Bacillus subtilis TerC Family Proteins Help Prevent ManganeseIntoxication

Srinand Paruthiyil,a Azul Pinochet-Barros,a Xiaojuan Huang,a John D. Helmanna

aDepartment of Microbiology, Cornell University, Ithaca, New York, USA

ABSTRACT Manganese (Mn) is an essential element and is required for the viru-lence of many pathogens. In Bacillus subtilis, Mn(II) homeostasis is regulated byMntR, a Mn(II)-responsive, DNA-binding protein. MntR serves as both a repressor ofMn(II) uptake transporters and as a transcriptional activator for expression of twocation diffusion facilitator Mn(II) efflux pumps, MneP and MneS. Mutants lacking ei-ther mntR or both mneP and mneS are extremely sensitive to Mn(II) intoxication. Us-ing transposon mutagenesis to select suppressors of Mn(II) sensitivity, we identifiedYceF, a TerC family membrane protein, as capable of providing Mn(II) resistance. An-other TerC paralog, YkoY, is regulated by a Mn(II)-sensing riboswitch and is partiallyredundant in function with YceF. YkoY is regulated in parallel with an unknownfunction protein YybP, also controlled by a Mn(II)-sensing riboswitch. Strains lackingbetween 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 assaysand 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 andMneS efflux transporters to Mn(II) intoxication. This sensitivity correlates with ele-vated intracellular Mn(II), consistent with the suggestion that TerC proteins functionin Mn(II) efflux.

IMPORTANCE Manganese homeostasis is primarily regulated at the level of trans-port. 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 in-tracellular Mn(II) also binds to Mn-sensing riboswitches to activate transcription ofyybP and ykoY, which encodes a TerC family member. Here, we demonstrate thattwo TerC family proteins, YceF and YkoY, help prevent Mn(II) intoxication. TerC fam-ily proteins are widespread in bacteria and may influence host-pathogen interac-tions, but their effects on Mn(II) homeostasis are unclear. Our results suggest thatTerC proteins work by Mn(II) export under Mn(II) overload conditions to help allevi-ate toxicity.

KEYWORDS Bacillus subtilis, TerC family, manganese, metal resistance,metalloregulation, regulation, riboswitch

Manganese (Mn) is an important nutrient metal ion critical for growth and is usedto support many cellular processes, including detoxification of reactive oxygen

species (ROS) and the production of deoxynucleoside triphosphates for DNA replica-tion, and in enzymes of central carbon metabolism (1–3). Escherichia coli is consideredto have an iron-centric metabolism and conditionally imports Mn(II) in response tooxidative stress (4). Some enzymes that use Fe(II) as a cofactor may be inactivated byoxidative processes or iron limitation, and binding of Mn(II) may help to sustain enzymefunction (5–8). Alternatively, Fe(II)-dependent enzymes may be functionally replaced byMn(II)-dependent isozymes under conditions of iron limitation (9–11).

In contrast to E. coli, Bacillus subtilis and many pathogenic Firmicutes have a more

Citation Paruthiyil S, Pinochet-Barros A, HuangX, Helmann JD. 2020. Bacillus subtilis TerCfamily proteins help prevent manganeseintoxication. J Bacteriol 202:e00624-19. https://doi.org/10.1128/JB.00624-19.

Editor Tina M. Henkin, Ohio State University

Copyright © 2020 American Society forMicrobiology. All Rights Reserved.

Address correspondence to John D. Helmann,jdh9@cornell.edu.

