Microlunatus speluncae sp. nov., a novel actinobacterium isolated from a Karstic subterranean environment sample

  • Yuan-Guo Xie
  • Bao-Zhu FangEmail author
  • Ming-Xian Han
  • Lan Liu
  • Jian-Yu Jiao
  • Xiao-Tong Zhang
  • Min Xiao
  • Wen-Jun LiEmail author
Original Paper


A novel actinobacterial strain, designated SYSU K12189T, was isolated from a soil sample collected from a Karst cave in Xingyi county, Guizhou province, south-western China. The taxonomic position of the strain was investigated using a polyphasic approach. Cells of the strain were observed to be aerobic and Gram-stain positive. On the basis of 16S rRNA gene sequence similarities and phylogenetic analysis, strain SYSU K12189T is closely related to the type strains of the genus Microlunatus, Microlunatus parietis 12-Be-011T (98.5% sequence similarity), Microlunatus nigridraconis CPCC 203993T (98.4%) and Microlunatus cavernae YIM C01117T (96.6%), and is therefore considered to represent a member of the genus Microlunatus. DNA–DNA hybridization values between strain SYSU K12189T and related type strains of the genus Microlunatus were < 70%. In addition, LL-diaminopimelic acid was found to be the diagnostic diamino acid in the cell wall peptidoglycan. The major isoprenoid quinone was identified as MK-9(H4), while the major fatty acids (> 10%) were found to be anteiso-C15:0, iso-C15:0, iso-C16:0 and iso-C14:0. The polar lipids were found to contain diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, three glycolipids and two unidentified lipids. The genomic DNA G+C content of strain SYSU K12189T was determined to be 69.4 mol%. On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU K12189T is concluded to represent a novel species of the genus Microlunatus, for which the name Microlunatus speluncae sp. nov. is proposed. The type strain is SYSU K12189T (= KCTC 39847T = DSM 103947T).


Microlunatus speluncae sp. nov. Limestone sample Karst cave Polyphasic taxonomy 


Author’s contributions

WJL, YGX and BZF designed research and project outline. MXH, LK LL and YGX performed isolation, deposition and identification. JYJ and XTZ performed genome analysis. BZF and WJL drafted the manuscript. All authors read and approved the final manuscript.


This research was supported by National Key R&D Program of China (2017YFD0200503), Natural Science Foundation of China (No. 31600015) and National Fundamental Fund Project Subsidy Funds of Personnel Training of China (No. J1310025).

Compliance with ethical standards

Conflict of interest

All the authors have declared no conflict of interest.

Ethical statement

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

Supplementary material

10482_2019_1321_MOESM1_ESM.docx (1.9 mb)
Supplementary file1 (DOCX 1958 kb)


