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

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.

Genus Pseudonocardia : What we know about its biological properties, abilities and current application in biotechnology

“…For example, in a study by Yamamoto et al (2018), medium pH was 7.0–7.3, and incubation was performed at 28℃ while shaking at 120 rpm. Besides, Mengyan et al reported that their conditions for growing Gram‐positive bacteria included fixation of washed cells in 1 ml of ethanol/phosphate‐buffered saline (PBS) (1:1, v/v) and incubation at room temperature for 2 h. Also, for Gram‐negative bacteria, resuspension was done in 1 ml of 4% paraformaldehyde and fixation was performed at 4℃ for 3 h. After fixation, all cells were washed twice with 500 μl of 1X PBS and stored at −20℃ (Li, Yang, et al, 2018). In a study by Shan et al, the isolation media were supplemented with 20 μg/ml of nystatin to suppress the fungal growth.…”

“…After fixation, all cells were washed twice with 500 μL of 1 X PBS and stored at -20°C (Li et al 2018b). In a study by Shan et al, the isolation media were supplemented with 20 g/mL of nystatin to suppress the fungal growth.…”

“…According to some studies, discrepancies and biases may occur between the results of culture-dependent and culture-independent techniques using the traditional isolation method. However, the use of fluorescence in situ hybridization (FISH) or modern flow cytometry (FCM) technology reduces biases, enabling the in situ identification of Pseudonocardia species (Li, Yang, et al, 2018).…”

Synchronic Biotransformation of 1,4-Dioxane and 1,1-Dichloroethylene by a Gram-Negative Propanotroph Azoarcus sp. DD4

“…Because of its extreme hydrophilicity and chemical stability, dioxane is resistant to conventional physical–chemical treatments . Though a plethora of dioxane-metabolizing microbes are available, − their application for in situ bioremediation has been greatly stymied by the facts that (1) trace levels of dioxane (parts per billion) in the field are insufficient to sustain the growth of the amended metabolizers or stimulate the expression of responsible enzymes, (2) most isolates are Gram-positive filamentous Actinomycetes (Figure A) that are inclined to form clumps (Figure S1) that presumably prevent efficient subsurface distribution, and (3) co-occurring chlorinated compounds, especially 1,1-DCE, inhibit or even cease the removal of dioxane by archetypic dioxane degraders (e.g., Pseudonocardia dioxanivorans CB1190). , In addition, 1,1-DCE is also a recalcitrant compound partially because of the cellular toxicity derived from its biotransformation metabolite, 1,1-DCE epoxide. , Given the high coexistence frequency and persistence of dioxane and 1,1-DCE, the development of an effective remediation approach to synchronize the removal of both contaminants is urgently needed.…”

Field Test of On-Site Treatment of 1,4-Dioxane-Contaminated Groundwater Using <i>Pseudonocardia</i> sp. D17

“…By contrast, in parallel with the findings of bacterial strains capable of degrading 1,4-dioxane in different environments over the last two decades [3,[15][16][17], previous studies have assessed the feasibility of several bioremediation strategies to treat 1,4-dioxane-contaminated groundwater, including natural attenuation, biostimulation, and bioaugmentation. The occurrence of natural attenuation, which depends on the intrinsic degrading abilities of indigenous microorganisms, has been verified in lab-scale microcosm studies [18][19][20] and in field surveys [21,22], indicating monitored natural attenuation as a potential remedial strategy.…”