Received 30 September 2019Accepted 29 October 2019

Accepted manuscript posted online 4November 2019Published

RESEARCH ARTICLE

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manganese-centric physiology and require comparatively high levels of Mn(II) forgrowth (2). Mammalian hosts may limit the bioavailability of Mn(II) for use by patho-gens, as part of the innate immune mechanism known as nutritional immunity (12, 13).The immune effector protein calprotectin is an important mediator of the Mn(II)-withholding response and is produced in high concentrations at sites of infection tolimit bacterial growth (14). The essential roles of Mn(II) in cell physiology likely varybetween organisms and depend on growth conditions. In B. subtilis the sole ribonu-cleotide reductase and the glycolytic enzyme phosphoglycerate mutase are bothMn(II)-dependent enzymes (15, 16). In Staphylococcus aureus, a Mn(II)-dependent phos-phoglycerate mutase is sensitive to inhibition by calprotectin, and the cell responds byproduction of a Mn(II)-independent isozyme (3). Nevertheless, Mn(II) can act as alimiting factor for infection, and simply increasing dietary Mn(II) can exacerbate S.aureus infections (17, 18).

As a result of host-imposed restrictions on metal ion availability, high-affinity Mn(II)uptake systems are important virulence determinants for many pathogens (1, 19–23).However, high-affinity Mn(II) import systems impose their own risks, since increasedMn(II) availability may lead to Mn(II) intoxication. Balancing intracellular Mn(II) levelstherefore requires pathways both for uptake and efflux. Indeed, Mn(II) efflux systemshave recently been described as critical for virulence in diverse pathogenic bacteria(24–29).

Bacillus subtilis displays an absolute requirement for Mn(II) (30) and has long servedas a model system for bacterial manganese homeostasis (2, 31–33). The B. subtilis MntRregulatory protein and its orthologs serve as the central regulator of Mn(II) homeostasisin many bacteria (1, 33–36). A role for MntR in Mn(II) homeostasis was inferred when itwas found that null mutants were exquisitely sensitive to Mn(II) (31). Whereas B. subtilisnormally grows well at up to 1 mM extracellular Mn(II), an mntR-null mutant is unableto grow with 10 �M Mn(II) (31, 37). This high sensitivity is due, in part, to derepressionof Mn(II) import in the mntR mutant. Subsequent studies revealed that Mn(II)-boundMntR represses expression of both a proton-coupled importer of the NRAMP family(MntH) and an ABC transporter (MntABC) (32). However, derepression of uptake isinsufficient to explain the high sensitivity of an mntR mutant to manganese: MntR alsoserves as a required activator for the expression of two, Mn(II)-inducible cation diffusionfacilitator (CDF) efflux pumps, MneP and MneS (37).

Mn(II) also regulates gene expression through the metal-sensing yybP-ykoY ribo-switch, named after the two genes associated with this regulatory element in B. subtilis(38–40). In response to elevated cytosolic levels of Mn(II), the B. subtilis yybP-ykoYriboswitches adopt an altered RNA structure that precludes formation of a transcriptiontermination structure (38, 39). However, the functions of the riboswitch-regulatedproteins, YybP and YkoY, in manganese homeostasis are unclear. YybP is a predictedmembrane protein of unknown function, whereas YkoY is a member of the widespreadTerC family of membrane proteins, named for their original association with telluriumresistance (41). Recently, Alx, an E. coli TerC homolog also regulated by a Mn(II)-sensingriboswitch, was suggested to be involved in facilitating Mn(II) import (42).

Here, we sought to better understand the molecular basis for Mn(II) intoxication byselecting mutations that increase tolerance in cells lacking the two known Mn(II) effluxpumps, MneP and MneS. Suppression of the Mn-sensitive phenotype of an mneP mneSparent strain was conferred by mutations that upregulated the expression of YceF, aTerC protein family member. Consistently, mutation of yceF in the mneP mneS parentstrain further increased Mn(II) sensitivity and led to a rise in intracellular Mn(II). YceF isa paralog of the Mn-inducible YkoY protein, and we demonstrate that these two TerCproteins have partially overlapping functions in manganese homeostasis, likely bycontributing to a secondary pathway for Mn(II) efflux.

