Applied Microbiology and Biotechnology

, Volume 103, Issue 11, pp 4483–4497 | Cite as

Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station

  • Nitin Kumar Singh
  • Jason M. Wood
  • Snehit S. Mhatre
  • Kasthuri VenkateswaranEmail author
Genomics, transcriptomics, proteomics


Several evolutionarily distinct, near full-length draft metagenome-resolved genomes (MRG), were assembled from sequences recovered from the International Space Station (ISS) environments. The retrieval of MRGs facilitated the exploration of a large collection of archived strains (~ 500 isolates) and assisted in isolating seven related strains. The whole genome sequences (WGS) of seven ISS strains exhibited 100% identity to the 4.85 × 106 bp of four MRGs. The “metagenome to phenome” approach led to the description of a novel bacterial genus from the ISS samples. The phylogenomics and traditional taxonomic approaches suggested that these seven ISS strains and four MRGs were not phylogenetically affiliated to any validly described genera of the family Erwiniaceae, but belong to a novel genus with the proposed name Kalamiella. Comparative genomic analyses of Kalamiella piersonii strains and MRGs showed genes associated with carbohydrate (348 genes), amino acid (384), RNA (59), and protein (214) metabolisms; membrane transport systems (108), pathways for biosynthesis of cofactors, vitamins, prosthetic groups, and pigments (179); as well as mechanisms for virulence, disease, and defense (50). Even though Kalamiella genome annotation and disc diffusion tests revealed multidrug resistance, the PathogenFinder algorithm predicted that K. piersonii strains are not human pathogens. This approach to isolating microbes allows for the characterization of functional pathways and their potential virulence properties that can directly affect human health. The isolation of novel strains from the ISS has broad applications in microbiology, not only because of concern for astronaut health but it might have a great potential for biotechnological relevance. The metagenome to phenome approach will help to improve our understanding of complex metabolic networks that control fundamental life processes under microgravity and in deep space.


Kalamiella piersonii Metagenome-resolved genomes Genome-inferred phenotype Phylogenomics MLSA 



We thank Aleksandra Checinska Sielaff for isolating the ISS strains, Arman Seuylemezian for MALDI-TOF analysis. Ganesh Babu Malli Mohan, Cynthia Ly, and Tristan Grams for their technical help rendered in reviving and isolating DNA of the ISS strains when needed. © 2019 California Institute of Technology. Government sponsorship is acknowledged.

Authors’ contributions

KV and NKS conceived and designed the experiments. NKS, JMW, and SSM performed the experiments. NKS analyzed the data. JMW performed analysis of the de novo assemblies, including contig alignment, and annotation checks. SSM carried out the phenotypic assays Biolog-based biochemical characterization. NKS, JMW, SSM, and KV wrote the paper. All authors read and approved the final manuscript.


Part of the research described in this publication was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with National Aeronautics and Space Administration. This research was funded by a 2012 Space Biology NNH12ZTT001N grant no. 19-12829-26 under Task Order NNN13D111T award to KV, which also funded the post-doctoral fellowships for NKS, JMW and SSM.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Not applicable.

Supplementary material

253_2019_9813_MOESM1_ESM.pdf (565 kb)
ESM 1 (PDF 565 kb)


