Using <em>Mycobacterium smegmatis</em> as a Bioindicator for Zinc-limited Growth Conditions in Mycobacteria

Many bacteria build alternative ribosomes in Zn²⁺-limiting growth conditions by replacing Zn²⁺-binding ribosomal proteins with Zn²⁺-independent paralogs. Defining a system to study these alternative ribosomes has proven difficult because Zn²⁺ contamination in the laboratory is common. To address this issue, chelating agents are sometimes added to growth media, but this approach convolutes the biological response to gradual Zn²⁺limitation and is associated with ribosome hibernation. Here, detailed instructions are outlined for preparing media and seeding cultures for Zn²⁺-limited growth without adding chelators. Following this method, the model bacterium, Mycobacterium smegmatis, undergoes morphogenesis, which depends on alternative ribosomes. Because morphogenesis is tractable and only occurs in Zn²⁺-limiting conditions, M. smegmatis can be used as a bioindicator to verify biologically relevant growth conditions. Three bioindicator phenotypes (cell density, cell length, and coenzyme F420 fluorescence) that indicate Zn²⁺ limitation in the wild-type are described, and changes in these bioindicators for a deletion mutant that cannot build alternative ribosomes are outlined. Since trace Zn²⁺ contamination is difficult to control for each batch of media, and precise quantification of Zn²⁺ in each media preparation is overly burdensome, following this bioindicator phenotype is an accessible way to validate the preparation of Zn²⁺-limited growth media. To help identify proper conditions for Zn²⁺-limiting growth and alternative ribosome production, changes in the bioindicator phenotypes were profiled for Zn²⁺-contaminated or severely Zn²⁺-depleted preparations of Zn²⁺-limited media as well. Further details to achieve Zn²⁺-limiting growth and alternative ribosome production in M. tuberculosis are presented, along with the associated bioindicator phenotype. Overall, the detailed instructions and bioindicator phenotypes described here will help standardize the production of translationally active alternative ribosomes in mycobacteria.