RESULTSElevated expression of YceF increases Mn(II) resistance in an efflux-deficient

strain. To identify genes that are involved in Mn(II) resistance, we took advantage of

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the high Mn(II) sensitivity of an mneP mneS double efflux mutant (37). We introduceda plasmid carrying the mariner transposon (mTn) into the mneP mneS strain andselected for growth on plates containing elevated levels of Mn(II) (100 and 150 �M).Subsequently, the location of the mariner insertion was identified by DNA sequencing.Multiple independent insertions were found across the yceC operon, including twoinsertions in yceC and one in yceE (Fig. 1a). In addition, we recovered a single insertiondisrupting yhdP, encoding a putative Mg(II) efflux pump homologous to the S. aureusmpfA gene (43, 44). Genomic DNA was isolated for each insertion and backcrossed intothe parental mneP mneS strain. All reconstructed strains maintained resistance to Mn(II)as judged by zone of inhibition assays. The mneP mneS yceC::mTn and mneP mneSyceE::mTn strains were as resistant to Mn(II) intoxication as the wild type (WT)(Fig. 1b).

The yceC operon includes yceCDEF-yceGH, with one transcriptional terminator afteryceF and a second after yceH (45). This operon is under complex transcriptional control

FIG 1 Transposon insertions in a mneP mneS mutant background confer resistance to Mn(II). (a)Schematic illustration of genes implicated in Mn(II) tolerance. The yceC operon is under complextranscriptional control and encodes the TerC homolog, YceF. The ykoY and yybP genes are Mn(II)inducible by virtue of a Mn(II)-sensing riboswitch, and YkoY is a paralog of YceF. MntR activatesexpression of two cation diffusion facilitator (CDF) family Mn(II) efflux pumps. The locations of themariner transposon (mTn) insertions in the yceC operon are shown. (b) Disk diffusion tests wereperformed to measure sensitivity to Mn(II). PS indicates the mneP mneS mutant strain. Mid-logarithmiccells (OD600, �0.4) were plated on LB agar plates and overlaid with 10 �l of 100 mM MnCl2 on a filterpaper disk. The zone of growth inhibition was measured after overnight growth. Data represent themeans � the SD for at least three biological replicates. (c) Mn(II) sensitivity as measured usingzone-of-inhibition assays. PS indicates an mneP mneS efflux defective strain. Complementation experi-ments indicate that the Mn(II) sensitivity of deletions in the yceC operon can be decreased to wild-typelevels only by induction of ectopic copies of YceF integrated at the amyE locus. Data represent means �the SD for three biological replicates. Experiments were performed with IPTG added to 100 �M. **, P �0.01; ***, P � 0.001; ****, P � 0.0001 (as determined using an unpaired two-tailed Student t test).

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by multiple stress-responsive � factors, including the �W, �M, and �X extracytoplasmicfunction (ECF) � factors (46–49) and the general stress � factor, �B (50). The yceCDEF lociare homologous to sequences in other organisms originally associated with telluriumresistance, but their precise functions are unknown (51). The YceCDE proteins are allrelated to TerD (the UniProt CAPAB TerDEXZ family), whereas YceF is related to TerC. Incontrast, yceGH are not related to defined tellurium resistance genes. Although origi-nally associated with an ability to reduce extracellular tellurite and thereby conferresistance, the physiological role of these tellurite resistance genes has remainedobscure (51).

To test whether inactivation of the yceC, yceD, and yceE genes contributed to Mn(II)resistance, null mutations were generated in the mneP mneS mutant background usingthe BKE collection of strains carrying erythromycin resistance (erm) cassettes replacingthe corresponding coding regions (52). In contrast to the original mariner insertions, thestrains with integrated erm cassettes had only a modestly increased Mn(II) resistancerelative to the mneP mneS parent strain, and this effect could not be complemented byexpression of the inactivated gene from an ectopic locus (Fig. 1C). Furthermore, whenthe erm cassettes were removed to leave behind unmarked, in-frame deletions (52), theincreased resistance to Mn(II) was lost (data not shown). These results led us tohypothesize that the increased Mn(II) resistance in the original transposon insertionmutants was due to a polar effect in which the integrated transposon led to an increasein expression of the downstream gene yceF.

