Extremophiles

, Volume 17, Issue 2, pp 265–275 | Cite as

Cell sorting analysis of geographically separated hypersaline environments

  • Olga Zhaxybayeva
  • Ramunas Stepanauskas
  • Nikhil Ram Mohan
  • R. Thane Papke
Original Paper

Abstract

Biogeography of microbial populations remains to be poorly understood, and a novel technique of single cell sorting promises a new level of resolution for microbial diversity studies. Using single cell sorting, we compared saturated NaCl brine environments (32–35 %) of the South Bay Salt Works in Chula Vista in California (USA) and Santa Pola saltern near Alicante (Spain). Although some overlap in community composition was detected, both samples were significantly different and included previously undiscovered 16S rRNA sequences. The community from Chula Vista saltern had a large bacterial fraction, which consisted of diverse Bacteroidetes and Proteobacteria. In contrast, Archaea dominated Santa Pola’s community and its bacterial fraction consisted of the previously known Salinibacter lineages. The recently reported group of halophilic Archaea, Nanohaloarchaea, was detected at both sites. We demonstrate that cell sorting is a useful technique for analysis of halophilic microbial communities, and is capable of identifying yet unknown or divergent lineages. Furthermore, we argue that observed differences in community composition reflect restricted dispersal between sites, a likely mechanism for diversification of halophilic microorganisms.

Keywords

Haloarchaea Cell sorting Genome amplification Biogeography Nanohaloarchaea Prokaryotic speciation 

Supplementary material

792_2013_514_MOESM1_ESM.pdf (195 kb)
Supplementary material 1 (PDF 195 kb)
792_2013_514_MOESM2_ESM.eps (5 mb)
Supplementary material 2 (EPS 5140 kb)
792_2013_514_MOESM3_ESM.eps (1.8 mb)
Supplementary material 3 (EPS 1889 kb)

