Several projects in our lab focus on creating approaches to detect growth differences within populations of bacteria. We plan to use these tools to determine if host-derived stresses are sufficient to the slow the growth rates of individual bacteria within host tissues, and if this impacts the antibiotic susceptibility of individual bacteria:
TIMER fluorescent protein
A slowly folding DsRed derivative is used to detect individual bacteria with slowed growth. TIMER has been optimized for Y. pseudotuberculosis through a series of mutations, so that red fluorescent signal accumulates in slow growing cells. We have been characterizing the accumulation of signal in bacteriological media to determine which conditions result in detectable signal, and have been comparing TIMER fluorescence to additional reporter signals. We have also been comparing TIMER fluorescence signal to reporter signals within tissues, to determine if the stressed cells are also slow-growing.
The tet operon has been modified so that addition of a tetracycline derivative results in repression of additional mCherry expression. Fluorescence dilution is used to detect bacterial cell division events after the addition of tetracycline. This construct has been characterized in bacteriological media, and we’re beginning in vivo experiments to detect cell division events within host tissues.
A ribosomal promoter (PS10) has been fused to a destabilized gfp so that ribosomal content can be assessed within individual cells. Faster growing cells should have heightened ribosomal content relative to slow growing cells, providing a way of also detecting a faster growing population. Additionally, because many antibiotics target ribosomes, this can also indicate how much target is present within individual cells, and potentially help explain susceptibility differences.