To test the role of yceF in Mn(II) resistance, we introduced a yceF::erm null mutationinto the mneP mneS mutant background. This led to a small but reproducible increasein Mn(II) sensitivity, and this effect was complemented by the ectopic expression of yceFintegrated at the amyE locus (Fig. 1c). These results suggest that YceF increases Mn(II)resistance, and the original selection of yceC::mTn and yceE::mTn transposants reflectedthe upregulation of this minor resistance determinant.

Next, we tested the phenotypes of yceF in an otherwise WT background. For thesestudies, we removed the erm cassette from the yceF::erm strain to generate a mutantstrain with a clean deletion at yceF (ΔyceF). Sensitivity to Mn(II), Fe(II), and Zn(II) wastested with zone of inhibition assays. A yceF mutation in a WT background has a smallbut statistically significant sensitivity to Mn(II), and a yceF mutation exacerbates theMn(II) sensitivity of the mneP mneS strain (Fig. 2). The ΔyceF mutant is also slightly moresensitive to Fe(II) intoxication in the WT but not in the mneP mneS mutant background.Interestingly, the mneP mneS strain also displayed a modest increase in Fe(II) sensitivityrelative to WT. This suggests that perhaps MneP and/or MneS may also have effluxactivity with respect to Fe(II). Finally, yceF did not confer Zn(II) sensitivity in a WT and

FIG 2 YceF confers increased Mn(II) tolerance, but has little effect on sensitivity to other tested metals.yceF mutant sensitivity to Mn(II) (10 �l of 100 mM MnCl2/1 M FeSO4/100 mM ZnCl2). PS indicates themneP mneS background. Mid-logarithmic cells (OD600, �0.4) were plated on LB agar plates and overlaidwith 10 �l of each metal on a filter paper disk. The zone of growth inhibition was measured afterovernight growth. Data represent the means � the SD for at least three biological replicates. *, P � 0.05;***, P � 0.001; ns, no statistical significance (as determined using an unpaired two-tailed Student t test).

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mneP mneS mutant background (Fig. 2). This suggests that yceF has activity primarilyrelated to Mn(II) resistance.

TerC family proteins YceF and YkoY are partially redundant. YceF is one of threeTerC family proteins in Bacillus subtilis, the others being YkoY and YjbE. YjbE is asporulation-specific protein, expressed as part of the �E regulon (53), and there is littleif any expression of yjbE during vegetative growth (45). Therefore, we focused ourattention here on the two paralogs expressed during growth, YceF and YkoY. The ykoYleader region contains a Mn(II)-responsive riboswitch (39, 40), consistent with a role forYkoY under conditions of high Mn(II). A homologous riboswitch precedes the yybPgene, encoding a predicted membrane protein of unknown function, and both ykoYand yybP are induced by Mn(II) (39). We hypothesized that there may be geneticredundancy between the yceF, ykoY, and yybP genes. Therefore, we constructed anarray of mutant strains lacking one or more of these genes. These mutants wereassayed using zones of inhibition with Mn(II) on the filter disk. In a WT background, nosingle deletion strain displayed an increased Mn(II) sensitivity (Fig. 3). However, boththe yceF ykoY double mutant and the yceF ykoY yybP triple mutant showed a statisticallysignificant increase in Mn(II) sensitivity (Fig. 3). Thus, there is redundancy between theYceF and YkoY TerC family proteins, and their role in Mn(II) resistance can be revealedeven in a strain able to express the two efflux pumps, MneP and MneS.

We also tested the role of these genes in the mneP mneS mutant background, whichis already sensitized to Mn(II) intoxication (Fig. 3). In this case, the yceF mutation, butnot the ykoY mutation, increased Mn(II) sensitivity. It is not obvious why the yybP geneappeared to contribute to Mn(II) resistance when combined with a ykoY single mutantin a WT background but not in the mneP mneS mutant background (Fig. 3). Finally, wenote that in both WT and mneP mneS mutant backgrounds, the combined inactivationof yybP, ykoY, and yceF led to the highest level of Mn(II) sensitivity.

