Multi-Omics Profiling Specifies Involvement of Alternative Ribosomal Proteins in Response to Zinc Limitation in Mycobacterium smegmatis.

Zinc ion (Zn2+) is an essential micronutrient and a potent antioxidant. However, Zn2+ is often limited in the environment. Upon Zn2+ limitation, Mycolicibacterium (basonym: Mycobacterium) smegmatis (Msm) undergoes a morphogenesis, which relies on alternative ribosomal proteins (AltRPs); i.e., Zn2+-independent paralogues of Zn2+-dependent ribosomal proteins. However, the underlying physiological changes triggered by Zn2+ 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 Zn2+ limitation, by comparing the transcriptomes and proteomes of Zn2+-limited and Zn2+-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 Zn2+ is limited in both mycobacteria. By comparing the multi-omics analysis of a knockout mutant lacking AltRPs (ΔaltRP) to the Msm wild type strain, we specify the involvement of AltRPs in the response to Zn2+ limitation. Our results show that AltRP expression in Msm does not affect the conserved oxidative stress response during Zn2+ 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 Zn2+-dependent paralogues; they are also involved in the transcriptomic response to the Zn2+-limited environment.

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis.

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 (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ 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, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ 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 Zn2+ 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 Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-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 Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

Phenotypic Assessment Suggests Multiple Start Codons for HetN, an Inhibitor of Heterocyst Differentiation, in Anabaena sp. Strain PCC 7120.

Multicellular organisms must carefully regulate the timing, number, and location of specialized cellular development. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are interspersed between vegetative cells in a periodic pattern to achieve an optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors, PatS and HetN. PatS functions to create a de novo pattern from a homogenous field of undifferentiated cells, while HetN maintains the pattern throughout subsequent growth. Both PatS and HetN harbor the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes the interpretation of inhibitory domains relatively simple, HetN is a 287-amino-acid protein with multiple functional regions. Previous work suggested the possibility of a truncated form of HetN containing the ERGSGR motif as the source of the HetN-derived inhibitory signal. In this work, we present evidence that the glutamate of the ERGSGR motif is required for proper HetN inhibition of heterocysts. Mutational analysis and subcellular localization indicate that the gene encoding HetN uses two methionine start codons (M1 and M119) to encode two protein forms: M1 is required for protein localization, while M119 is primarily responsible for inhibitory function. Finally, we demonstrate that patS and hetN are not functionally equivalent when expressed from the other gene's regulatory sequences. Taken together, these results help clarify the functional forms of HetN and will help refine future work defining a HetN-derived inhibitory signal in this model of one-dimensional periodic patterning.IMPORTANCE The proper placement of different cell types during a developmental program requires the creation and maintenance of a biological pattern to define the cells that will differentiate. Here we show that the HetN inhibitor, responsible for pattern maintenance of specialized nitrogen-fixing heterocyst cells in the filamentous cyanobacterium Anabaena, may be produced from two different start methionine codons. This work demonstrates that the two start sites are individually involved in a different HetN function, either membrane localization or inhibition of cellular differentiation.

Alternative ribosomal proteins are required for growth and morphogenesis of Mycobacterium smegmatis under zinc limiting conditions.

Zinc is an essential micronutrient required for proper structure and function of many proteins. Bacteria regularly encounter zinc depletion and have evolved diverse mechanisms to continue growth when zinc is limited, including the expression of zinc-independent paralogs of zinc-binding proteins. Mycobacteria have a conserved operon encoding four zinc-independent alternative ribosomal proteins (AltRPs) that are expressed when zinc is depleted. It is unknown if mycobacterial AltRPs replace their primary paralogs in the ribosome and maintain protein synthesis under zinc-limited conditions, and if such replacements contribute to their physiology. This study shows that AltRPs from Mycobacterium smegmatis are essential for growth when zinc ion is scarce. Specifically, the deletion mutant of this operon (ΔaltRP) is unable to grow in media containing a high-affinity zinc chelator, while growth of the wild type strain is unaffected under the same conditions. However, when zinc is gradually depleted during growth in zinc-limited medium, the ΔaltRP mutant maintains the same growth rate as seen for the wild type strain. In contrast to M. smegmatis grown with sufficient zinc supplementation that forms shorter cells when transitioning from logarithmic to stationary phase, M. smegmatis deficient for zinc elongates after the expression of AltRPs in late logarithmic phase. These zinc-depleted bacteria also exhibit a remarkable morphology characterized by a condensed chromosome, increased number of polyphosphate granules, and distinct appearance of lipid bodies and the cell wall compared to the zinc-replete cells. However, the ΔaltRP cells fail to elongate and transition into the zinc-limited morphotype, resembling the wild type zinc-replete bacteria instead. Therefore, the altRP operon in M. smegmatis has a vital role in continuation of growth when zinc is scarce and in triggering specific morphogenesis during the adaptation to zinc limitation, suggesting that AltRPs can functionally replace their zinc-dependent paralogs, but also contribute to mycobacterial physiology in a unique way.

Zinc regulates a switch between primary and alternative S18 ribosomal proteins in Mycobacterium tuberculosis.

The Mycobacterium tuberculosis genome encodes five putative 'alternative' ribosomal proteins whose expression is repressed at high Zn(2+) concentration. Each alternative protein has a primary homologue that is predicted to bind Zn(2+). We hypothesized that zinc triggers a switch between these paired homologous proteins and therefore chose one of these pairs, S18-1/S18-2, to study mechanisms of the predicted competition for their incorporation into ribosomes. Our data show that Zn(2+)-depletion causes accumulation of both S18-2 mRNA and protein. In contrast, S18-1 mRNA levels are unchanged to slightly elevated under Zn(2+)-limited conditions. However, the amount of S18-1 protein is markedly decreased. We further demonstrate that both S18 proteins interact with ribosomal protein S6, a committed step in ribosome biogenesis. Zn(2+) is absolutely required for the S18-1/S6 interaction while it is dispensable for S18-2/S6 dimer formation. These data suggest a model in which S18-1 is the dominant ribosome constituent in high zinc conditions, e.g. inside of phagosomes, but that it can be replaced by S18-2 when zinc is deficient, e.g. in the extracellular milieu. Consequently, Zn(2+)-depletion may serve as a signal for building alternative ribosomes when M. tuberculosis is released from macrophages, to allow survival in the extracellular environment.