Abstract
It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed42, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed42 accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show DsRed42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed42 signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed42 variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.
Highlights
Identifying differences in the growth rates of individual cells can provide significant information in many biological systems
We have generated a variant of TIMER that can be used to mark slow-growing subsets of Yersinia pseudotuberculosis, which has a relatively short division time, similar to E. coli
New DsRed variant to detect slowed growth in bacteria sufficient to promote DsRed42 signal accumulation in culture, within host tissues, DsRed42 signal correlates with a stationary phase reporter
Summary
Identifying differences in the growth rates of individual cells can provide significant information in many biological systems. Many research groups have begun to develop tools to differentiate between faster and slower dividing cells, which enable downstream studies to identify additional differences within subpopulations. Many of these tools, such as slow-folding fluorescent proteins, were initially developed to study eukaryotic systems [4,5]. Several of these approaches have been adapted to study bacterial populations, primarily to better understand the pathways that lead to the formation of slow-growing subsets of bacteria with reduced antibiotic susceptibility. Bacterial population numbers can contract and expand dramatically during infection [13,14], which can dilute fluorescent signals or plasmids very early after inoculation and result in a complete loss of signal
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