We next measured growth curves of these strains with increasing levels of MnCl2(Fig. 4; see also Fig. S1 in the supplemental material). Again, mutation of yceF conferredan increase in sensitivity in the mneP mneS mutant background. This can be seen as anincreased lag phase during subculture in lysogeny broth (LB) supplemented with 5 to10 �M Mn(II) (Fig. 4; see also Fig. S1). A ykoY mutation had no noticeable effect on

FIG 3 Mn(II) tolerance results from an additive effect of multiple genes. Mn(II) sensitivity was measuredby using zone-of-inhibition assays. PS indicates the mneP mneS background. The del5 strain (ΔmnePΔmneS ΔykoY ΔyceF yybP::erm) is described in Table S1 in the supplemental material. Data representmeans � the SD for three biological replicates. Multipronged significance bars indicate the statisticalsignificance for a given strain in reference to mneP and mneS. **, P � 0. 01; ***, P � 0.001; ****, P � 0.0001(as determined using an unpaired two-tailed Student t test).

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growth in the mneP mneS mutant background but exacerbated the growth defectwhen combined with the yceF mutation (the mneP mneS yceF ykoY mutant versus thecorresponding mneP mneS yceF and mneP mneS ykoY triple mutants [Fig. 4]). Thiscontrasts with the lack of an apparent additive effect in zone of inhibition assays(Fig. 3).

Cells lacking YceF and YkoY accumulate manganese. Since YceF and YkoYcontribute to Mn(II) resistance in a mneP mneS mutant background, we next testedwhether the loss of these proteins affects the intracellular accumulation of Mn(II). Cellsgrowing in LB medium were shocked by addition of 100 �M MnCl2 and intracellularmetal ion levels were measured using inductively coupled plasma mass spectrometry(ICP-MS) before shock and 30 min after shock. The Mn(II) levels of all strains were similarbefore shock, and WT cells maintained Mn(II) homeostasis even after shock (Fig. 5). Incontrast, the mneP mneS strain accumulated 12-fold more Mn(II), as reported previously(37). In a mneP mneS ykoY mutant a similar increase was noted, whereas in a yceF mnePmneS mutant Mn(II) levels increased �26-fold. In the yceF ykoY mneP mneS quadruplemutant, Mn(II) accumulation increased slightly above that of a yceF mneP mneS mutant,although this difference did not reach our defined level of statistical significance (P �

0.05). The intracellular levels of Fe(II), Zn(II), and Cu(II) stayed relatively stable for allstrains despite the Mn(II) shock (Fig. S2). These results suggest that YceF and YkoY are

FIG 4 Growth curves highlighting the role of TerC family proteins in Mn(II) homeostasis. Strains were grown in aBioscreen multiwell growth analyzer in LB broth amended or not with 10 �M MnCl2, and cell growth wasdetermined as a function of cell density (OD600) over the course of 25 h. PS indicates the mneP mneS mutations.All curves were monitored at least three times with consistent results. The yceF and ykoY single mutants grew aswell as the wild type under these conditions. Complete results, including a range of Mn(II) concentrations, areprovided in Fig. S1 in the supplemental material.

FIG 5 yceF and ykoY mutants in a mneP mneS background (PS) confer Mn(II) tolerance by reducinginternal Mn(II) accumulation. ICP-MS analysis was used to measure intracellular Mn(II) accumulation afterMn(II) shock. Wild-type and mutant cells were grown to mid-logarithmic phase (OD600, �0.4), 100 �MMnCl2 was added to the LB medium, and samples were collected before shock (“0”) and 30 min aftershock (“30”). Mn(II) levels were normalized against total protein. Data represent means for two biologicalreplicates performed at different times, and error bars indicate the ranges. ***, P � 0.001; ****, P � 0.0001;ns, no statistical significance (as determined using an unpaired two-tailed Student t test).

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important for adaptation to excess Mn(II) in mneP mneS strains and that in theirabsence intracellular Mn(II) accumulates to even higher levels than that seen with themneP mneS mutant alone.