  1. Adeolu M, Alnajar S, Naushad S, SG R (2016) Genome-based phylogeny and taxonomy of the ‘Enterobacteriales’: proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov. Int J Syst Evol Microbiol 66(12):5575–5599. Google Scholar
  2. Aksoy S (1995) Wigglesworthia gen. nov. and Wigglesworthia glossinidia sp. nov., taxa consisting of the mycetocyte-associated, primary endosymbionts of tsetse flies. Int J Syst Bacteriol 45(4):848–851. Google Scholar
  3. Andersen MH, McIlroy SJ, Nierychlo M, Nielsen PH, Albertsen M (2019) Genomic insights into Candidatus Amarolinea aalborgensis gen. Nov., sp. nov., associated with settleability problems in wastewater treatment plants. Syst Appl Microbiol 42(1):77–84. Google Scholar
  4. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75. Google Scholar
  5. Barber MF, Elde NC (2015) Buried treasure: evolutionary perspectives on microbial iron piracy. Trends Genet 31(11):627–636. Google Scholar
  6. Bargoma E, La Duc MT, Kwan K, Vaishampayan P, Venkateswaran K (2013) Differential recovery of phylogenetically disparate microbes from spacecraft-qualified metal surfaces. Astrobiology 13(2):189–202. Google Scholar
  7. Barnum TP, Figueroa IA, Carlström CI, Lucas LN, Engelbrektson AL, Coates JD (2018) Genome-resolved metagenomics identifies genetic mobility, metabolic interactions, and unexpected diversity in perchlorate-reducing communities. ISME J 12(6):1568–1581. Google Scholar
  8. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. Google Scholar
  9. Bonetta S, Pignata C, Bonetta S, Meucci L, Giacosa D, Marino E, Gilli G, Carraro E (2017) Viability of Legionella pneumophila in water samples: a comparison of Propidium Monoazide (PMA) treatment on membrane filters and in liquid. Int J Environ Res Public Health 14(5):467. Google Scholar
  10. Bowers RM, Kyrpides NC, Stepanauskas R, Harmon-Smith M, Doud D, Reddy TBK, Schulz F, Jarett J, Rivers AR, Eloe-Fadrosh EA, Tringe SG, Ivanova NN, Copeland A, Clum A, Becraft ED, Malmstrom RR, Birren B, Podar M, Bork P, Weinstock GM, Garrity GM, Dodsworth JA, Yooseph S, Sutton G, Glockner FO, Gilbert JA, Nelson WC, Hallam SJ, Jungbluth SP, Ettema TJG, Tighe S, Konstantinidis KT, Liu WT, Baker BJ, Rattei T, Eisen JA, Hedlund B, McMahon KD, Fierer N, Knight R, Finn R, Cochrane G, Karsch-Mizrachi I, Tyson GW, Rinke C, Lapidus A, Meyer F, Yilmaz P, Parks DH, Eren AM, Schriml L, Banfield JF, Hugenholtz P, Woyke T (2017) Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea. Nat Biotechnol 35(8):725–731. Google Scholar
  11. Braymer JJ, Giedroc DP (2014) Recent developments in copper and zinc homeostasis in bacterial pathogens. Curr Opin Chem Biol 19:59–66. Google Scholar
  12. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421. Google Scholar
  13. Checinska A, Probst AJ, Vaishampayan P, White JR, Kumar D, Stepanov VG, Fox GE, Nilsson HR, Pierson DL, Perry J, Venkateswaran K (2015) Microbiomes of the dust particles collected from the International Space Station and Spacecraft Assembly Facilities. Microbiome 3(1).
  14. Checinska Sielaff A, Kumar RM, Pal D, Mayilraj S, Venkateswaran K (2017) Solibacillus kalamii sp. nov., isolated from a high-efficiency particulate arrestance filter system used in the International Space Station. Int J Syst Evol Microbiol 67(4):896–901. Google Scholar
  15. Chen C, Xin K, Liu H, Cheng J, Shen X, Wang Y, Zhang L (2017) Pantoea alhagi, a novel endophytic bacterium with ability to improve growth and drought tolerance in wheat. Sci Rep 7:41564. Google Scholar
  16. Chun J, Rainey FA (2014) Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea. Int J Syst Evol Microbiol 64(2):316–324. Google Scholar
  17. Cosentino S, Voldby Larsen M, Møller Aarestrup F, Lund O (2013) PathogenFinder—distinguishing friend from foe using bacterial whole genome sequence data. PLoS One 8(10):e77302. Google Scholar
