Halalkalicoccus subterraneus sp. nov., an extremely halophilic archaeon isolated from a subterranean halite deposit

  • Shaoxing ChenEmail author
  • Yao Xu
  • Siqi Sun
  • Feilong Chen
  • Jingwen Liu
Original Paper


An extremely halophilic archaeon, designated strain GSM28T, was isolated from a subterranean halite deposit in a Yunnan salt mine, China. Cells of the strain were observed to be cocci, non-motile and Gram-variable, and to require at least 15% (w/v) NaCl for growth (optimum 20%). Growth was found to occur in the ranges of 20–45 °C (optimum 42 °C) and pH 6.0–8.5 (optimum 7.5). Cells did not lyse in distilled water. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain belongs to the genus Halalkalicoccus and shows 99.1% similarities with its close phylogenetic relative Halalkalicoccus paucihalophilus DSM 24557T. Genomic ANI analysis showed that the DNA–DNA relatedness between strain GSM28T and the closely related species Hac. paucihalophilus DSM 24557T and Halalkalicoccus jeotgali B3T was 83.7% and 83.1%, respectively. The major polar lipids were determined to be phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl-glucosyl-glycerol diether-1 and two unidentified glycolipids. The DNA G + C content was determined to be 61.8 mol %. On the basis of physiological, biochemical tests and phylogenetic differentiations, strain GSM28T is concluded to represent a novel species in the genus Halalkalicoccus, for which the name Halalkalicoccus subterraneus sp. nov. is proposed. The type strain is GSM28T (= CGMCC 1.16344T = NBRC 113432T).


Salt mine Halite deposit Haloarchaea Halalkalicoccus Polyphasic taxonomy 



2-Morpholinoethanesulfonic acid


1, 4-Piperazine bis (ethanesulfonic acid)


2-(Cyclohexylamino) ethanesulfonic acid


N-Cyclohexyl-3-aminopropanesulfonic acid




Optical density


Average nucleotide identity


Multilocus sequence analysis


DNA–DNA hybridization


Thin-layer chromatography




Phosphatidylglycerol phosphate methyl ester


Sulfated mannosyl-glucosyl-glycerol diether-1



We thank Professor Zhu L. Yang from the Kunming Institute of Botany, Chinese Academy of Sciences for the help in sample collection, and we also thank Dr. Hao Yan from the College of Life Sciences, Anhui Normal University, for his technical assistance.

Author’s contribution

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


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), and the Department of Education Anhui Province, China.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Ethical statement

No specific ethical or institutional permits were required to conduct sampling and the experimental studies did not involve endangered or protected species.

Supplementary material

10482_2019_1241_MOESM1_ESM.docx (12.8 mb)
Supplementary material 1 (DOCX 13121 kb)


