Halorubrum amylolyticum sp. nov., a novel halophilic archaeon isolated from a salt mine


A pleomorphic and non-motile halophilic archaeon forming light-red pigmented colonies, strain ZC67T, was isolated from the Yuanyongjing Salt Mine, Yunnan, China. Based on similarity search and phylogenetic analysis of the 16S rRNA gene sequence, strain ZC67T belongs to the genus Halorubrum and is closely related to the species of Halorubrum (Hrr.) saccharovorum JCM 8865T, Hrr. persicum C49T, Hrr. halophilum B8T, Hrr. lipolyticum 9-3T, Hrr. salsamenti Y69T and Hrr. depositum Y78T with 16S rRNA gene sequence similarities of 99.0%, 98.7%, 98.5%, 98.4%, 98.1% and 97.7%, respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain ZC67T and its close relatives were less than 90.5% and 89.3%, respectively. In silico DNA-DNA hybridization (DDH) analysis showed that DNA-DNA relatedness between strain ZC67T and its relatives is less than 45%. Values of ANI, AAI and in silico DDH were clearly below the thresholds used for the delineation of a new species. The major polar lipids of strain ZC67T were similar to other neutrophilic members in the genus Halorubrum containing phosphatidylglycerol, phosphatidylglycerolphosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl-glucosyl-glycerol diether-1. The DNA G+C content was determined to be 66.3 mol% (based on the draft genome). Combined with other diagnostic characteristics, e.g. phenotypic and chemotaxonomic differences, strain ZC67T is concluded to represent a novel species in the genus Halorubrum, for which the name Halorubrum amylolyticum sp. nov. is proposed. The type strain is ZC67T (=CGMCC 1.15718T = JCM 31850T).

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2-Morpholinoethanesulfonic acid


1, 4-Piperazine bis (ethanesulfonic acid)


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid


2-(Cyclohexylamino) ethanesulfonic acid




China General Microbiological Culture Collection Center


Japan Collection of Microorganisms


International Unit of enzyme activity


Molecular Evolutionary Genetics Analysis


  1. Auch AF, von Jan M, Klenk HP, Göker M (2010) Digital DNA–DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2:117–134

    Article  Google Scholar 

  2. Bankevich A, Nurk S, Antipov D, Gurevich AA, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477

    CAS  Article  Google Scholar 

  3. Boucher Y, Douady CJ, Sharma AK, Kamekura M, Doolittle WF (2004) Intragenomic heterogeneity and intergenomic recombination among haloarchaeal rRNA genes. J Bacteriol 186:3980–3990

    CAS  Article  Google Scholar 

  4. Chen S, He J, Zhang J, Xu Y, Huang J, Ke LX (2017a) Halorubrum salsamenti sp. nov., a novel halophilic archaeon isolated from a brine of salt mine. Curr Microbiol 74:1358–1364

    CAS  Article  Google Scholar 

  5. Chen S, Xu Y, Ke LX (2017b) Halorubrum trueperi sp. nov., a halophilic archaeon isolated from a salt mine. Int J Syst Evol Microbiol 67:1564–1570

    CAS  Article  Google Scholar 

  6. Chen S, Xu Y, Sun S, Chen F, Liu J (2019) Halalkalicoccus subterraneus sp. nov., an extremely halophilic archaeon isolated from a subterranean halite deposit. Antonie Van Leeuwenhoekvan Leeuwenhoek. https://doi.org/10.1007/s10482-019-01241-3

    Article  Google Scholar 

  7. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, Rooney AP, Yi H, Xu XW, De Meyer S, Trujillo ME (2018) Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 68:461–466

    CAS  Article  Google Scholar 

  8. Cui HL, Tohty D, Zhou PJ, Liu SJ (2006) Halorubrum lipolyticum sp. nov. and Halorubrum aidingense sp. nov., isolated from two salt lakes in Xin-Jiang, China. Int J Syst Evol Microbiol 56:1631–1634

    CAS  Article  Google Scholar 

  9. Cui HL, Lin ZY, Dong Y, Zhou PJ, Liu SJ (2007) Halorubrum litoreum sp. nov., an extremely halophilic archaeon from a solar saltern. Int J Syst Evol Microbiol 57:2204–2206

    CAS  Article  Google Scholar 

  10. Cui HL, Gao X, Yang X, Xu XW (2010) Halorussus rarus gen. nov., sp. nov., a new member of the family Halobacteriaceae isolated from a marine solar saltern. Extremophiles 14:493–499

