Bacillus subtilis TerC Family Proteins Help Prevent Manganese Intoxication

2020

J Bacteriol

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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.

Section: Discussionmentioning

confidence: 99%

Dysregulation of Magnesium Transport Protects Bacillus subtilis against Manganese and Cobalt Intoxication

Wendel

2020

J Bacteriol

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“…Increased intracellular Mg is postulated to protect one or more targets from Mn mismetallation, as proposed also for Bradyrhizobium japonicum (Hohle & O’Brian, 2014). Selection for Mn resistant suppressors of the efflux deficient PS strain led to the identification of strains with upregulated expression of YceF, a the TerC family membrane protein implicated in Mn efflux (Paruthiyil et al., 2020). While these results highlight the diverse ways in which cells can balance metal ion levels and suggest an interplay between Mg and Mn homeostasis, they do not identify the causes of Mn intoxication.…”

Section: Introductionmentioning

confidence: 99%

Manganese impairs the QoxABCD terminal oxidase leading to respiration‐associated toxicity

Sachla

Luo

2021

Molecular Microbiology

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Cell physiology relies on metalloenzymes and can be easily disrupted by imbalances in metal ion pools. Bacillus subtilis requires manganese for growth and has highly regulated mechanisms for import and efflux that help maintain homeostasis. Cells defective for manganese (Mn) efflux are highly sensitive to intoxication, but the processes impaired by Mn excess are often unknown. Here, we employed a forward genetics approach to identify pathways affected by manganese intoxication. Our results highlight a central role for the membrane‐localized electron transport chain in metal intoxication during aerobic growth. In the presence of elevated manganese, there is an increased generation of reactive radical species associated with dysfunction of the major terminal oxidase, the cytochrome aa3 heme‐copper menaquinol oxidase (QoxABCD). Intoxication is suppressed by diversion of menaquinol to alternative oxidases or by a mutation affecting heme A synthesis that is known to convert QoxABCD from an aa3 to a bo3‐type oxidase. Manganese sensitivity is also reduced by derepression of the MhqR regulon, which protects cells against reactive quinones. These results suggest that dysfunction of the cytochrome aa3‐type quinol oxidase contributes to metal‐induced intoxication.

“…For a fourth application, we introduced yceF * (an allele of yceF with an Ile206Thr substitution) on a 2.17 kb repair template cloned into pAJS23 to generate pAJS27. The yceF gene encodes a membrane protein from the TerC family and is part of the yceCDEFGH operon (19). We were again able to recover the desired recombinant strain with high yield (Table 1, Fig 2D).…”

Section: Resultsmentioning

confidence: 99%

A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis

Sachla

Alfonso

2021

Preprint

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The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any non-essential gene, and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be co-transformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps.