Analysis of the expression of membrane protein inseratses YidC1 and YidC2 in Streptococcus mutans

Dilan Patel

Authors:  Dilan Patel, Surabhi Mishra, Joyce Morales, L. Jeannine Brady

Faculty Mentor: Jeannine Brady

College:  College of Dentistry

Abstract

YidC belongs to a family of membrane-localized chaperone-insertases present in bacteria, chloroplasts and mitochondria. It was discovered during study of the cariogenic bacterium Streptococcus mutans, that Gram-positive organisms encode two YidC paralogs. Identifying the respective functions of the dual paralogs is an important research goal. Elimination of Escherichia coli yidC is lethal. Elimination of S. mutans yidC1 compared to yidC2 has different phenotypic consequences, but both cannot be deleted simultaneously. S. mutans yidC1 and yidC2 reside at different loci under the control of separate promoters. The purpose of this work is to study the relative expression of yidC1 compared to yidC2 under various environmental conditions. To measure gene expression under the control of the yidC1 and yidC2 promoters, respectively, each promoter region was amplified by polymerase chain reaction using S. mutans genomic DNA as the template. We are now constructing ~700 bp promoter fusions in frame with the lacZ reporter gene in the pMZ plasmid. The engineered plasmids will next be transformed into S. mutans strain UA159, using competence stimulating peptide with selection on 1 mg/ml kanamycin. This will enable b-galactosidase assays of the engineered reporters strains to be performed under defined experimental conditions to reflect specific expression from the yidC1 compared to yidC2 gene promoter.

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Yusuf Mehkri
Yusuf Mehkri (@guest_1090)
1 year ago

If I understood correctly, the main goal is to understand the relative expression of YIDC1 and YIDC2 because these proteins are highly involved in bacterial membrane biogenesis. Let’s say you figure this out for different environmental conditions. How can these findings now be translated clinically in the future.

Dilan Patel
Dilan Patel (@guest_5816)
Reply to  Yusuf Mehkri
1 year ago

Yes exactly! One of the main goals of this study is to analyze the relative expression of yidC1 and yidC2 in the UA159 strain of S. mutans. This bacterium has a dual yidC paralog system and as a result is able to survive in conditions where one of the yidC paralogs is deleted, contrary to the lethal outcome in E.coli. Both yidC1 and yidC2 and integral to the bacterium’s membrane biogenesis and its overall pathogenicity, therefore determining the relationship between yidC1 and yidC2 within S. mutans under different environmental conditions has large clinical potential. Although this research is still far from being translated clinically, creating a safe oral medication that disrupts the membrane biogenesis of S. mutans while mimicking uncomfortable environmental conditions for the bacterium would be the most intuitive clinal goal for my research.

Dilan Patel
Dilan Patel (@guest_5868)
Reply to  Dilan Patel
1 year ago

If you have any other questions please feel free to contact me. My email is dilanpatel@ufl.edu.

Kelly Rice
Kelly Rice (@guest_1518)
1 year ago

A very interesting project! Have the yidC promoters been mapped for their regulatory elements? Also are you trying to generate transcriptional or translational fusions? Nice presentation!

Dilan Patel
Dilan Patel (@guest_6434)
Reply to  Kelly Rice
1 year ago

The structure/function relationship of membrane protein insertases YidC1 and YidC2 is actually being studied by another individual in the Brady Lab at the College of Dentistry. Surabhi Mishra’s most recent paper, “Membrane proteomic analysis reveals overlapping and independent functions of Streptococcus mutans Ffh, YidC1, and YidC2”, revels a more in depth study in regards to YidC1 and YidC2 structure and function. Please feel free to read her paper and contact me if you have any other questions that I may be able to help with.

As far as your second question my study involved generating a transcriptional fusion in which my constructed plasmid incorporated a yidC1/C2 promoter region (upstream) in frame with the lacZ gene (downstream) in a pMZ plasmid to measure and assess the relative expression of the promoter.

Elena Yarmla
Elena Yarmla (@guest_3364)
1 year ago

Good job!

Jeannine Brady
Jeannine Brady (@guest_4194)
1 year ago

Thank you for helping out with this project Dilan. Good job.

Rohan Gupta
Rohan Gupta (@guest_7594)
1 year ago

Great work Dilan!