Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria

Area of Science:

• Microbiology
• Bacterial Pathogenesis
• Molecular Biology

Background:

• Bacterial populations display heterogeneous growth rates, impacting antibiotic efficacy.
• Identifying slow-growing bacteria within host tissues is challenging, hindering research into growth-slowing pathways.

Purpose of the Study:

• To develop a novel fluorescent reporter to identify and study slowly dividing bacteria within host tissues.
• To overcome limitations of existing reporters like TIMER for fast-dividing bacterial species.

Main Methods:

• Development of a novel TIMER variant, DsRed42, a slow-folding fluorescent protein.
• Testing DsRed42 fluorescence accumulation in late stationary phase cultures of Escherichia coli and Yersinia pseudotuberculosis.
• Assessing DsRed42 signal accumulation during nitric oxide (NO) exposure.
• In vivo analysis of DsRed42 signal in a mouse model of Y. pseudotuberculosis deep tissue infection.

Main Results:

• DsRed42 accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis.
• DsRed42 signal accumulates upon exposure to nitric oxide (NO), indicating detection of growth-arrested cells.
• In vivo, DsRed42 signal was detected in Y. pseudotuberculosis infections, primarily in stationary phase bacteria.
• No significant overlap was observed between DsRed42 signal and NO-exposed bacteria in vivo, suggesting transient NO stress.

Conclusions:

• The novel DsRed42 variant enables identification of slow-growing bacterial subpopulations.
• DsRed42 is a valuable tool for studying bacterial heterogeneity in fast-dividing species within host environments.
• Findings suggest transient NO stress allows bacteria to recover and resume replication.