  18. Crucian B, Babiak-Vazquez A, Johnston S, Pierson DL, Ott CM, Sams C (2016) Incidence of clinical symptoms during long-duration orbital spaceflight. Int J Gen Med 9:383–391. Google Scholar
  19. Doijad S, Imirzalioglu C, Yao Y, Pati NB, Falgenhauer L, Hain T, Foesel BU, Abt B, Overmann J, Mirambo MM, Mshana SE, Chakraborty T (2016) Enterobacter bugandensis sp. nov., isolated from neonatal blood. Int J Syst Evol Microbiol 66(2):968–974. Google Scholar
  20. Felsenstein J (2004) PHYLIP (phylogeny inference package), version 3.6, vol 5, distributed by the author. Department of Genome Sciences, University of Washington, SeattleGoogle Scholar
  21. Galtier N, Gouy M, Gautier C (1996) SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12(6):543–548Google Scholar
  22. Gardan L, Christen R, Achouak W, Prior P (2004) Erwinia papayae sp. nov., a pathogen of papaya (Carica papaya). Int J Syst Evol Microbiol 54(Pt 1):107–113. Google Scholar
  23. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57(1):81–91. Google Scholar
  24. Gurevich A, Saveliev V, Vyahhi N, Tesler G (2013) QUAST: quality assessment tool for genome assemblies. Bioinformatics 29(8):1072–1075. Google Scholar
  25. Halpern M, Fridman S, Aizenberg-Gershtein Y, Izhaki I (2013) Transfer of Pseudomonas flectens Johnson 1956 to Phaseolibacter gen. nov., in the family Enterobacteriaceae, as Phaseolibacter flectens gen. nov., comb. nov. Int J Syst Evol Microbiol 63(1):268–273.
  26. Hauben L, Moore ER, Vauterin L, Steenackers M, Mergaert J, Verdonck L, Swings J (1998) Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst Appl Microbiol 21(3):384–397. Google Scholar
  27. Hollis DG, Hickman FW, Fanning GR, Farmer JJ, Weaver RE, Brenner DJ (1981) Tatumella ptyseos gen. nov., sp. nov., a member of the family Enterobacteriaceae found in clinical specimens. J Clin Microbiol 14(1):79–88Google Scholar
  28. Jones DM (1981) Manual of methods for general bacteriology. J Clin Pathol 34(9):1069–1069Google Scholar
  29. Kang DD, Froula J, Egan R, Wang Z (2015) MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities. PeerJ 3:e1165. Google Scholar
  30. Kibbee RJ, Örmeci B (2017) Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent. J Microbiol Methods 132:139–147. Google Scholar
  31. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62(Pt 3):716–721. Google Scholar
  32. Krishnamurthi S, Chakrabarti T, Stackebrandt E (2009) Re-examination of the taxonomic position of Bacillus silvestris Rheims et al. 1999 and proposal to transfer it to Solibacillus gen. nov. as Solibacillus silvestris comb. nov. Int J Syst Evol Microbiol 59(Pt 5:1054–1058. Google Scholar
  33. La Duc MT, Satomi M, Agata N, Venkateswaran K: gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group. J Microbiol Methods 2004, 56(3):383–394Google Scholar
  34. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Ponten T, Ussery DW, Aarestrup FM, Lund O (2012) Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 50(4):1355–1361. Google Scholar
  35. Leon MJ, Fernandez AB, Ghai R, Sanchez-Porro C, Rodriguez-Valera F, Ventosa A (2014) From metagenomics to pure culture: isolation and characterization of the moderately halophilic bacterium Spiribacter salinus gen. nov., sp. nov. Appl Environ Microbiol 80(13):3850–3857. Google Scholar
  36. Meier-Kolthoff JP, Auch AF, Klenk H-P, Goker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60. Google Scholar
  37. Mergaert J, Hauben L, Cnockaert MC, Swings J (1999) Reclassification of non-pigmented Erwinia herbicola strains from trees as Erwinia billingiae sp. nov. Int J Syst Bacteriol 49(Pt 2):377–383. Google Scholar
  38. Müller KD, Schmid EN, Kroppenstedt RM (1998) Improved identification of mycobacteria by using the microbial identification system in combination with additional trimethylsulfonium hydroxide pyrolysis. J Clin Microbiol 36(9):2477–2480Google Scholar
