Identifying Difficidin in the Supernatant of Bacillus velezensis using High Resolution Mass Spectrometry

Abstract

Difficidin is a naturally occurring extracellular antibiotic produced by Bacillus velezensis. The dfn operon encodes for the difficidin biosynthesis proteins. This operon (dfn) is transcriptionally regulated by intrinsic transcription termination sites that are spread throughout the 70 kilobase operon and that limit dfn expression. LoaP is a specialized transcription elongation protein that promotes readthrough of these terminators. We hypothesize that LoaP associates with RNA polymerase to promote readthrough of the termination sites; however, the LoaP regulatory mechanism is still unknown. We have constructed different bacterial strains containing mutations in LoaP. Yet, we do not have an assay for measuring the impact of these mutations on the regulation of the dfn operon. This study seeks to address this problem by developing a method to quantify difficidin. Specifically, we will establish a mass spectrometry-based detection assay to identify the levels of difficidin under various conditions. Three strains - wild-type, ΔloaP, and Δdfn - were utilized to test native levels, low levels, and absence of difficidin, respectively. Strains were grown overnight to an OD600 of 2 in 150 mL of rich medium (LB broth). Cells were pelleted and extraction of difficidin was performed by incubation of the supernatant at 4°C overnight with Amberlite XAD-16 resin. Metabolites were then eluted from the resin using 8 mL of methanol and analyzed by mass spectrometry. Data for the three strains were compared against each other to identify the retention time of difficidin. The WT strain had an intense cluster of peaks at a retention time of 17-19 minutes. These peaks completely disappeared in the Δdfn strain. The disappearance of a peak in the Δdfn strain that is present in the WT strain is a strong indication that difficidin has been identified. Further tests using the fragmentation of the metabolites at the retention time determined are required to confirm whether difficidin has been identified by mass spectrometry.