  1. 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 68:461–466CrossRefGoogle Scholar
  2. Cui HL, Zhou PJ, Oren A, Liu SJ (2009) Intraspecific polymorphism of 16S rRNA genes in two halophilic archaeal genera, Haloarcula and Halomicrobium. Extremophiles 13:31–37CrossRefGoogle Scholar
  3. Dussault HP (1955) An improved technique for staining red halophilic bacteria. J Bacteriol 70:484–485Google Scholar
  4. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  5. Gutiérrez C, González C (1972) Method for simultaneous detection of proteinase and esterase activities in extremely halophilic bacteria. Appl Microbiol 24:516–517Google Scholar
  6. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  7. Hartmann R, Sickinger HD, Oesterhelt D (1980) Anaerobic growth of halobacteria. Proc Natl Acad Sci USA 77:3821–3825CrossRefGoogle Scholar
  8. Kamekura M (1993) Lipids of extreme halophiles. In: Vreeland RH, Hochstein LI (eds) The biology of halophilic bacteria. CRC Press, Boca Raton, pp 135–161Google Scholar
  9. Liu BB, Tang SK, Zhang YG, Lu XH, Li L, Cheng J, Zhang YM, Zhang LL, Li WJ (2013) Halalkalicoccus paucihalophilus sp. nov., a halophilic archaeon from Lop Nur region in Xinjiang, northwest of China. Antonie Van Leeuwenhoek 103:1007–1014CrossRefGoogle Scholar
  10. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218CrossRefGoogle Scholar
  11. 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 60:2398–2408CrossRefGoogle Scholar
  12. Mukhtar S, Mirza BS, Mehnaz S, Mirza MS, Mclean J, Malik KA (2018) Impact of soil salinity on the microbial structure of halophyte rhizosphere microbiome. World J Microbiol Biotechnol 34:136CrossRefGoogle Scholar
  13. Mwirichia R, Cousin S, Muigai AW, Boga HI, Stackebrandt E (2010) Archaeal diversity in the haloalkaline Lake Elmenteita in Kenya. Curr Microbiol 60:47–52CrossRefGoogle Scholar
  14. Nurk S, Bankevich A, Antipov D, Gurevich AA, Korobeynikov A, Lapidus A, Prjibelski AD, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, Clingenpeel SR, Woyke T, McLean JS, Lasken R, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477CrossRefGoogle Scholar
  15. Oren A, Ventosa A, Grant WD (1997) Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47:233–238CrossRefGoogle Scholar
  16. Ozcan B, Ozcengiz G, Coleri A, Cokmus C (2007) Diversity of halophilic archaea from six hypersaline environments in Turkey. J Microbiol Biotechnol 17:985–992Google Scholar
  17. Papke RT, White E, Reddy P, Weigel G, Kamekura M, Minegishi H, Usami R, Ventosa A (2011) A multilocus sequence analysis approach to the phylogeny and taxonomy of the Halobacteriales. Int J Syst Evol Microbiol 61:2984–2995CrossRefGoogle Scholar
  18. Roh SW, Nam YD, Chang HW, Sung Y, Kim KH, Oh HM, Bae JW (2007) Halalkalicoccus jeotgali sp. nov., a halophilic archaeon from shrimp jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 57:2296–2298CrossRefGoogle Scholar
  19. Rosenzweig WD, Peterson J, Woish J, Vreeland RH (2000) Development of a protocol to retrieve microorganisms from ancient salt crystals. Geomicrobiol J 17:185–192CrossRefGoogle Scholar
  20. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P (ed) Methods for general and molecular bacteriology. American Society for Microbiology, Washington DC, pp 607–654Google Scholar
  21. Sun W, Li J, Jiang L, Sun Z, Fu M, Peng X (2015) Profiling microbial community structures across six large oilfields in China and the potential role of dominant microorganisms in bioremediation. Appl Microbiol Biotechnol 99:8751–8764CrossRefGoogle Scholar
  22. 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 28:2731–2739CrossRefGoogle Scholar
  23. Valenzuela-Encinas C, Neria-González I, Alcántara-Hernández RJ, Enríquez-Aragón JA, Estrada-Alvarado I, Hernández-Rodríguez C, Dendooven L, Marsch R (2008) Phylogenetic analysis of the archaeal community in an alkaline-saline soil of the former lake Texcoco (Mexico). Extremophiles 12:247–254CrossRefGoogle Scholar
  24. Xiao W, Wang ZG, Wang YX, Schneegurt MA, Li ZY, Lai YH, Zhang SY, Wen ML, Cui XL (2013) Comparative molecular analysis of the prokaryotic diversity of two salt mine soils in southwest China. J Basic Microbiol 53:942–952CrossRefGoogle Scholar
  25. Xue Y, Fan H, Ventosa A, Grant WD, Jones BE, Cowan DA, Ma Y (2005) Halalkalicoccus tibetensis gen. nov., sp. nov., representing a novel genus of haloalkaliphilic archaea. Int J Syst Evol Microbiol 55:2501–2505CrossRefGoogle Scholar
  26. Yoon SH, Ha SM, Lim JM, Kwon SJ, Chun J (2017) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281–1286CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.College of Life SciencesAnhui Normal UniversityWuhuPeople’s Republic of China
  2. 2.College of Life SciencesHonghe UniversityMengziPeople’s Republic of China

Personalised recommendations