    CAS  Article  Google Scholar 

  11. de la Haba RR, Corral P, Sánchez-Porro C, Infante-Domínguez C, Makkay AM, Amoozegar MA, Ventosa A, Papke RT (2018) Genotypic and lipid analyses of strains from the archaeal genus Halorubrum reveal insights into their taxonomy, divergence, and population structure. Front Microbiol 9:512

    Article  Google Scholar 

  12. De Ley J, Cattoir H, Reynaerts A (1970) The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12:133–142

    Article  Google Scholar 

  13. Dussault HP (1955) An improved technique for staining red halophilic bacteria. J Bacteriol 70:484–485

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376

    CAS  Article  Google Scholar 

  15. Feng J, Zhou PJ, Liu SJ (2004) Halorubrum xinjiangense sp. nov., a novel halophile isolated from saline lakes in China. Int J Syst Evol Microbiol 54:1789–1791

    CAS  Article  Google Scholar 

  16. Fullmer MS, Soucy SM, Swithers KS, Makkay AM, Wheeler R, Ventosa A, Goqarten JP, Papke RT (2014) Population and genomic analysis of the Halorubrum. Front Microbiol 5:140

    Article  Google Scholar 

  17. Gerhardt P, Murray RGE, Wood WA, Krieg NR (1994) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C.

    Google Scholar 

  18. Gupta RS, Naushad S, Fabros R, Adeolu M (2016) A phylogenomic reappraisal of family-level divisions within the class Halobacteria: proposal to divide the order Halobacteriales into the families Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov., and the order Haloferacales into the families, Haloferacaceae and Halorubraceae fam nov. Antonie Van Leeuwenhoek 109:1521–1523

    Article  Google Scholar 

  19. Gutiérrez C, González C (1972) Method for simultaneous detection of proteinase and esterase activities in extremely halophilic bacteria. Appl Microbiol 24:516–517

    PubMed  PubMed Central  Google Scholar 

  20. Gutiérrez MC, Castillo AM, Pagaling E, Heaphy S, Kamekura M, Xue Y, Ma Y, Cowan DA, Jones BE, Grant WD, Ventosa A (2008) Halorubrum kocurii sp. nov., an archaeon isolated from a saline lake. Int J Syst Evol Microbiol 58:2031–2035

    Article  Google Scholar 

  21. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  22. Kharroub K, Quesada T, Ferrer R, Fuentes S, Aguilera M, Boulahrouf A, Ramos-Cormenzana A, Monteoliva-Sánchez M (2006) Halorubrum ezzemoulense sp. nov., a halophilic archaeon isolated from Ezzemoul sabkha, Algeria. Int J Syst Evol Microbiol 56:1583–1588

    CAS  Article  Google Scholar 

  23. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNAgenes in genomic sequence. Nucleic Acids Res 25:955–964

    CAS  Article  Google Scholar 

  24. Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, Tang J, Wu G, Zhang H, Shi Y, Liu Y, Yu C, Wang B, Lu Y, Han C, Cheung DW, Yiu SM, Peng S, Xiaoqian Z, Liu G, Liao X, Li Y, Yang H, Wang J, Lam TW, Wang J (2012) SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1:18

    Article  Google Scholar 

  25. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218

    CAS  Article  Google Scholar 

  26. McGenity TJ, Grant WD (1995) Transfer of Halobacterium saccharovorum, Halobacterium sodomense, Halobacterium trapanicum NRC 34041 and Halobacterium lacusprofundi to the genus Halorubrum gen. nov., as Halorubrum saccharovorum comb. nov., Halorubrum sodomense comb. nov., Halorubrum trapanicum comb. nov., and Halorubrum lacusprofundi comb. nov. Syst Appl Microbiol 18:237–243

    Article  Google Scholar 

  27. McGenity TJ, Grant WD (2015) Halorubrum. In: Whitman WB (ed) Bergey’s manual of systematics of Archaea and bacteria. Association with Bergey’s Manual Trust, Wiley, pp 1–11

  28. McGenity TJ, Oren A (2012) Life in saline environments. In: Bell E (ed) Life at extremes: environments, organisms, and strategies for survival. CABI International, Wallingford, pp 402–437

  29. Medlar AJ, Toronen P, Holm L (2018) AAI-profiler: fast proteome-wide exploratory analysis reveals taxonomic identity, misclassification and contamination. Nucleic Acids Res 46:W479–W485

