Isolation and Molecular Characterization of a Fluorescence-Emitting Bacterium from the Gut of Earthworm, Perionyx excavatus (Perrier, 1872), and Its Symbiotic Association in Earthworm Regeneration

Abstract

Background: Earthworms are the most important soil invertebrate and have been used for exploring potent therapeutic agents. Perionyx excavatus (Perrier, 1872) is a tropical earthworm species widely used in vermicomposting worldwide, which has a prodigious regeneration capability. Objectives: This study was carried out to isolate fluorescence-emitting microbiota from the gut of earthworm, P. excavatus, and characterize the fluorophores along with finding the symbiotic association in regenerating worms. Methods: Gram staining was performed to selectively identify the fluorescence-emitting bacterial colonies and further confirmed using 16S ribosomal RNA (rRNA) sequencing. Thin layer chromatography (TLC), ultraviolet-visible spectroscopy (UV-Vis), and Fourier-transform infrared spectroscopy (FTIR) were carried out to separate and characterize the isolated fluorophores. Further, fluorescence microscopy was used to examine the autofluorescence property. Results: Gram staining results showed that the isolated fluorescence-emitting gut bacterium is Gram-positive and it is rod-shaped. Molecular characterization confirmed that the isolated fluorescence-emitting bacterium is Priestia filamentosa. Moreover, two fluorophores responsible for emitting fluorescence, known as unknown fluorescent compound 1 (CF1), and fluorescent compound 2 (CF2) were separated and partially characterized. Anti-inflammatory assays confirm that the fluorophores have very good anti-inflammatory properties. In addition, these fluorophores were expressed in the epithelial cell layer of earthworm, P. excavatus, during regeneration. Notably, bright, intense fluorescence was observed high in regenerating worms compared to control worms. Conclusions: Hence, these two fluorophores will be studied in-depth in the context of structural conformation, chemical composition, and functional characteristics for potential biotechnological applications.