  39. Munson MA, Baumann P, Kinsey MG (1991) Buchnera gen. nov. and Buchnera aphidicola sp. nov., a taxon consisting of the mycetocyte-associated, primary endosymbionts of aphids. Int J Syst Evol Microbiol 41(4):566–568. Google Scholar
  40. Nies DH (1992) Resistance to cadmium, cobalt, zinc, and nickel in microbes. Plasmid 27(1):17–28. Google Scholar
  41. Norgan AP, Freese JM, Tuin PM, Cunningham SA, Jeraldo PR, Patel R (2016) Carbapenem- and colistin-resistant Enterobacter cloacae from Delta, Colorado, in 2015. Antimicrob Agents Chemother 60(5):3141–3144. Google Scholar
  42. Nurk S, Bankevich A, Antipov D, Gurevich A, Korobeynikov A, Lapidus A, Prjibelsky A, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, McLean J, Lasken R, Clingenpeel SR, Woyke T, Tesler G, Alekseyev MA, Pevzner PA (2013) Assembling genomes and mini-metagenomes from highly chimeric reads. Research in Computational Molecular Biology. Springer, Berlin, p 158–170Google Scholar
  43. Nurk S, Meleshko D, Korobeynikov A, Pevzner PA (2017) metaSPAdes: a new versatile metagenomic assembler. Genome Res 27(5):824–834. Google Scholar
  44. Palmer M, Steenkamp ET, Coetzee MPA, Avontuur JR, Chan WY, van Zyl E, Blom J, Venter SN (2018) Mixta gen. nov., a new genus in the Erwiniaceae. Int J Syst Evol Microbiol 68(4):1396–1407. Google Scholar
  45. Pandey KK, Mayilraj S, Chakrabarti T (2002) Pseudomonas indica sp. nov., a novel butane-utilizing species. Int J Syst Evol Microbiol 52(Pt 5:1559–1567. Google Scholar
  46. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW (2015) CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25(7):1043–1055. Google Scholar
  47. Parks DH, Rinke C, Chuvochina M, Chaumeil PA, Woodcroft BJ, Evans PN, Hugenholtz P, Tyson GW (2017) Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life. Nat Microbiol 2(11):1533–1542. Google Scholar
  48. Patel RK, Jain M (2012) NGS QC toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 7(2):e30619. Google Scholar
  49. Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS One 5(3):e9490. Google Scholar
  50. Rezzonico F, Smits THM, Born Y, Blom J, Frey JE, Goesmann A, Cleenwerck I, de Vos P, Bonaterra A, Duffy B, Montesinos E (2016) Erwinia gerundensis sp. nov., a cosmopolitan epiphyte originally isolated from pome fruit trees. Int J Syst Evol Microbiol 66(3):1583–1592. Google Scholar
  51. Roach DJ, Burton JN, Lee C, Stackhouse B, Butler-Wu SM, Cookson BT, Shendure J, Salipante SJ (2015) A year of infection in the intensive care unit: prospective whole genome sequencing of bacterial clinical isolates reveals cryptic transmissions and novel microbiota. PLoS Genet 11(7):e1005413. Google Scholar
  52. Sangwan N, Xia F, Gilbert JA (2016) Recovering complete and draft population genomes from metagenome datasets. Microbiome 4(1):8. Google Scholar
  53. Schumann P, Maier T (2014) Chapter 13—MALDI-TOF mass spectrometry applied to classification and identification of Bacteria. In: Michael Goodfellow IS, Jongsik C (eds) Methods in microbiology, volume 41. Academic Press, pp 275–306Google Scholar
  54. Seuylemezian A, Aronson HS, Tan J, Lin M, Schubert W, Vaishampayan P (2018) Development of a custom MALDI-TOF MS database for species-level identification of bacterial isolates collected from spacecraft and associated surfaces. Front Microbiol 9(780):780. Google Scholar
  55. Seuylemezian A, Singh NK, Vaishampayan P, Venkateswaran K (2017) Draft genome sequence of Solibacillus kalamii, isolated from an air filter aboard the International Space Station. Genome Announc 5(35):e00696–e00617. Google Scholar
  56. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. Google Scholar
  57. Singh NK, Bezdan D, Sielaff AC, Wheeler K, Mason CE, Venkateswaran K (2018a) Multi-drug resistant Enterobacter bugandensis species isolated from the International Space Station and comparative genomic analyses with human pathogenic strains. BMC Microbiol 18(1):175. Google Scholar
