Undergraduate Research Day 2024

Permanent URI for this collectionhttp://hdl.handle.net/1903/31825

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Author: search.filters.author.Jenkins, Conor

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    Identifying Difficidin in the Supernatant of Bacillus velezensis using High Resolution Mass Spectrometry
    (2024) Nasisi, Benjamin; Jenkins, Conor; Jermain, Madison; Winkler, Wade
    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.
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    Investigating the effect of biofilm pathways on Bacillus subtilis anti-termination
    (2024-04-14) Robertello, Nathan; Tran, Thao; Jenkins, Conor; Winkler, Wade
    Biofilms are communities of bacteria ensconced in a blanket of exopolysaccharides and proteins. They can form in a variety of clinically important contexts such as the surfaces of teeth, contact lenses, and medical implants. Biofilm communities are more resistant to antibiotics than planktonic cells. Hence, it is important to understand the underlying mechanisms which promote biofilm formation. Bacillus subtilis is an important model system for studying biofilm-synthesis genes. The biosynthetic genes for B. subtilis exopolysaccharide are found in one, unusually long, operon (eps). A previously discovered processive anti-termination (PA) mechanism promotes readthrough of transcription termination sites within this operon. PA occurs when regulatory factor(s) modify the transcription elongation complex (TEC) such that it becomes resistant to downstream termination events. The PA mechanism of the eps operon requires a highly conserved RNA element called eps-associated RNA (EAR). It is not currently known how EAR exerts its influence on the TEC. The eps operon itself is only one of many biosynthetic genes necessary to biofilm formation and maintenance. In this study, we investigate the impact of a few key regulatory pathways on EAR anti-termination. We combined deletions of genes involved in these pathways with a genetic reporter assay for EAR anti-termination activity. Our preliminary data do not support a connection between these genes and the PA efficiency of EAR. These data suggest that anti-termination of the eps operon is not necessarily coupled to other known biofilm-regulatory pathways.