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Bio 215:403–410CrossRefGoogle Scholar
  2. An DS, Im WT, Yoon MH (2008) Microlunatus panaciterrae sp. nov., a b-glucosidase-producing bacterium, isolated from soil in a ginseng field. Int J Syst Evol Microbiol 58:2734–2738CrossRefPubMedGoogle Scholar
  3. Athalye M, Goodfellow M, Lacey J, White RP (1985) Numerical classification of Actinomadura and Nocardiopsis. Int J Syst Bacteriol 35:86–98CrossRefGoogle Scholar
  4. Cheng J, Chen W, Zhang BH, Nimaichand S, Zhou EM, Lu XH, Klenk HP, Li WJ (2013) Microlunatus cavernae sp. nov. a novel actinobacterium isolated from Alu ancient cave, Yunnan, south-west China. Antonie van Leeuwenhoek 104(1):95–101.CrossRefPubMedGoogle Scholar
  5. Christensen H, Angen O, Mutters R, Olsen JE, Bisgaard M (2000) DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA. Int J Syst Evol Microbiol 50:1095–1102CrossRefPubMedGoogle Scholar
  6. Collins MD, Pirouz T, Goodfellow M, Minnikin DE (1977) Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100:221–230CrossRefGoogle Scholar
  7. Collins MD, Jones D (1980) Lipids in the classification and identification of coryneform bacteria containing peptidoglycan based on 2, 4-diaminobutyric acid. J Appl Bacteriol 48:459–470CrossRefGoogle Scholar
  8. Cui YS, Im WT, Yin CR, Yang DC, Lee ST (2007) Microlunatus ginsengisoli sp. nov., isolated from soil of a ginseng field. Int J Syst Evol Microbiol 57:713–716CrossRefPubMedGoogle Scholar
  9. Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39:224–229CrossRefGoogle Scholar
  10. Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376CrossRefGoogle Scholar
  11. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefPubMedPubMedCentralGoogle Scholar
  12. Fitch WM (1971) Toward defining the course of evolution: minimum change for a specific tree topology. Syst Biol 20:406–416CrossRefGoogle Scholar
  13. Gordon RE, Barnett DA, Handerhan JE, Pang CHN (1974) Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol 24:54–63CrossRefGoogle Scholar
  14. 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 57:81–91CrossRefPubMedGoogle Scholar
  15. Hanada S, Nakamura K (2012) Genus Microlunatus. Nakamura, Hiraishi, Yoshimi, Kawaharasaki, Masuda and Kamagata 1995, 21VP. In: Goodfellow M, Kämpfer P, Busse HP, Trujillo ME, Suzuki K, Ludwig W, Whitman WB (eds) Bergey’s manual of systematic bacteriology: the Actinobacteria, Part B. 2nd edn, vol. 5. Springer, New York, pp 1168–1172.Google Scholar
  16. Harrison PG, Strulo B (2000) SPADES-a process algebra for discrete event simulation. J Logic Comput 10:3–42CrossRefGoogle Scholar
  17. Hayakawa M, Nonomura H (1987) Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J Ferment Technol 65:501–509CrossRefGoogle Scholar
  18. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW (2010) Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform 11:119CrossRefGoogle Scholar
  19. Kämpfer P, Young CC, Busse HJ, Chu JN, Schumann P, Arun AB, Shen FT, Rekha PD (2010a) Microlunatus soli sp. nov., isolated from soil. Int J Syst Evol Microbiol 60:824–827CrossRefPubMedGoogle Scholar
  20. Kämpfer P, Schäfer J, Lodders N, Martin K (2010b) Microlunatus parietis sp. nov., isolated from an indoor wall. Int J Syst Evol Microbiol 60:2420–2423CrossRefPubMedGoogle Scholar
  21. Kelly KL (1964) Inter-society color Council-National Bureau of Standards Color-Name charts illustrated with centroid colors. US Government Printing Office, WashingtonGoogle Scholar
  22. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kimura M (1985) The neutral theory of molecular evolution. Cambridge University Press, CambridgeGoogle Scholar
  24. Kroppenstedt RM (1982) Separation of bacterial menaquinones by HPLC using reverse phase (RP18) and a silver loaded ion exchanger as stationary phases. J Liq Chromatogr 5:2359–2367CrossRefGoogle Scholar
  25. Kumar S, Stecher G, Tamura K (2016) Mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  26. Lee JJ, Kim MK (2012) Microlunatus terrae sp. nov. a bacterium isolated from soil. J Microbiol 50(3):547–552.CrossRefPubMedGoogle Scholar
  27. Li SH, Yu XY, Park DJ, Hozzein WN, Kim CJ, Shu WS, Wadaan MA, Ding LX, Li WJ (2015) Rhodococcus soli sp. nov., an actinobacterium isolated from soil using a resuscitative technique. Antonie Van Leeuwenhoek 107:357–366CrossRefPubMedGoogle Scholar
  28. Li WJ, Xu P, Schumann P, Zhang YQ, Pukall R, Xu LH, Stackebrandt E, Jiang CL (2007) Georgenia ruanii sp. nov., a novel actinobacterium isolated from forest soil in Yunnan (China), and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57:1424–1428CrossRefPubMedGoogle Scholar
