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Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

ABSTRACT: Zinc homeostasis is crucial for bacterial cells, since imbalances affect viability. However, in mycobacteria, knowledge of zinc metabolism is incomplete. Mycobacterium smegmatis (MSMEG) is an environmental, nonpathogenic Mycobacterium that is widely used as a model organism to study mycobacterial metabolism and pathogenicity. How MSMEG maintains zinc homeostasis is largely unknown. SmtB and Zur are important regulators of bacterial zinc metabolism. In mycobacteria, these regulators are encoded by an operon, whereas in other bacterial species, SmtB and Zur are encoded on separate loci. Here, we show that the smtB-zur operon is consistently present within the genus Mycobacterium but otherwise found only in Nocardia, Saccharothrix, and Corynebacterium diphtheriae By RNA deep sequencing, we determined the Zur and SmtB regulons of MSMEG and compared them with transcriptional responses after zinc starvation or excess. We found an exceptional genomic clustering of genes whose expression was strongly induced by zur deletion and zinc starvation. These genes encoded zinc importers such as ZnuABC and three additional putative zinc transporters, including the porin MspD, as well as alternative ribosomal proteins. In contrast, only a few genes were affected by deletion of smtB and zinc excess. The zinc exporter ZitA was most prominently regulated by SmtB. Moreover, transcriptional analyses in combination with promoter and chromatin immunoprecipitation assays revealed a special regulation of the smtB-zur operon itself: an apparently zinc-independent, constitutive expression of smtB-zur resulted from sensitive coregulation by both SmtB and Zur. Overall, our data revealed yet unknown peculiarities of mycobacterial zinc homeostasis.IMPORTANCE Zinc is crucial for many biological processes, as it is an essential cofactor of enzymes and a structural component of regulatory and DNA binding proteins. Hence, all living cells require zinc to maintain constant intracellular levels. However, in excess, zinc is toxic. Therefore, cellular zinc homeostasis needs to be tightly controlled. In bacteria, this is achieved by transcriptional regulators whose activity is mediated via zinc-dependent conformational changes promoting or preventing their binding to DNA. SmtB and Zur are important antagonistically acting bacterial regulators in mycobacteria. They sense changes in zinc concentrations in the femtomolar range and regulate transcription of genes for zinc acquisition, storage, and export. Here, we analyzed the role of SmtB and Zur in zinc homeostasis in Mycobacterium smegmatis Our results revealed novel insights into the transcriptional processes of zinc homeostasis in mycobacteria and their regulation.

SUBMITTER: Goethe E

PROVIDER: S-EPMC7174638 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

Goethe Elke E Laarmann Kristin K Lührs Janita J Jarek Michael M Meens Jochen J Lewin Astrid A Goethe Ralph R

mSystems 20200421 2

Zinc homeostasis is crucial for bacterial cells, since imbalances affect viability. However, in mycobacteria, knowledge of zinc metabolism is incomplete. <i>Mycobacterium smegmatis</i> (MSMEG) is an environmental, nonpathogenic <i>Mycobacterium</i> that is widely used as a model organism to study mycobacterial metabolism and pathogenicity. How MSMEG maintains zinc homeostasis is largely unknown. SmtB and Zur are important regulators of bacterial zinc metabolism. In mycobacteria, these regulators ...[more]

PMID: 32317393

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Project description:Zinc uptake in bacteria is essential to maintain cellular homeostasis and survival. ZnuABC is an important zinc importer of numerous bacterial genera, which is expressed to restore zinc homeostasis when the cytosolic concentration decreases beyond a critical threshold. Upon zinc limitation the fast-growing nonpathogenic organism Mycobacterium smegmatis (MSMEG) as well as the ruminant pathogen M. avium subsp. paratuberculosis (MAP) increases expression of genes encoding ZnuABC homologues, but also of genes encoding other transporters. This suggests an involvement of these transporters in zinc homeostasis. Here we characterized the putative zinc transporters of MSMEG (ZnuABC and ZnuABC2) and MAP (ZnuABC, MptABC, and MAP3774-76). Deletion of either ZnuABC or ZnuABC2 in MSMEG did not lead to growth defects, but to an increased expression of zinc marker genes in MSMEGΔznuABC, indicating cytosolic zinc limitation. However, chromatin immunoprecipitation proved direct binding of the global zinc regulator Zur to promoter regions of both znuABC and znuABC2. Simultaneous deletion of both transporters caused severe growth defects, which could be restored either by homologous complementation with single ZnuABC transporters or supplementation of growth media with zinc but not iron, manganese, cobalt, or magnesium. Heterologous complementation of the double mutant with MAP transporters also resulted in reconstitution of growth. Nonradioactive FluoZinTM-3AM zinc uptake assays directly revealed the competence of all transporters to import zinc. Finally, structural and phylogenetic analyses provided evidence of a novel class of ZnuABC transporters represented by the ZnuABC2 of MSMEG, which is present only in actinobacteria, mainly in the genera Nocardia, Streptomyces and fast growing Mycobacteria IMPORTANCEZinc is necessary for bacterial growth but simultaneously toxic when in excess. Hence, bacterial cells have developed systems to alter intracellular concentration. Regulation of these systems is primarily executed at transcriptional level by regulator proteins which sense femtomolar changes in the zinc level. In environmental and pathogenic mycobacteria zinc starvation induces expression of common zinc import systems such as the ZnuABC transporter, but also of other additional not yet characterized transport systems. In this study, we characterized the role of such systems in zinc transport. We showed that transport systems of both species whose transcription is induced upon zinc starvation can exchangeably restore cellular zinc homeostasis in transporter deficient mutants by transporting zinc into the cell.

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Dataset Information

Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

Publications

Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

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