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Detection and cell sorting of Pseudonocardia species by fluorescence in situ hybridization and flow cytometry using 16S rRNA-targeted oligonucleotide probes

  • Environmental biotechnology
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Abstract

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.

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Acknowledgements

The authors thank Joel M. Sederstrom (Baylor College of Medicine) for the assistance with flow cytometry.

Funding

This study was funded by SERDP (Grant # ER-2301).

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  1. Mengyan Li and Yu Yang co-first authors with equal contribution

Authors and Affiliations

  1. Department of Chemistry and Environmental Science, New Jersey Institute of Technology, University Heights, Newark, NJ, 07102, USA

    Mengyan Li

  2. Department of Civil and Environmental Engineering, Rice University, 6100 Main Street, Houston, 77005, TX, USA

    Mengyan Li, Yu Yang, Ya He, Jacques Mathieu, Cong Yu, Qilin Li & Pedro J. J. Alvarez

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  1. Mengyan Li
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Li, M., Yang, Y., He, Y. et al. Detection and cell sorting of Pseudonocardia species by fluorescence in situ hybridization and flow cytometry using 16S rRNA-targeted oligonucleotide probes. Appl Microbiol Biotechnol 102, 3375–3386 (2018). https://doi.org/10.1007/s00253-018-8801-3

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