Project Title

A Stem-Loop Secondary Structure Influencing Expression Of The Post-Transcriptional Regulator, RsmA, In Pseudomonas aeruginosa

Authors' Affiliations

Ian Miller- Department of Health Sciences, East Tennessee State University Christopher Pritchett- Department of Health Sciences, East Tennessee State University

Location

RIPSHIN MTN. ROOM 130

Start Date

4-4-2018 11:40 AM

End Date

4-4-2018 11:55 AM

Name of Project's Faculty Sponsor

Christopher Pritchett

Faculty Sponsor's Department

Department of Health Sciences

Type

Oral Presentation

Classification of First Author

Graduate Student-Master’s

Project's Category

Biomedical and Health Sciences

Abstract Text

Pseudomonas aeruginosa is an infectious Gram-negative bacillus that is found in environments ranging from aerobic to anaerobic, soil to water, plant tissues to human tissues, and even found thriving on plastics and medical implant devices. P. aeruginosa is a major concern for individuals who have cystic fibrosis, chronic obstructive pulmonary disorder, diabetes, have recently undergone surgery, have recently experienced severe burns, or have experienced other ailments that resulted in a compromised immune system, such as Human Immunodeficiency Virus (HIV). P. aeruginosa evades the host immune response by expressing a myriad of virulence factors, and it is through stringent gene regulation of virulence factors that allow P. aeruginosa to initiate acute infections and persist as a chronic infection of its host. The expression of virulence factors is controlled by a complex regulatory system comprised of Two-Component Systems (TCS), post-transcriptional regulators, small non-coding RNAs (sRNA), and others. A significant post-transcriptional regulator involved in this regulatory network is the Regulator of Secondary Metabolites (RsmA). RsmA belongs to the CsrA family of mRNA binding proteins found in many Gram-negative bacteria. Much is known about the targets of RsmA and its functions; however, little is known about how RsmA itself is regulated. Leader sequences, 5’ and 3’, have been demonstrated to have regulatory roles. Using bioinformatics, we have observed potential for the formation of a stem-loop secondary structure in the 5’ leader sequence of rsmA. We propose that this stem-loop plays an important role in the expression of RsmA in P. aeruginosa. In this study, we constructed rsmA leader fusions using the lacUV5 promoter and lacZ reporter to measure translation with and without the secondary structure present. Secondly, we introduced point mutations in the stem of the stem-loop of the leader fusions to disrupt the formation of the stem-loop. Finally, we performed Site-Directed Mutagenesis on the rsmA leader to examine protein levels in vivo via western blot analysis using an HA-tagged rsmA. Our data shows that when the stem-loop formation is disrupted or deleted, translation of RsmA increases. This data suggests that the stem-loop provides a regulatory function in the expression of RsmA.

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Apr 4th, 11:40 AM Apr 4th, 11:55 AM

A Stem-Loop Secondary Structure Influencing Expression Of The Post-Transcriptional Regulator, RsmA, In Pseudomonas aeruginosa

RIPSHIN MTN. ROOM 130

Pseudomonas aeruginosa is an infectious Gram-negative bacillus that is found in environments ranging from aerobic to anaerobic, soil to water, plant tissues to human tissues, and even found thriving on plastics and medical implant devices. P. aeruginosa is a major concern for individuals who have cystic fibrosis, chronic obstructive pulmonary disorder, diabetes, have recently undergone surgery, have recently experienced severe burns, or have experienced other ailments that resulted in a compromised immune system, such as Human Immunodeficiency Virus (HIV). P. aeruginosa evades the host immune response by expressing a myriad of virulence factors, and it is through stringent gene regulation of virulence factors that allow P. aeruginosa to initiate acute infections and persist as a chronic infection of its host. The expression of virulence factors is controlled by a complex regulatory system comprised of Two-Component Systems (TCS), post-transcriptional regulators, small non-coding RNAs (sRNA), and others. A significant post-transcriptional regulator involved in this regulatory network is the Regulator of Secondary Metabolites (RsmA). RsmA belongs to the CsrA family of mRNA binding proteins found in many Gram-negative bacteria. Much is known about the targets of RsmA and its functions; however, little is known about how RsmA itself is regulated. Leader sequences, 5’ and 3’, have been demonstrated to have regulatory roles. Using bioinformatics, we have observed potential for the formation of a stem-loop secondary structure in the 5’ leader sequence of rsmA. We propose that this stem-loop plays an important role in the expression of RsmA in P. aeruginosa. In this study, we constructed rsmA leader fusions using the lacUV5 promoter and lacZ reporter to measure translation with and without the secondary structure present. Secondly, we introduced point mutations in the stem of the stem-loop of the leader fusions to disrupt the formation of the stem-loop. Finally, we performed Site-Directed Mutagenesis on the rsmA leader to examine protein levels in vivo via western blot analysis using an HA-tagged rsmA. Our data shows that when the stem-loop formation is disrupted or deleted, translation of RsmA increases. This data suggests that the stem-loop provides a regulatory function in the expression of RsmA.