    CAS  Article  Google Scholar 

  30. Meier-Kolthoff JP, Göker M, Spröer C, Klenk HP (2013) When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol 195:413–418

    CAS  Article  Google Scholar 

  31. Minegishi H, Kamekura M, Itoh T, Echigo A, Usami R, Hashimoto T (2010) Further refinement of the phylogeny of the Halobacteriaceae based on the full-length RNA polymerase subunit B’ (rpoB’) gene. Int J Syst Evol Microbiol 60:2398–2408

    Article  Google Scholar 

  32. Nawrocki EP, Eddy SR (2013) Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29:2933–2935

    CAS  Article  Google Scholar 

  33. Nawrocki EP, Burge SW, Bateman A, Daub J, Eberhardt RY, Eddy SR, Flodden EW, Gardner PP, Jones TA, Tate J, Finn RD (2014) Rfam 12.0: updates to the RNA families database. Nucleic Acids Res 43:D130–137

    Article  Google Scholar 

  34. Ochsenreiter T, Pfeifer F, Schleper C (2002) Diversity of Archaea in hypersaline environments characterized by molecular-phylogenetic and cultivation studies. Extremophiles 6:267–274

    CAS  Article  Google Scholar 

  35. Oren A, Ventosa A, Grant WD (1997) Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47:233–238

    Article  Google Scholar 

  36. Oren A, Arahal DR, Ventosa A (2009) Emended descriptions of genera of the family Halobacteriaceae. Int J Syst Evol Microbiol 59:637–642

    Article  Google Scholar 

  37. Parte AC (2019) List of prokaryotic names with standing in nomenclature. https://www.bacterio.net

  38. Ram Mohan N, Fullmer MS, Makky AM, Wheeler R, Ventosa A, Naor A, Goqarten JP, Papke RT (2014) Evidence from phylogenetic and genome fingerprinting analyses suggests rapidly changing variation in Halorubrum and Haloarcula populations. Front Microbiol 5:143

    Article  Google Scholar 

  39. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  Google Scholar 

  40. Schbert M, Lindgreen S, Orlando L (2016) AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res Notes 9:88

    Article  Google Scholar 

  41. Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849

    CAS  Article  Google Scholar 

  42. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    CAS  Article  Google Scholar 

  43. Yim KJ, Cha IT, Lee HW, Song HS, Kim KN, Lee SJ, Nam YD, Hyun DW, Bae JW, Rhee SK, Seo MJ, Choi JS, Choi HJ, Roh SW, Kim D (2014) Halorubrum halophilum sp. nov., an extremely halophilic archaeon isolated from a salt-fermented seafood. Antonie Van Leeuwenhoek 105:603–612

    CAS  Article  Google Scholar 

  44. Yoon SH, Ha SM, Lim J, Kwon S, Chun J (2017) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281–1286

    CAS  Article  Google Scholar 

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We thank Professor Zhu L. Yang from the Kunming Institute of Botany, Chinese Academy of Sciences for the help in sample collection.


This work was supported by grants from the National Natural Science Foundation of China (31460003), the Anhui Provincial Key Lab. of the Conservation and Exploitation of Biological Resources (591601), the China Scholarship Council (201808340054) and the Department of Education Anhui Province, China.

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SC conceived the project. SS, FC, YX, JL and SC performed the experiments. SS and SC analysed the data, and drafted the manuscript. All authors read and approved the final manuscript.

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Correspondence to Shaoxing Chen.

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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rpoB′ genes of strain ZC67T are KX376720 and KX595316, respectively. The accession numbers for the genome sequences of strains ZC67T, Hrr. salsamenti Y69T and Hrr. depositum Y78T are SDJP00000000, VCNL00000000 and VCNM00000000, respectively.

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Sun, S., Chen, F., Xu, Y. et al. Halorubrum amylolyticum sp. nov., a novel halophilic archaeon isolated from a salt mine. Antonie van Leeuwenhoek 112, 1849–1861 (2019). https://doi.org/10.1007/s10482-019-01313-4

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  • Haloarchaea
  • Salt mine
  • Halorubrum
  • Polyphasic taxonomy
  • In silico DNA-DNA hybridization
  • Average nucleotide identity (ANI)
  • Average amino-acid identity (AAI)
  • Multilocus sequence analysis (MLSA)