Altered Siderophore-production in a Pseudomonas aeruginosa lrp Mutant
Disciplines
Microbiology
Abstract (300 words maximum)
In Pseudomonas aeruginosa, Lrp has only been confirmed to regulate dadAX, the alanine biosynthesis operon, classifying it as a local regulator. This differs from the global Lrp regulation observed in E. coli. Unpublished data from the Griffin lab suggests that Lrp may be involved in global regulation in P. aeruginosa under conditions of nutrient depletion and acquisition. The two major siderophores of P. aeruginosa are pyoverdine and pyochelin. These siderophores scavenge for iron, which is essential for the bacteria’s survival, and transport it back to the cell when in low-iron environments. P. aeruginosa will produce pyochelin under moderate iron limitation and switch to pyoverdine under severe iron limitation. We grew wild-type PAO1 and lrp- mutant PW9942 in an iron-deficient medium for 24 hours. To detect pyochelin production, the cells are subjected to an ethyl acetate extraction then detected by spectral absorbance. Pyoverdine can be detected directly in the supernatant of P. aeruginosa cultures when grown in succinate media. After 24 hours, PAO1 and PW9942 cultures are OD standardized and centrifuged. The cell-free supernatant is carefully collected for analysis. The siderophores are detected by their reported optical absorbance and fluorescence. Pyoverdine is measured at 400nm and pyochelin at 350nm. The spectral analysis is conducted using a SpectraMax iD5. Duplicate samples are collected and averaged for each experimental trial done in triplicate. The lrp mutant exhibits a statistically significant decrease in pyoverdine fluorescence and absorbance indicative of reduced pyoverdine production. On the other hand, there does not appear to be a significant change in the production of pyochelin, reinforcing the “feast or famine” characterization of Lrp studied in other organisms as well as the need to identify the complete Lrp regulon in P. aeruginosa.
Academic department under which the project should be listed
Molecular and Cellular Biology
Primary Investigator (PI) Name
Melanie Griffin
Altered Siderophore-production in a Pseudomonas aeruginosa lrp Mutant
In Pseudomonas aeruginosa, Lrp has only been confirmed to regulate dadAX, the alanine biosynthesis operon, classifying it as a local regulator. This differs from the global Lrp regulation observed in E. coli. Unpublished data from the Griffin lab suggests that Lrp may be involved in global regulation in P. aeruginosa under conditions of nutrient depletion and acquisition. The two major siderophores of P. aeruginosa are pyoverdine and pyochelin. These siderophores scavenge for iron, which is essential for the bacteria’s survival, and transport it back to the cell when in low-iron environments. P. aeruginosa will produce pyochelin under moderate iron limitation and switch to pyoverdine under severe iron limitation. We grew wild-type PAO1 and lrp- mutant PW9942 in an iron-deficient medium for 24 hours. To detect pyochelin production, the cells are subjected to an ethyl acetate extraction then detected by spectral absorbance. Pyoverdine can be detected directly in the supernatant of P. aeruginosa cultures when grown in succinate media. After 24 hours, PAO1 and PW9942 cultures are OD standardized and centrifuged. The cell-free supernatant is carefully collected for analysis. The siderophores are detected by their reported optical absorbance and fluorescence. Pyoverdine is measured at 400nm and pyochelin at 350nm. The spectral analysis is conducted using a SpectraMax iD5. Duplicate samples are collected and averaged for each experimental trial done in triplicate. The lrp mutant exhibits a statistically significant decrease in pyoverdine fluorescence and absorbance indicative of reduced pyoverdine production. On the other hand, there does not appear to be a significant change in the production of pyochelin, reinforcing the “feast or famine” characterization of Lrp studied in other organisms as well as the need to identify the complete Lrp regulon in P. aeruginosa.