References

  1. Andam CP, Harlow TJ, Papke RT, Gogarten JP (2012) Ancient origin of the divergent forms of leucyl-tRNA synthetases in Halobacteriales. BMC Evol Biol 12:85PubMedCrossRefGoogle Scholar
  2. Anton J, Llobet-Brossa E, Rodriguez-Valera F, Amann R (1999) Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds. Environ Microbiol 1:517–523PubMedCrossRefGoogle Scholar
  3. Anton J, Oren A, Benlloch S, Rodriguez-Valera F, Amann R, Rossello-Mora R (2002) Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. Int J Syst Evol Microbiol 52:485–491PubMedGoogle Scholar
  4. Baati H, Guermazi S, Gharsallah N, Sghir A, Ammar E (2010) Novel prokaryotic diversity in sediments of Tunisian multipond solar saltern. Res Microbiol 161:573–582PubMedCrossRefGoogle Scholar
  5. Benlloch S, Acinas SG, Anton J, Lopez-Lopez A, Luz SP, Rodriguez-Valera F (2001) Archaeal biodiversity in crystallizer ponds from a solar saltern: culture versus PCR. Microb Ecol 41:12–19PubMedGoogle Scholar
  6. Bidle K, Amadio W, Oliveira P, Paulish T, Hicks S, Earnest C (2005) A phylogenetic analysis of haloarchaea found in a solar saltern. Bios 76:89–96CrossRefGoogle Scholar
  7. Boucher Y, Douady CJ, Sharma AK, Kamekura M, Doolittle WF (2004) Intragenomic heterogeneity and intergenomic recombination among haloarchaeal rRNA genes. J Bacteriol 186:3980–3990PubMedCrossRefGoogle Scholar
  8. Breuert S, Allers T, Spohn G, Soppa J (2006) Regulated polyploidy in halophilic archaea. PLoS One 1:e92PubMedCrossRefGoogle Scholar
  9. Burns DG, Camakaris HM, Janssen PH, Dyall-Smith ML (2004) Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable. Appl Environ Microbiol 70:5258–5265PubMedCrossRefGoogle Scholar
  10. Casamayor EO, Schafer H, Baneras L, Pedros-Alio C, Muyzer G (2000) Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. Appl Environ Microbiol 66:499–508PubMedCrossRefGoogle Scholar
  11. Casamayor EO, Massana R, Benlloch S, Ovreas L, Diez B, Goddard VJ et al (2002) Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ Microbiol 4:338–348PubMedCrossRefGoogle Scholar
  12. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ et al (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:D141–D145PubMedCrossRefGoogle Scholar
  13. Cuadros-Orellana S, Martin-Cuadrado AB, Legault B, D’Auria G, Zhaxybayeva O, Papke RT, Rodriguez-Valera F (2007) Genomic plasticity in prokaryotes: the case of the square haloarchaeon. ISME J 1:235–245PubMedCrossRefGoogle Scholar
  14. de la Haba RR, Marquez MD, Papke RT, Ventosa A (2011) Multilocus sequence analysis (MLSA) of the family Halomonadaceae. Int J Syst Evol Microbiol 62:520–538PubMedCrossRefGoogle Scholar
  15. Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P et al (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci USA 99:5261–5266PubMedCrossRefGoogle Scholar
  16. Dyall-Smith ML, Pfeiffer F, Klee K, Palm P, Gross K, Schuster SC et al (2011) Haloquadratum walsbyi: limited diversity in a global pond. PLoS One 6:e20968PubMedCrossRefGoogle Scholar
  17. Fenchel T (2003) Microbiology. Biogeography for bacteria. Science 301:925–926PubMedCrossRefGoogle Scholar
  18. Ferris MJ, Muyzer G, Ward DM (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 62:340–346PubMedGoogle Scholar
  19. Finlay BJ (2002) Global dispersal of free-living microbial eukaryote species. Science 296:1061–1063PubMedCrossRefGoogle Scholar
  20. Finsinger K, Scholz I, Serrano A, Morales S, Uribe-Lorio L, Mora M et al (2008) Characterization of true-branching cyanobacteria from geothermal sites and hot springs of Costa Rica. Environ Microbiol 10:460–473PubMedCrossRefGoogle Scholar
  21. Fleming EJ, Langdon AE, Martinez-Garcia M, Stepanauskas R, Poulton NJ, Masland ED, Emerson D (2011) What’s new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH. PLoS One 6:e17769PubMedCrossRefGoogle Scholar
  22. Ghai R, Pasic L, Fernandez AB, Martin-Cuadrado AB, Mizuno CM, McMahon KD et al (2011) New abundant microbial groups in aquatic hypersaline environments. Sci Rep 1:135PubMedCrossRefGoogle Scholar
  23. Grant S, Grant WD, Jones BE, Kato C, Li L (1999) Novel archaeal phylotypes from an East African alkaline saltern. Extremophiles 3:139–145PubMedCrossRefGoogle Scholar
  24. Gunde-Cimerman N, Ramos J, Plemenitas A (2009) Halotolerant and halophilic fungi. Mycol Res 113:1231–1241PubMedCrossRefGoogle Scholar
  25. Heywood JL, Sieracki ME, Bellows W, Poulton NJ, Stepanauskas R (2011) Capturing diversity of marine heterotrophic protists: one cell at a time. ISME J 5:674–684PubMedCrossRefGoogle Scholar
