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

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.

Rapid and Sensitive Quantification of Anammox Bacteria by Flow Cytometric Analysis Based on Catalyzed Reporter Deposition Fluorescence In Situ Hybridization

“…Two forms of CARD-FISH were performed to evaluate the feasibility of the method, including in-solution and embedded in agarose, which was used in previous studies to prepare solid samples compatible with FCM. , In this section, only the in-solution Flow-CARD-FISH was described. As the detection accuracy of CARD-FISH is largely determined by processing conditions of TSA, several key factors were systematically evaluated; namely, HRP labeling temperature, incubation time of fluorescent tyramide labeling and fluorescent substrate concentration.…”

Presence of a Novel Methanogenic Archaeal Lineage in Anaerobic Digesters Inferred from <I>mcrA</I> and 16S rRNA Gene Phylogenetic Analyses

“…This approach was based on a combination of FISH and FACS analysis that has been used for the linking of phylogeny and metabolic activity (Mota et al, 2012).…”

CARD-FISH for Environmental Microorganisms: Technical Advancement and Future Applications

“…More recently, sequential mRNA FISH followed by fluorescence-assisted cell sorting (SmRFF) has been developed, and active nitrite reducers in an activated sludge sample were successfully identified (50). This technique is useful to identify phylogenetically unknown mRNA-expressers in environments.…”

“…The ideal conditions may differ based on the sample of interest. Recently, cell sorting following mRNA-FISH was reported, as described above (50). In that report, mRNA-FISH was carried out for cells embedded in agarose on a glass slide, and the cells were recovered from the slide into suspension in pre-warmed PBS buffer.…”