Transcription of the yceC operon is not strongly regulated by Mn(II). Since mnePand mneS are both transcriptionally induced by Mn(II) acting through the MntRtranscription factor (37), and ykoY and yybP are regulated by a Mn(II)-sensing riboswitch(39), we wanted to explore whether transcription of the yceC operon might also beinduced by Mn(II). We generated promoter-lacZ fusion strains for yybP, ykoY, and theyceC operon and compared their expression with mneP and mneS promoter fusions30 min after resuspension of washed, mid-logarithmic-phase cells in LB mediumamended with various concentrations of Mn(II) (Fig. 6). As reported, mneP and mneS areresponsive to Mn(II) (37), with maximal induction achieved at 300 �M Mn(II) for mnePand at 400 �M Mn(II) for mneS. The yybP and ykoY riboswitches were also responsive toMn(II) concentration [maximal induction by �200 �M Mn(II)], although in both casesthere was a substantial basal level of expression (�50% full induction). Expression ofthe yceC operon was constitutive and did not appear to respond to Mn(II) under theseconditions. However, prior studies suggest that this operon may be induced by a shiftof cells to conditions of elevated Mn(II), which can lead to a transient spike inintracellular Mn(II) levels (32).

YceF and YkoY share conserved sequence features with other TerC homologs.A previous study aligned TerC family proteins from various bacterial species to defineconserved motifs, although B. subtilis was not included (42). Here, we aligned the threeTerC family proteins encoded in B. subtilis (YceF, YkoY, and YjbE) with the Escherichiacoli homolog (Alx) (Fig. 7a). All four proteins share six predicted transmembranesegments with conserved aspartic acid residues present in segments 1 and 4, as notedpreviously (42). These conserved acidic motifs (Fig. 7b) are predicted to be locatedwithin the membrane, and were previously postulated to form part of an ion transportchannel (42). This is consistent with the observations here that cells lacking YceFaccumulate elevated levels of Mn(II) after shock (Fig. 5). In contrast with previous results(42), this suggests a likely function in Mn(II) export.

DISCUSSION

Cells rely on essential transition metals as cofactors for an estimated 30% ofenzymes. Managing the acquisition, storage, and efflux of metal ions is critical for thehealth of the cell and helps to sustain cytosolic metals at levels needed to metallatecritical enzymes, while avoiding toxicity associated with metal ion overload (33). Mn(II)is an essential element for B. subtilis and related bacteria, and sequestration of Mn(II) by

FIG 6 Expression of ykoY, yybP, mneS, and mneP and the yceC operon as a function of Mn(II) concen-tration. Promoter activity was monitored using �-galactosidase assays for mneP-lacZ, mneS-lacZ, yceC-lacZ, yybP-lacZ, and ykoY-lacZ transcriptional fusions in the WT as a function of added Mn(II). Cells weregrown to mid-logarithmic phase (OD600, �0.4) with Mn(II) added to the specified concentration. Aproportion of 1 is equal to the fully activated value in Miller units (21.2 for yceC, 32 for mneP, 8.3 for mneS,3.5 for ykoY, and 4.3 for yybP). The results shown are representative of experiments performed at leastthree different times. Open shapes represent MntR-regulated loci, gray shapes are riboswitch regulated,and the triangle represents uncharacterized regulation. Data represent means � the SD for threebiological replicates.

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the mammalian innate immune protein calprotectin is important in the ability of hoststo prevent infection (1, 14). Because of the wide variation and the dynamic nature ofMn(II) pools, both in the host and in the environment, mechanisms for sustaining Mn(II)homeostasis are critical for cell function.