  26. Hongmei J, Aitchison JC, Lacap DC, Peerapornpisal Y, Sompong U, Pointing SB (2005) Community phylogenetic analysis of moderately thermophilic cyanobacterial mats from China, the Philippines and Thailand. Extremophiles 9:325–332PubMedCrossRefGoogle Scholar
  27. Ionescu D, Hindiyeh M, Malkawi H, Oren A (2010) Biogeography of thermophilic cyanobacteria: insights from the Zerka Ma’in hot springs (Jordan). FEMS Microbiol Ecol 72:103–113PubMedCrossRefGoogle Scholar
  28. Jiang H, Dong H, Zhang G, Yu B, Chapman LR, Fields MW (2006) Microbial diversity in water and sediment of Lake Chaka, an Athalassohaline lake in northwestern China. Appl Environ Microbiol 72:3832–3845PubMedCrossRefGoogle Scholar
  29. Jousset A, Schulz W, Scheu S, Eisenhauer N (2011) Intraspecific genotypic richness and relatedness predict the invasibility of microbial communities. ISME J 5:1108–1114PubMedCrossRefGoogle Scholar
  30. Kellogg CA, Griffin DW (2006) Aerobiology and the global transport of desert dust. Trends Ecol Evol 21:638–644PubMedCrossRefGoogle Scholar
  31. Langenheder S, Szekely AJ (2011) Species sorting and neutral processes are both important during the initial assembly of bacterial communities. ISME J 5:1086–1094PubMedCrossRefGoogle Scholar
  32. Lau MC, Aitchison JC, Pointing SB (2009) Bacterial community composition in thermophilic microbial mats from five hot springs in central Tibet. Extremophiles 13:139–149PubMedCrossRefGoogle Scholar
  33. Lozupone C, Hamady M, Knight R (2006) UniFrac—an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7:371PubMedCrossRefGoogle Scholar
  34. Lueders T, Friedrich MW (2003) Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl Environ Microbiol 69:320–326PubMedCrossRefGoogle Scholar
  35. Martin AP (2002) Phylogenetic approaches for describing and comparing the diversity of microbial communities. Appl Environ Microbiol 68:3673–3682PubMedCrossRefGoogle Scholar
  36. Martinez-Garcia M, Swan BK, Poulton NJ, Gomez ML, Masland D, Sieracki ME, Stepanauskas R (2011) High-throughput single-cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton. ISME J 6:113–123PubMedCrossRefGoogle Scholar
  37. Maturrano L, Santos F, Rossello-Mora R, Anton J (2006) Microbial diversity in Maras salterns, a hypersaline environment in the Peruvian Andes. Appl Environ Microbiol 72:3887–3895PubMedCrossRefGoogle Scholar
  38. Mutlu MB, Martinez-Garcia M, Santos F, Pena A, Guven K, Anton J (2008) Prokaryotic diversity in Tuz Lake, a hypersaline environment in inland Turkey. FEMS Microbiol Ecol 65:474–483PubMedCrossRefGoogle Scholar
  39. Naor A, Lapierre P, Mevarech M, Papke RT, Gophna U (2012) Low species barriers in halophilic archaea and the formation of recombinant hybrids. Curr Biol 22:1444–1448PubMedCrossRefGoogle Scholar
  40. Narasingarao P, Podell S, Ugalde JA, Brochier-Armanet C, Emerson JB, Brocks JJ et al (2011) De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities. ISME J 6:81–93PubMedCrossRefGoogle Scholar
  41. Oh D, Porter K, Russ B, Burns D, Dyall-Smith M (2010) Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds. Extremophiles 14:161–169PubMedCrossRefGoogle Scholar
  42. Oren A (1994) The ecology of the extremely halophilic archaea. FEMS Microbiol Rev 13:415–440CrossRefGoogle Scholar
  43. Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1:2PubMedCrossRefGoogle Scholar
  44. Oren A, Kuhl M, Karsten U (1995) An endoevaporitic microbial mat within a gypsum crust: zonation of phototrophs, photopigments, and light penetration. Mar Ecol Prog Ser 128:151–159CrossRefGoogle Scholar
  45. Page KA, Connon SA, Giovannoni SJ (2004) Representative freshwater bacterioplankton isolated from Crater Lake, Oregon. Appl Environ Microbiol 70:6542–6550PubMedCrossRefGoogle Scholar
  46. Papke RT (2009) A critique of prokaryotic species concepts. Methods Mol Biol 532:379–395CrossRefGoogle Scholar
  47. Papke RT, Ramsing NB, Bateson MM, Ward DM (2003) Geographical isolation in hot spring cyanobacteria. Environ Microbiol 5:650–659PubMedCrossRefGoogle Scholar
  48. Papke RT, Zhaxybayeva O, Feil EJ, Sommerfeld K, Muise D, Doolittle WF (2007) Searching for species in haloarchaea. Proc Natl Acad Sci USA 104:14092–14097PubMedCrossRefGoogle Scholar
  49. Price MN, Dehal PS, Arkin AP (2009) FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26:1641–1650PubMedCrossRefGoogle Scholar
