Significance: Mycobacteria as well as other bacteria remodel their ribosomes in response to zinc depletion by replacing zinc-binding ribosomal proteins with zinc-free paralogues, releasing zinc for other metabolic processes. In this study, we show that the remodeled ribosome acquires a structurally stable but functionally inactive and aminoglycoside-resistant state in zinc-starvedMycobacterium smegmatis. Conversely,M. smegmatiscells that are growth arrested in zinc-rich conditions have unstable ribosomes and reduced survival. We further provide evidence for ribosome remodeling inMycobacterium tuberculosisin host tissues, suggesting that ribosome hibernation occurs during TB infections. Our findings could offer insights into mechanisms of persistence and antibiotic tolerance of mycobacterial infections.

Mycobacterium tuberculosis (Mtb) spontaneously grows at the air-medium interface forming pellicle biofilms, which harbor more drug tolerant persisters than planktonic cultures. The underlying basis for increased persisters in Mtb biofilms is unknown. Using a Tn-seq approach, we show here that multiple genes that are necessary for fitness of Mtb cells within biofilms, but not in planktonic cultures, are also implicated in tolerance of bacilli to a diverse set of stressors and antibiotics. Thus, development of Mtb biofilms appears to be associated with an enrichment of population, in which challenging growth conditions within biofilm architecture select for cells that maintain intrinsic tolerance to exogenous stresses including antibiotic exposure. We further observed that the intrinsic drug tolerance of constituent cells of a biofilm determines the frequency of persisters. These findings together allow us to propose that the selection of elite cells during biofilm development promotes the frequency of persisters. Furthermore, probing the possibility that the population enrichment is an outcome of unique environment within biofilms, we demonstrate biofilm-specific induction in the synthesis of isonitrile lipopeptides (INLP). Mutation analysis indicates that INLP is necessary for the architecture development of Mtb biofilms. In summary, the study offers an insight into persistence of Mtb biofilms under antibiotic exposure, while identifying INLP as a potential biomarker for further investigation of this phenomenon.

Under detergent-free in vitro conditions, Mycobacterium tuberculosis , the etiological agent of tuberculosis in humans, spontaneously forms organized multicellular structures called biofilms. Moreover, in vitro biofilms of M. tuberculosis are more persistent against antibiotics than their single-cell planktonic counterparts, thereby raising questions about the occurrence of biofilms in the host tissues and their significance in persistence during chemotherapy of tuberculosis. In this article, we present arguments that extracellular M. tuberculosis in necrotizing lesions likely grows as biofilms.

Significance: Mycobacterium tuberculosis ( Mtb ) is the etiological agent of human tuberculosis (TB). Mtb can persist inside host macrophages by successfully adapting to intracellular conditions. Acquisition of balanced amounts of essential micronutrients is one such important process. Our studies have identified a metallophore produced on demand to restore Mtb zinc metabolic imbalance. These diacyl-diisonitrile lipopeptides, named kupyaphores, are specifically induced during infection and move in and out of cells to protect bacteria from host-mediated nutritional deprivation and intoxication. Furthermore, we identify an Mtb isonitrile hydratase homolog , expressed in low-zinc conditions, which probably facilitates zinc release from kupyaphores. Identification of this zinc acquisition strategy could provide opportunities in future to understand systemic zinc dysbiosis and associated manifestations in TB patients.

Summary: Most mycobacterial species spontaneously form biofilms, inducing unique growth physiologies and reducing drug sensitivity. Biofilm growth progresses through three genetically programmed stages: substratum attachment, intercellular aggregation and architecture maturation. Growth of Mycobacterium smegmatis biofilms requires multiple factors including a chaperonin (GroEL1) and a nucleoid‐associated protein (Lsr2), although how their activities are linked remains unclear. Here it is shown that Lsr2 participates in intercellular aggregation, but substratum attachment of Lsr2 mutants is unaffected, thereby genetically distinguishing these developmental stages. Further, a suppressor mutation in a glycopeptidolipid synthesis gene (mps) that results in hyperaggregation of cells and fully restores the form and functions of Δlsr2 mutant biofilms was identified. Suppression by the mps mutation is specific to Δlsr2; it does not rescue the maturation‐deficient biofilms of a ΔgroEL1 mutant, thereby differentiating the process of aggregation from maturation. Gene expression analysis supports a stepwise process of maturation, highlighted by temporally separated, transient inductions of iron and nitrogen import genes. Furthermore, GroEL1 activity is required for induction of nitrogen, but not iron, import genes. Together, the findings begin to define molecular checkpoints during development of mycobacterial biofilms.

Significance: We previously reported that hibernation of 70S ribosomes in mycobacteria is induced as a response to zinc starvation. Because zinc limitation also induces ribosome remodeling, our findings raise questions about the conditions for ribosome remodeling and hibernation. Here, we show that the two processes are induced at different concentrations of zinc and that the caseinolytic protease system plays a crucial role in zinc-dependent inhibition of hibernation during remodeling. The findings offer insights into the molecular pathway underlying the transition from remodeling of ribosomes to hibernation in response to progressive zinc depletion in mycobacteria. This study is also a demonstration of reactivation of hibernating ribosomes by zinc. Finally, this study correlates ribosome hibernation with streptomycin tolerance inMycobacterium tuberculosisduring infection.