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The Bacillus subtilis yqgC-sodA operon protects magnesium-dependent enzymes by supporting manganese efflux.

ABSTRACT: Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the yqgC-sodA deletion mutants results from distinct enzymatic vulnerabilities.

SUBMITTER: Sachla AJ

PROVIDER: S-EPMC10888875 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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The <i>Bacillus subtilis yqgC-sodA</i> operon protects magnesium-dependent enzymes by supporting manganese efflux.

Sachla Ankita J AJ Soni Vijay V Piñeros Miguel M Luo Yuanchan Y Im Janice J JJ Rhee Kyu Y KY Helmann John D JD

bioRxiv : the preprint server for biology 20240215

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the <i>Bacillus subtilis yqgC-sodA</i> (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese ( ...[more]

PMID: 38405924

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Project description:Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function, and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations, they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the YS deletion mutants results from distinct enzymatic vulnerabilities.IMPORTANCEBacteria require multiple trace metal ions for survival. Metal homeostasis relies on the tightly regulated expression of metal uptake, storage, and efflux proteins. Metal intoxication occurs when metal homeostasis is perturbed and often results from enzyme mis-metalation. In Bacillus subtilis, Mn-dependent superoxide dismutase (MnSOD) is the most abundant Mn-containing protein and is important for oxidative stress resistance. Here, we report novel roles for MnSOD and a co-regulated membrane protein, YqgC, in Mn homeostasis. Loss of both MnSOD and YqgC (but not the individual proteins) prevents the efficient expression of Mn efflux proteins and leads to a large-scale perturbation of the metabolome due to inhibition of Mg-dependent enzymes, including key chorismate-utilizing MST (menaquinone, siderophore, and tryptophan) family enzymes.

Project description:Transition metals are essential for life but are toxic when in excess. Metal ion intoxication may result from the mismetallation of essential metal-dependent enzymes with a noncognate metal. To begin to identify enzymes and processes that are susceptible to mismetallation, we have selected for strains with increased resistance to Mn(II) and Co(II). In Bacillus subtilis, cells lacking the MntR metalloregulator are exquisitely sensitive to Mn(II) but can easily become resistant by acquiring mutations affecting the MntH Mn(II) importer. Using transposon mutagenesis, and starting with an mntR mntH strain, we recovered mariner insertions that inactivated the mpfA gene encoding a putative Mg(II) efflux system. Loss of MpfA leads to elevated intracellular Mg(II), increased sensitivity to high Mg(II), and reduced Mn(II) sensitivity. Consistently, we also recovered an insertion disrupting the mgtE riboswitch, which normally restricts expression of the major Mg(II) importer. These results suggest that Mn(II) intoxication results from disruption of a Mg(II)-dependent enzyme or process. Mutations that inactivate MpfA were also recovered in a selection for Co(II) resistance beginning with sensitized strains lacking the major Co(II) efflux pump, CzcD. Since both Mn(II) and Co(II) may mismetallate iron-dependent enzymes, we repeated the selections under conditions of iron depletion imposed by expression of the Listeria monocytogenes FrvA iron exporter. Under conditions of iron depletion, a wider variety of suppressor mutations were recovered, but they still point to a central role for Mg(II) in maintaining metal ion homeostasis.IMPORTANCE Cellular metal ion homeostasis is tightly regulated. When metal ion levels are imbalanced, or when one metal is at toxic levels, enzymes may bind to the wrong metal cofactor. Enzyme mismetallation can impair metabolism, lead to new and deleterious reactions, and cause cell death. Beginning with Bacillus subtilis strains genetically sensitized to metal intoxication through loss of efflux or by lowering intracellular iron, we identified mutations that suppress the deleterious effects of excess Mn(II) or Co(II). For both metals, mutations in mpfA, encoding a Mg(II) efflux pump, suppressed toxicity. These mutant strains have elevated intracellular Mg(II), suggesting that Mg(II)-dependent processes are very sensitive to disruption by transition metals.

Project description:By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates the division frequency of Bacillus subtilis without affecting its growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than do those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media as well as in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but this occurred only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 4.0 mM, with little or no detectable change being observed in labile, intracellular Mg2+, based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with the increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed an enhanced expression of a large number of SigB-regulated genes and the activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple the growth rate from the division frequency and identify transcriptional changes that suggest that cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase the uptake of iron, Mn2+, and Zn2+. IMPORTANCE The signals that cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating that cycles of division can be skipped. Here, we show that by manipulating a single nutrient, namely, Mg2+, cell division can be uncoupled from the growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

Project description:Manganese (Mn) is an essential element and is required for the virulence of many pathogens. In Bacillus subtilis, Mn(II) homeostasis is regulated by MntR, a Mn(II)-responsive, DNA-binding protein. MntR serves as both a repressor of Mn(II) uptake transporters and as a transcriptional activator for expression of two cation diffusion facilitator Mn(II) efflux pumps, MneP and MneS. Mutants lacking either mntR or both mneP and mneS are extremely sensitive to Mn(II) intoxication. Using transposon mutagenesis to select suppressors of Mn(II) sensitivity, we identified YceF, a TerC family membrane protein, as capable of providing Mn(II) resistance. Another TerC paralog, YkoY, is regulated by a Mn(II)-sensing riboswitch and is partially redundant in function with YceF. YkoY is regulated in parallel with an unknown function protein YybP, also controlled by a Mn(II)-sensing riboswitch. Strains lacking between one and five of these known or putative Mn(II) tolerance proteins (MneP, MneS, YceF, YkoY, and YybP) were tested for sensitivity to Mn(II) in growth assays and for accumulation of Mn(II) using inductively coupled plasma mass spectrometry. Loss of YceF and, to a lesser extent, YkoY, sensitizes cells lacking the MneP and MneS efflux transporters to Mn(II) intoxication. This sensitivity correlates with elevated intracellular Mn(II), consistent with the suggestion that TerC proteins function in Mn(II) efflux.IMPORTANCE Manganese homeostasis is primarily regulated at the level of transport. Bacillus subtilis MntR serves as a Mn(II)-activated repressor of importer genes (mntH and mntABC) and an activator of efflux genes (mneP and mneS). Elevated intracellular Mn(II) also binds to Mn-sensing riboswitches to activate transcription of yybP and ykoY, which encodes a TerC family member. Here, we demonstrate that two TerC family proteins, YceF and YkoY, help prevent Mn(II) intoxication. TerC family proteins are widespread in bacteria and may influence host-pathogen interactions, but their effects on Mn(II) homeostasis are unclear. Our results suggest that TerC proteins work by Mn(II) export under Mn(II) overload conditions to help alleviate toxicity.

Dataset Information

The Bacillus subtilis yqgC-sodA operon protects magnesium-dependent enzymes by supporting manganese efflux.

Publications

The <i>Bacillus subtilis yqgC-sodA</i> operon protects magnesium-dependent enzymes by supporting manganese efflux.

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