Mars exploration has revealed potential habitable environments, while research into life in extreme environments on Earth has PHOENIX_PF4J-3 PH04B1_2.1

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PH03A6_1 expanded our concept of habitability. The next generation of missions may carry life detection instruments, and have targets with PH03A6_2.1 PHOENIX_Ph-03A2 the potential to support life, such as Europa or the subsurface of Mars. But the scientific integrity of any life detection depends on PhoeniX_Ph-03A6-3

PHOENIX_Ph-03A1-2-1 PHOENIX_Ph-04A3-1 Figure 1: Phylogeny of our ability to avoid – or at least recognize – false positives that may result from terrestrial organisms carried by the spacecraft. Because of the necessary compromise between spacecraft material integrity and bioburden reduction methods, current PHOENIX_Ph-03A5-2-3a Phoenix-associated isolates. technology does not allow for a mission to be completely sterile at launch. Characterizing spacecraft-associated microorganisms is of interest to the field of planetary protection for three reasons: 1) Information on their resilience to sterilization methods, as well as their potential to survive and reproduce under Mars or other planetary conditions, may guide technology development and mission planning. 2) New technologies are being developed to supersede the NASA Standard Assay, but must first be validated against the legacy method. An understanding of the diversity captured by the Standard Assay is therefore desirable for comparison. 3) The archive can serve as a false-positives library in case of candidate life detection, especially for a potential Mars Acknowledgements Sample Return. Funding for this work was provided by NASA through the JPL/Caltech Mars Program Engineering Office, and the California Institute of Technology Summer Undergraduate Research Fellowship (SURF). We would like to thank Aleksandra Checinska for The isolates described here nearly complete the study of archived isolates from the Phoenix mission. In total, good-quality 16S advice and lab support. The authors would also like to acknowledge 2014 summer interns Ryan Hillary, Ryan Hendrickson, Erik rDNA sequences were obtained from 211 isolates. Based on BLAST identification, most of these were spore-forming species, such McFarland and Kyla Bradylong for mutual support and discussion. The authors wish to acknowledge Garrett Smith, as Bacillus (67%) or Paenibacillus (6%). Staphylococcus was common, at 20%. Several extreme-associated species were identified. Nicholas Sanchez, Neil Schwalb, Farah Zerehi and Anabel Carigo for their laboratory and DNA sequence work for Phoenix These included Bacillus aryabhatti, originally described from high-altitude aerial samples, and Sporosarcina aquimarina, a microbial isolates. halophilic facultative anaerobe. Five potentially novel Bacillus species were identified on the basis of <97% sequence identity to The research described here was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a their closest match, with two being highly divergent (93% identity). Future work will characterize the phenotypes of the potentially contract with the National Aeronautics and Space Administration. novel species identified here. © 2014 California Institute of Technology. Government sponsorship acknowledged.