Dataset Information

A moonlighting function of Mycobacterium smegmatis Ku in zinc homeostasis?

ABSTRACT: Ku protein participates in DNA double-strand break repair via the nonhomologous end-joining pathway. The three-dimensional structure of eukaryotic Ku reveals a central core consisting of a ?-barrel domain and pillar and bridge regions that combine to form a ring-like structure that encircles DNA. Homologs of Ku are encoded by a subset of bacterial species, and they are predicted to conserve this core domain. In addition, the bridge region of Ku from some bacteria is predicted from homology modeling and sequence analyses to contain a conventional HxxC and CxxC (where x is any residue) zinc-binding motif. These potential zinc-binding sites have either deteriorated or been entirely lost in Ku from other organisms. Using an in vitro metal binding assay, we show that Mycobacterium smegmatis Ku binds two zinc ions. Zinc binding modestly stabilizes the Ku protein (by ?3°C) and prevents cysteine oxidation, but it has little effect on DNA binding. In vivo, zinc induces significant upregulation of the gene encoding Ku (?sixfold) as well as a divergently oriented gene encoding a predicted zinc-dependent MarR family transcription factor. Notably, overexpression of Ku confers zinc tolerance on Escherichia coli. We speculate that zinc-binding sites in Ku proteins from M. smegmatis and other mycobacterial species have been evolutionarily retained to provide protection against zinc toxicity without compromising the function of Ku in DNA double-strand break repair.

SUBMITTER: Kushwaha AK

PROVIDER: S-EPMC4315663 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A moonlighting function of Mycobacterium smegmatis Ku in zinc homeostasis?

Kushwaha Ambuj K AK Deochand Dinesh K DK Grove Anne A

Protein science : a publication of the Protein Society 20141226 2

Ku protein participates in DNA double-strand break repair via the nonhomologous end-joining pathway. The three-dimensional structure of eukaryotic Ku reveals a central core consisting of a β-barrel domain and pillar and bridge regions that combine to form a ring-like structure that encircles DNA. Homologs of Ku are encoded by a subset of bacterial species, and they are predicted to conserve this core domain. In addition, the bridge region of Ku from some bacteria is predicted from homology model ...[more]

PMID: 25450225

Similar Datasets

Project description: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.

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.

Project description: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.

Dataset Information

A moonlighting function of Mycobacterium smegmatis Ku in zinc homeostasis?

Publications

A moonlighting function of Mycobacterium smegmatis Ku in zinc homeostasis?

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets