Project Title

Role of STM1987 and ArtI in Arginine Response of Salmonella Typhimurium

Authors' Affiliations

Deeba Mohseni, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN. Joseph Headrick, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN. Erik Petersen, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN.

Location

Culp Room 217

Start Date

4-6-2022 10:15 AM

End Date

4-6-2022 10:30 AM

Faculty Sponsor’s Department

Health Sciences

Name of Project's Faculty Sponsor

Erik Petersen

Additional Sponsors

Sean Fox

Classification of First Author

Undergraduate Student

Competition Type

Non-Competitive

Type

Boland Symposium

Project's Category

Microbiology

Abstract or Artist's Statement

Antibiotic resistance is becoming an increasing problem in healthcare. Cyclic-di-GMP, a common bacterial second messenger, has been thought to help develop virulence and biofilms in bacteria. By being able to dysregulate cyclic-di-GMP production, antibiotic resistance may be better combatted. STM1987, an arginine responsive diguanylate cyclase, dimerizes and generates the bacterial second messenger cyclic-di-GMP in response to the amino acid L-arginine in a pathway that requires the periplasmic arginine-binding protein ArtI. Their responses to arginine and when they dimerize could help clarify this pathway, so I sought to develop a periplasmic dimerization sensor to better monitor these biochemical interactions. Similar to STM1987, the ToxR transcriptional regulator from Vibrio cholera is also activated by dimerization. By switching out the periplasmic domain of ToxR for the periplasmic regions of interest, I can better evaluate the cyclic-di-GMP response to L-arginine. Using a technique called Gibson cloning, a ToxR fusion and dimerization-responsive promoter was constructed and then electroporated into wild-type Salmonella Typhimurium. Overnight cultures of Salmonella with the appropriate plasmid were grown and then restarted in media without amino acids. I began by optimizing induction conditions for the ToxR-periplasmic domain fusion to determine the appropriate level of fusion protein to monitor dimerization. Different vectors for the transcriptional reporter were also tested to identify a readable level. Using an mCherry fluorescent reporter and different L-arginine additions, responses were measured on a plate reader by checking the fluorescence of every sample. Any change in response to L-arginine is compared to a negative control ToxR fusion that does not contain a periplasmic component, allowing me to identify the STM1987- or ArtI-specific responses. While I am still working on optimizing the appropriate testing conditions, the results so far are consistent with the addition of L-arginine increasing fluorescence in the STM1987-ToxR fusion strain. This suggests that the periplasmic domain of STM1987 is dimerizing in response to L-arginine, activating ToxR-mediated mCherry expression. Future plans for the system are to finalize testing conditions for the L-arginine response, determine the ideal L-arginine concentration for signaling, and begin testing point mutants of ArtI and STM1987 to further define this interaction.

This document is currently not available here.

Share

COinS
 
Apr 6th, 10:15 AM Apr 6th, 10:30 AM

Role of STM1987 and ArtI in Arginine Response of Salmonella Typhimurium

Culp Room 217

Antibiotic resistance is becoming an increasing problem in healthcare. Cyclic-di-GMP, a common bacterial second messenger, has been thought to help develop virulence and biofilms in bacteria. By being able to dysregulate cyclic-di-GMP production, antibiotic resistance may be better combatted. STM1987, an arginine responsive diguanylate cyclase, dimerizes and generates the bacterial second messenger cyclic-di-GMP in response to the amino acid L-arginine in a pathway that requires the periplasmic arginine-binding protein ArtI. Their responses to arginine and when they dimerize could help clarify this pathway, so I sought to develop a periplasmic dimerization sensor to better monitor these biochemical interactions. Similar to STM1987, the ToxR transcriptional regulator from Vibrio cholera is also activated by dimerization. By switching out the periplasmic domain of ToxR for the periplasmic regions of interest, I can better evaluate the cyclic-di-GMP response to L-arginine. Using a technique called Gibson cloning, a ToxR fusion and dimerization-responsive promoter was constructed and then electroporated into wild-type Salmonella Typhimurium. Overnight cultures of Salmonella with the appropriate plasmid were grown and then restarted in media without amino acids. I began by optimizing induction conditions for the ToxR-periplasmic domain fusion to determine the appropriate level of fusion protein to monitor dimerization. Different vectors for the transcriptional reporter were also tested to identify a readable level. Using an mCherry fluorescent reporter and different L-arginine additions, responses were measured on a plate reader by checking the fluorescence of every sample. Any change in response to L-arginine is compared to a negative control ToxR fusion that does not contain a periplasmic component, allowing me to identify the STM1987- or ArtI-specific responses. While I am still working on optimizing the appropriate testing conditions, the results so far are consistent with the addition of L-arginine increasing fluorescence in the STM1987-ToxR fusion strain. This suggests that the periplasmic domain of STM1987 is dimerizing in response to L-arginine, activating ToxR-mediated mCherry expression. Future plans for the system are to finalize testing conditions for the L-arginine response, determine the ideal L-arginine concentration for signaling, and begin testing point mutants of ArtI and STM1987 to further define this interaction.