  29. Minnikin DE, Collins MD, Goodfellow M (1979) Fatty acid and polar lipid composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl Bacteriol 47:87–95CrossRefGoogle Scholar
  30. Minnikin DE, Hutchinson IG, Caldicott AB, Goodfellow M (1980) Thin-layer chromatography of methanolysates of mycolic acid-containing bacteria. J Chromatogr A 188:221–233CrossRefGoogle Scholar
  31. Minnikin D, O'Donnell A, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Appl Bacteriol 2:233–241Google Scholar
  32. Nakamura K, Hiraishi A, Yoshimi Y, Kawaharasaki M, Masuda K, Kamagata Y (1995) Microlunatus phosphovorus gen. nov., sp. nov, a new Gram-positive polyphosphate-accumulating bacterium isolated from activated sludge. Int J Syst Bacteriol 45:17–22CrossRefPubMedGoogle Scholar
  33. Nie GX, Ming H, Li S, Zhou EM, Cheng J, Tang X, Feng HG, Tang SK, Li WJ (2012) Amycolatopsis dongchuanensis sp. nov., a novel actinobacterium isolated from dry-hot valley in Yunnan, south-west China. Int J Syst Evol Microbiol 62:2650–2656CrossRefPubMedGoogle Scholar
  34. Nouioui I, Carro L, García-López M, Meier-Kolthoff JP, Woyke T, Kyrpides NC, Pukall R, Klenk HP, Goodfellow M, Göker M (2018) Genome-based taxonomic classification of the phylum actinobacteria. Front in Microbiol 9:2007CrossRefGoogle Scholar
  35. Pridham TG, Gottlieb G (1948) The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J Bacteriol 56:107–114PubMedPubMedCentralGoogle Scholar
  36. Reasoner DJ, Geldreich EE (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 49:1–7PubMedPubMedCentralGoogle Scholar
  37. Richter M, Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106:19126–19131CrossRefGoogle Scholar
  38. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  39. Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Microbial ID, Inc, NewarkGoogle Scholar
  40. Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477PubMedPubMedCentralGoogle Scholar
  41. Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340CrossRefGoogle Scholar
  42. Tamaoka J, Katayama-Fujimura Y, Kuraishi H (1983) Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J Appl Bacteriol 54:31–36CrossRefGoogle Scholar
  43. Tang SK, Wang Y, Chen Y, Luo K, Cao LL, Xu LH, Li WJ (2009) Zhihengliuella alba sp. nov., and emended description of the genus Zhihengliuella. Int J Syst Bacteriol 59:2025–2032CrossRefGoogle Scholar
  44. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  45. Tuo L, Li J, Liu SW, Liu Y, Hu L, Chen L, Jiang MG, Sun CH (2016) Microlunatus endophyticus sp. nov., an endophytic actinobacterium isolated from bark of Bruguiera sexangular. Int J Syst Evol Microbiol 666:481–486.CrossRefGoogle Scholar
  46. Wang YX, Cai M, Zhi XY, Zhang YQ, Tang SK, Xu LH, Cui XL, Li WJ (2008) Microlunatus aurantiacus sp. nov., a novel actinobacterium isolated from a rhizosphere soil sample. Int J Syst Evol Microbiol 58:1873–1877CrossRefPubMedGoogle Scholar
  47. Waksman SA (1967) The actinomycetes, a summary of current knowledge. Ronald Press, New YorkGoogle Scholar
  48. Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE (1987) International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464CrossRefGoogle Scholar
  49. Williams ST, Goodfellow M, Alderson G (1989) Genus Streptomyces Waksman and Henrici 1943, 339AL. In: Williams ST, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol. 4, Williams & Willkins, Baltimore, pp 2453–2492.Google Scholar
  50. Xu P, Li WJ, Tang SK, Zhang YQ, Chen GZ, Chen HH, Xu LH, Jiang CL (2005) Naxibacter alkalitolerans gen. nov., sp. nov., a novel member of the family ‘Oxalobacteraceae’ isolated from China. Int J Syst Evol Microbiol 55:1149–1153CrossRefPubMedGoogle Scholar
  51. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017a) Introducing EzBioCloud: a taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol 67:1613–1617CrossRefPubMedPubMedCentralGoogle Scholar
  52. Yoon SH, Ha SM, Lim JM, Kwon SJ, Chun J (2017b) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281–1286CrossRefGoogle Scholar
  53. Zhang CF, Ai MJ, Zhao LL, Liu HY, Yu LY, Zhang YQ (2016) Microlunatus nigridraconis sp. nov., an actinobacterium from rhizosphere soil. Int J Syst Evol Microbiol 66: 3614–3618CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life SciencesSun Yat-Sen UniversityGuangzhouPeople’s Republic of China
  2. 2.State Key Laboratory of Biogeology and Environmental GeologyChina University of GeosciencesWuhanPeople’s Republic of China

Personalised recommendations