Detection and cell sorting of Pseudonocardia species by fluorescence in situ hybridization and flow cytometry using 16S rRNA-targeted oligonucleotide probes
Pseudonocardia spp. are receiving increasing attention due to their ability to biodegrade recalcitrant cyclic ether pollutants (e.g., 1,4-dioxane and tetrahydrofuran), as well as for their distinctive ecological niches (e.g., symbiosis with ants/plants and production of antibiotics). Isolating and characterizing Pseudonocardia spp. is thus important to discern their metabolic and physiological idiosyncrasies and advance their potential applications. However, slow growth, low cell yield, and dissimilar colony morphology hinder efficient isolation of Pseudonocardia using conventional plating methods. Here, we develop the first fluorescent probe (Pse631) targeting the 16S rRNA of Pseudonocardia members. In combination with flow cytometry and cell sorting, in situ hybridization with this probe enables sensitive and specific detection of Pseudonocardia cells in mixed cultures and enriched environmental samples without significant false positives, using Escherichia coli, Bacillus subtilis, and Mycobacterium spp. as negative controls. Pseudonocardia dioxanivorans CB1190 cells labeled with Pse631 as a positive control were detected when their relative abundance in the total bacterial community was as low as 0.1%. Effective separation of Pseudonocardia cells from the mixed consortium was confirmed by quantitative PCR analysis of sorted cells. This study provides a culture-independent high-throughput molecular approach enabling effective separation of Pseudonocardia populations from complex microbial communities. This approach will not only facilitate subsequent molecular analyses including species identification and quantification, but also advance understanding of their catabolic capacities and functional molecular diversity.
KeywordsPseudonocardia Fluorescence in situ hybridization Flow cytometry 1,4-Dioxane Tetrahydrofuran
The authors thank Joel M. Sederstrom (Baylor College of Medicine) for the assistance with flow cytometry.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Davenport RJ, Curtis TP, Goodfellow M, Stainsby FM, Bingley M (2000) Quantitative use of fluorescent in situ hybridization to examine relationships between mycolic acid-containing Actinomycetes and foaming in activated sludge plants. Appl Environ Microbiol 66(3):1158–1166. https://doi.org/10.1128/aem.66.3.1158-1166.2000 CrossRefPubMedPubMedCentralGoogle Scholar
- Dekker KA, Inagaki T, Gootz TD, Huang LH, Kojima Y, Kohlbrenner WE, Matsunaga Y, McGuirk PR, Nomura E, Sakakibara T, Sakemi S, Suzuki Y, Yamauchi Y, Kojima N (1998) New quinolone compounds from Pseudonocardia sp. with selective and potent anti-Helicobacter pylori activity: taxonomy of producing strain, fermentation, isolation, structural elucidation and biological activities. J Antibiotics 51(2):145–152CrossRefGoogle Scholar
- Embley T (1992) The family Pseudonocardiaceae. The Prokaryotes 1:996–1027Google Scholar
- Hoshino T, Schramm A (2010) Detection of denitrification genes by in situ rolling circle amplification-fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells. Environ Microbiol 12(9):2508–2517. https://doi.org/10.1111/j.1462-2920.2010.02224.x CrossRefPubMedGoogle Scholar
- Keller GH, Manak MM (1989) DNA probes. Macmillan Publishers Ltd, LondonGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and clustal X version 2.0. Bioinformatics 23(21):2947–2948. https://doi.org/10.1093/bioinformatics/btm404 CrossRefPubMedGoogle Scholar
- Li J, Zhao G-Z, Varma A, Qin S, Xiong Z, Huang H-Y, Zhu W-Y, Zhao L-X, Xu L-H, Zhang S, Li W-J (2012) An endophytic Pseudonocardia species induces the production of artemisinin in Artemisia annua. PLOS ONE 7(12):e51410. https://doi.org/10.1371/journal.pone.0051410 CrossRefPubMedPubMedCentralGoogle Scholar
- Li M, Mathieu J, Liu Y, Van Orden ET, Yang Y, Fiorenza S, Alvarez PJ (2013) The abundance of tetrahydrofuran/dioxane monooxygenase genes (thmA/dxmA) and 1, 4-dioxane degradation activity are significantly correlated at various impacted aquifers. Environ Sci Tech Lett 1(1):122–127CrossRefGoogle Scholar
- Mohr T, Stickney J, DiGuiseppi W (2010) Environmental investigation and remediation: 1,4-dioxane and other solvent stabilizers. CRC Press.Google Scholar
- Mueller UG, Ishak H, Lee JC, Sen R, Gutell RR (2010) Placement of attine ant-associated Pseudonocardia in a global Pseudonocardia phylogeny (Pseudonocardiaceae, Actinomycetales): a test of two symbiont-association models. Antonie van Leeuwenhoek 98(2):195–212. https://doi.org/10.1007/s10482-010-9427-3 CrossRefPubMedPubMedCentralGoogle Scholar
- Pozhitkov A, Noble PA, Domazet-Lošo T, Nolte AW, Sonnenberg R, Staehler P, Beier M, Tautz D (2006) Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted. Nucleic Acids Res 34(9):e66–e66CrossRefPubMedPubMedCentralGoogle Scholar
- Reichert K, Lipski A, Pradella S, Stackebrandt E, Altendorf K (1998) Pseudonocardia asaccharolytica sp. nov. and Pseudonocardia sulfidoxydans sp. nov., two new dimethyl disulfide-degrading actinomycetes and emended description of the genus Pseudonocardia. Int J Syst Evol Microbiol 48(2):441–449Google Scholar
- Sales CM, Mahendra S, Grostern A, Parales RE, Goodwin LA, Woyke T, Nolan M, Lapidus A, Chertkov O, Ovchinnikova G, Sczyrba A, Alvarez-Cohen L (2011) Genome sequence of the 1,4-dioxane-degrading Pseudonocardia dioxanivorans strain CB1190. J Bacteriol 193(17):4549–4550. https://doi.org/10.1128/Jb.00415-11 CrossRefPubMedPubMedCentralGoogle Scholar
- Urzi C, La Cono V, Stackebrandt E (2004) Design and application of two oligonucleotide probes for the identification of Geodermatophilaceae strains using fluorescence in situ hybridization (FISH). Environ Microbiol 6(7):678–685. https://doi.org/10.1111/j.1462-2920.2004.00619.x CrossRefPubMedGoogle Scholar
- Werckenthin C, Gey A, Straubinger RK, Poppert S (2012) Rapid identification of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes by fluorescence in situ hybridization (FISH). Vet Microbiol 156(3–4):330–335. https://doi.org/10.1016/j.vetmic.2011.10.007 CrossRefPubMedGoogle Scholar
- Whittenbury R, Phillips K, Wilkinson J (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. Microbiol 61(2):205–218Google Scholar
- Wright ES, Yilmaz LS, Corcoran AM, Okten HE, Noguera DR (2014) Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification. Appl Environ Microbiol 80(16):5124–5133. https://doi.org/10.1128/AEM.01685-14 CrossRefPubMedPubMedCentralGoogle Scholar
- Yilmaz LS, Parnerkar S, Noguera DR (2011) mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl Environ Microbiol 77(3):1118–1122. https://doi.org/10.1128/AEM.01733-10 CrossRefPubMedGoogle Scholar
- Zhou J, He Z, Yang Y, Deng Y, Tringe SG, Alvarez-Cohen L (2015) High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats. mBio 6(1). https://doi.org/10.1128/mBio.02288-14