Visualization of dormancy induced by ribosome hibernation in zinc-starved mycobacteria
Jamie Corro1,2 [graduate student]; Yunlong Li2; Richard Cole1,2
Biomedical Sciences Department, State University at Albany1
Wadsworth Center, New York State Department of Health, Albany, New York2
Ribosomes are essential biomolecular machines utilized for protein synthesis in every kingdom of life. The 70S bacterial ribosome is composed of two subunits: the small 30S subunit with 21 proteins and 16S rRNA, and the large 50S subunit with 36 proteins, 23S rRNA, and 5S rRNA. In many bacterial species, including mycobacteria, zinc differentially regulates ribosome assembly: the ribosomal proteins with the zinc-binding CXXC motif, called C+ r-proteins, are replaced by their motif-free C- paralogues under zinc starvation conditions. Previous studies discovered that the C- ribosome is targeted for hibernation by a mycobacterial-specific protein Y (Mpy), which is recruited to the ribosome by an Mpy recruitment factor (Mrf). Mrf expression is transcriptionally and post-transcriptionally regulated by zinc in a manner that Mrf is stabilized at a lower zinc concentration than that inducing C+ to C- replacements. To further understand the physiological consequences of ribosome hibernation we constructed a reporter strain of Mycobacterium smegmatis that expresses Dendra2 (a green fluorescent protein) fusion of Mrf when cells experienced conditions for ribosome hibernation. This reporter construct allowed us to microscopically visualize the behavior of cells with hibernating ribosomes in real-time on a microfluidic platform at a single-cell level. Our findings suggest that mycobacterial cells with hibernating ribosome remain growth-arrested but viable. The results offer a new molecular framework for studying dormancy in mycobacteria.