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Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting

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Abstract

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.

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Acknowledgments

We would like to thank Jose Trinidad Ascencio-Ibanez, Larissa Benavente, and Mariana Franco for their assistance with expression cloning; Janet Dow for assistance with flow cytometry; and Eva Johannes for assistance with CLSM. Funding was provided by the National Science Foundation (CBET 0853864).

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Authors and Affiliations

  1. School of Civil Engineering and Geosciences, Newcastle University, Cassie Building, Room 1.06, Newcastle, NE1 7RU, UK

    Cesar R. Mota

  2. Department of Civil, Construction, and Environmental Engineering, North Carolina State University, Campus Box 7908, Raleigh, NC, 27695-7908, USA

    Mark Jason So & Francis L. de los Reyes III

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  1. Cesar R. Mota
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  2. Mark Jason So
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  3. Francis L. de los Reyes III
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Correspondence to Francis L. de los Reyes III.

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Mota, C.R., So, M.J. & de los Reyes, F.L. Identification of Nitrite-Reducing Bacteria Using Sequential mRNA Fluorescence In Situ Hybridization and Fluorescence-Assisted Cell Sorting. Microb Ecol 64, 256–267 (2012). https://doi.org/10.1007/s00248-012-0018-x

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