  58. Singh NK, Blachowicz A, Checinska A, Wang C, Venkateswaran K (2016) Draft genome sequences of two Aspergillus fumigatus strains, isolated from the International Space Station. Genome Announc 4(4):e00553–e00516. Google Scholar
  59. Singh NK, Wood JM, Karouia F, Venkateswaran K (2018b) Succession and persistence of microbial communities and antimicrobial resistance genes associated with International Space Station environmental surfaces. Microbiome 6(1):214. Google Scholar
  60. Skerman VBD (1967) A guide to the identification of the genera of bacteria, 2nd edn. Williams & Wilkins, BaltimoreGoogle Scholar
  61. Stackebrandt E, Ebers J (2006) Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 33(4):152–155Google Scholar
  62. Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321–346. Google Scholar
  63. Stewart RD, Auffret MD, Warr A, Wiser AH, Press MO, Langford KW, Liachko I, Snelling TJ, Dewhurst RJ, Walker AW, Roehe R, Watson M (2018) Assembly of 913 microbial genomes from metagenomic sequencing of the cow rumen. Nat Commun 9(1):870. Google Scholar
  64. Suzuki MT, Beja O, Taylor LT, Delong EF (2001) Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton. Environ Microbiol 3(5):323–331Google Scholar
  65. Tracz DM, Gilmour MW, Mabon P, Beniac DR, Hoang L, Kibsey P, Van Domselaar G, Tabor H, Westmacott GR, Corbett CR et al: Tatumella saanichensis sp. nov., isolated from a cystic fibrosis patient. Int J Syst Evol Microbiol 2015, 65(Pt 6):1959–1966Google Scholar
  66. Troxell B, Hassan HM (2013) Transcriptional regulation by ferric uptake regulator (fur) in pathogenic bacteria. Front Cell Infect Microbiol 3:59. Google Scholar
  67. Urbaniak C, Sielaff AC, Frey KG, Allen JE, Singh N, Jaing C, Wheeler K, Venkateswaran K (2018) Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces. Nat Sci Rep 8(1):814. Google Scholar
  68. Uritskiy GV, DiRuggiero J, Taylor J (2018) MetaWRAP-a flexible pipeline for genome-resolved metagenomic data analysis. Microbiome 6(1):158. Google Scholar
  69. Vaishampayan P, Probst AJ, La Duc MT, Bargoma E, Benardini JN, Andersen GL, Venkateswaran K (2013) New perspectives on viable microbial communities in low-biomass cleanroom environments. ISME J 7(2):312–324. Google Scholar
  70. Venkateswaran K (2017) Team I-MOE Microbial Characteristics of ISS Environmental Surfaces. In: 47th International Conference on Environmental Systems; ICES-2017-177, Charleston, South CarolinaGoogle Scholar
  71. Vesper S, McKinstry C, Hartmann C, Neace M, Yoder S, Vesper A (2008) Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA). J Microbiol Methods 72(2):180–184. Google Scholar
  72. Walterson AM, Stavrinides J (2015) Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiol Rev 39(6):968–984. Google Scholar
  73. Weinmaier T, Probst AJ, Duc MT, Ciobanu D, Cheng JF, Ivanova N, Rattei T, Vaishampayan P (2015) A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses. Microbiome 3:6. Google Scholar
  74. Wragg P, Randall L, Whatmore AM (2014) Comparison of Biolog GEN III MicroStation semi-automated bacterial identification system with matrix-assisted laser desorption ionization-time of flight mass spectrometry and 16S ribosomal RNA gene sequencing for the identification of bacteria of veterinary interest. J Microbiol Methods 105:16–21. Google Scholar
  75. Xu P, Li WJ, Tang SK, Zhang YQ, Chen GZ, Chen HH, Xu LH, Jiang CL: Naxibacter alkalitolerans gen. nov., sp. nov., a novel member of the family 'Oxalobacteraceae' isolated from China. Int J Syst Evol Microbiol 55(Pt 3):1149–1153Google Scholar

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© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply  2019

Authors and Affiliations

  1. 1.Jet Propulsion Laboratory, Biotechnology and Planetary Protection Group, M/S 89-2California Institute of TechnologyPasadenaUSA

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