  50. Purdy KJ, Cresswell-Maynard TD, Nedwell DB, McGenity TJ, Grant WD, Timmis KN, Embley TM (2004) Isolation of haloarchaea that grow at low salinities. Environ Microbiol 6:591–595PubMedCrossRefGoogle Scholar
  51. Radax C, Gruber C, Stan-Lotter H (2001) Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt. Extremophiles 5:221–228PubMedCrossRefGoogle Scholar
  52. Raghunathan A, Ferguson HR Jr, Bornarth CJ, Song W, Driscoll M, Lasken RS (2005) Genomic DNA amplification from a single bacterium. Appl Environ Microbiol 71:3342–3347PubMedCrossRefGoogle Scholar
  53. Rhodes ME, Spear JR, Oren A, House CH (2011) Differences in lateral gene transfer in hypersaline versus thermal environments. BMC Evol Biol 11:199PubMedCrossRefGoogle Scholar
  54. Rodriguez-Brito B, Li L, Wegley L, Furlan M, Angly F, Breitbart M et al (2010) Viral and microbial community dynamics in four aquatic environments. ISME J 4:739–751PubMedCrossRefGoogle Scholar
  55. Rodriguez-Valera F, Ruiz-Berraquero F, Ramos-Cormenzana A (1981) Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations. Microb Ecol 7:235–243CrossRefGoogle Scholar
  56. Rollins DM, Colwell RR (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl Environ Microbiol 52:531–538PubMedGoogle Scholar
  57. Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S et al (2007) The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol 5:e77PubMedCrossRefGoogle Scholar
  58. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541PubMedCrossRefGoogle Scholar
  59. Sieracki ME, Poulton NJ, Crosbie N (2005) Automated isolation techniques for microalgae. In: Anderson R (ed) Algal culturing techniques. Elsevier Academic, New YorkGoogle Scholar
  60. Sorensen KB, Canfield DE, Teske AP, Oren A (2005) Community composition of a hypersaline endoevaporitic microbial mat. Appl Environ Microbiol 71:7352–7365PubMedCrossRefGoogle Scholar
  61. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690PubMedCrossRefGoogle Scholar
  62. Stepanauskas R, Sieracki ME (2007) Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time. Proc Natl Acad Sci USA 104:9052–9057PubMedCrossRefGoogle Scholar
  63. Suzuki MT, Giovannoni SJ (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol 62:625–630PubMedGoogle Scholar
  64. Swan BK, Martinez-Garcia M, Preston CM, Sczyrba A, Woyke T, Lamy D et al (2011) Potential for chemolithoautotrophy among ubiquitous bacteria lineages in the dark ocean. Science 333:1296–1300PubMedCrossRefGoogle Scholar
  65. Trigui H, Masmoudi S, Brochier-Armanet C, Barani A, Gregori G, Denis M et al (2011) Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis. Extremophiles 15:347–358PubMedCrossRefGoogle Scholar
  66. Walsh DA, Papke RT, Doolittle WF (2005) Archaeal diversity along a soil salinity gradient prone to disturbance. Environ Microbiol 7:1655–1666PubMedCrossRefGoogle Scholar
  67. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267PubMedCrossRefGoogle Scholar
  68. Ward BB, Martino DP, Diaz MC, Joye SB (2000) Analysis of ammonia-oxidizing bacteria from hypersaline Mono Lake, California, on the basis of 16S rRNA sequences. Appl Environ Microbiol 66:2873–2881PubMedCrossRefGoogle Scholar
  69. Whitaker RJ, Grogan DW, Taylor JW (2003) Geographic barriers isolate endemic populations of hyperthermophilic archaea. Science 301:976–978PubMedCrossRefGoogle Scholar
  70. Whitaker RJ, Grogan DW, Taylor JW (2005) Recombination shapes the natural population structure of the hyperthermophilic archaeon Sulfolobus islandicus. Mol Biol Evol 22:2354–2361PubMedCrossRefGoogle Scholar
  71. Woyke T, Xie G, Copeland A, Gonzalez JM, Han C, Kiss H et al (2009) Assembling the marine metagenome, one cell at a time. PLoS One 4:e5299PubMedCrossRefGoogle Scholar
  72. Yoon HS, Price DC, Stepanauskas R, Rajah VD, Sieracki ME, Wilson WH et al (2011) Single-cell genomics reveals organismal interactions in uncultivated marine protists. Science 332:714–717PubMedCrossRefGoogle Scholar
  73. Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J, Chisholm SW, Church GM (2006) Sequencing genomes from single cells by polymerase cloning. Nat Biotechnol 24:680–686PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2013

Authors and Affiliations

  • Olga Zhaxybayeva
    • 1
  • Ramunas Stepanauskas
    • 2
  • Nikhil Ram Mohan
    • 3
  • R. Thane Papke
    • 3
  1. 1.Department of Biological SciencesDartmouth CollegeHanoverUSA
  2. 2.Bigelow Laboratory for Ocean SciencesEast BoothbayUSA
  3. 3.Department of Molecular and Cell BiologyUniversity of ConnecticutStorrsUSA

Personalised recommendations