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Zinc ion (Zn 2+ ) is an essential micronutrient and a potent antioxidant. However, Zn 2+ is often limited in the environment. Upon Zn 2+ limitation, Mycolicibacterium (basonym: Mycobacterium ) smegmatis (Msm) undergoes a morphogenesis, which relies on alternative ribosomal proteins (AltRPs); i.e., Zn 2+ -independent paralogues of Zn 2+ -dependent ribosomal proteins. However, the underlying physiological changes triggered by Zn 2+ limitation and how AltRPs contribute to these changes were not known. In this study, we expand the knowledge of mechanisms utilized by Msm to endure Zn 2+ limitation, by comparing the transcriptomes and proteomes of Zn 2+ -limited and Zn 2+ -replete Msm . We further compare, corroborate and contrast our results to those reported for the pathogenic mycobacterium, M. tuberculosis , which highlighted conservation of the upregulated oxidative stress response when Zn 2+ is limited in both mycobacteria. By comparing the multi-omics analysis of a knockout mutant lacking AltRPs (Δ altRP ) to the Msm wild type strain, we specify the involvement of AltRPs in the response to Zn 2+ limitation. Our results show that AltRP expression in Msm does not affect the conserved oxidative stress response during Zn 2+ limitation observed in mycobacteria, but AltRPs do significantly impact expression patterns of numerous genes that may be involved in morphogenesis or other adaptive responses. We conclude that AltRPs are not only important as functional replacements for their Zn 2+ -dependent paralogues; they are also involved in the transcriptomic response to the Zn 2+ -limited environment. 

Mycobacterium tuberculosis ( Mtb ) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn 2+ ) availability as a likely driver of bacterial phenotypic heterogeneity in vivo . Zn 2+ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn 2+ -limitation is an environmental cue sensed by Mtb , as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn 2+ -limited Mtb in vivo . Prolonged Zn 2+ limitation leads to numerous physiological changes in vitro , including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn 2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn 2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn 2+ -limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn 2+ -limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn 2+ availability likely plays a key role during early interactions with host cells.