In B. subtilis, the MntR metalloregulatory protein is the central regulator of Mn(II)homeostasis (31). The homodimeric MntR protein binds two Mn(II) ions per protomer,with the critical sensing site displaying Mn(II)-selective, heptacoordinate ligation (54).Interestingly, the Mn(II) selective riboswitches of the yybP-ykoY family also bind andrespond to Mn(II) with high selectivity (40), and this also involves a heptacoordinatesensing site (55). Once activated by Mn(II), MntR represses two operons encoding Mn(II)import systems: the ATP-dependent MntABC system and the proton-coupled NRAMPfamily protein MntH (32). In addition, MntR serves as a transcriptional activator for twocation diffusion facilitator (CDF) proteins, MneP and MneS, that function to reduceintracellular Mn(II) levels (37). The ykoY and yybP genes are both regulated by the

FIG 7 Alignment of TerC family proteins and putative structure of YceF. (a) Multisequence alignment of Alx (E. coli), YceF (B. subtilis), YkoY (B. subtilis), and YjbE(B. subtilis) protein sequences derived from NCBI. Proteins were aligned using COBALT with default parameters and “identity” conservation settings. Conservedresidues across species are in red. Transmembrane helices (green cylinders) are depicted as previously described (42). (b) Sequence logos showing the degreeof residue conservation within the TerC superfamily of transporters. The two sequences shown here align with sections within helix 1 and helix 4 as shown inpanel a. Sequence logos were obtained from Pfam. (c) Depiction of B. subtilis YceF. Six transmembrane helices are represented with the conserved DN and DSresidues in helices 1 and 4.

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eponymous Mn(II)-sensing riboswitch, as documented for B. subtilis, Lactobacillus, andE. coli (39, 40). However, the functions of YkoY and YybP in Mn(II) homeostasis remainundefined.

Here, we used a forward genetics approach to identify mutations that canalleviate the toxicity associated with growth of cells lacking Mn(II) efflux systems inmedium with excess Mn(II). Analysis of the resulting transposants identified up-regulation of the unknown function YceF protein as the key determinant ofincreased Mn(II) resistance. We were intrigued by the observation that YceF is aTerC homolog, and therefore a paralog of the YkoY protein known to be regulatedby a Mn(II)-sensing riboswitch. We postulated that YceF and YkoY have overlappingfunctions in Mn(II) homeostasis. To test this idea, we generated strains lackingvarious Mn(II) resistance determinants.

Our data indicate that YceF functions to help maintain Mn(II) homeostasis asrevealed in cells lacking the two Mn(II) exporting CDF proteins, MneP and MneS (themneP mneS mutant). Cells lacking mneP mneS and additionally lacking YceF accu-mulate much greater amounts of Mn(II) after shift to excess Mn(II) conditions (Fig.5). The yceF gene is cotranscribed with poorly understood genes encoding TerDfamily proteins as part of the yceC operon. This operon is under complex transcrip-tional control that likely involves the general stress response regulator �B (50) andthe extracytoplasmic function factors �W and �M (46, 47). Interestingly, the �B

regulon has been previously shown to be responsive to changes in Mn(II) availabil-ity (32). Indeed, the yceC operon is modestly induced by a shift of Mn(II) limited cells[grown with 50 nM Mn(II)] to replete conditions [2.5 �M Mn(II)], and this inductionis much more dramatic in an mntR-null mutant (32). However, the yceC operon didnot seem to respond to elevated Mn(II) under the steady-state growth conditionsmonitored here (Fig. 6).

We suggest that YceF has a more general function in cellular physiology and thatthis function may involve export of Mn(II) from the cytosol as part of its mechanism.This function appears to be at least partly overlapping with the role of theparalogous protein YkoY, as revealed by the additive effect of yceF and ykoYmutations in growth studies (Fig. 4). Thus, these two TerC homologs may beinvolved in pathways involved in translocation of Mn(II) across the membrane.Indeed, it was recently noted that TerC proteins are distantly related to knownMn(II) transporters, as part of the LysE superfamily, and are frequently associatedwith Mn(II)-sensing riboswitches (42). This superfamily of transporters includesdocumented Mn(II) efflux pumps of the MntP family, a cadmium resistance trans-porter (Cad), UPF0016 proteins, and iron-lead transporters (ILT), as well as TerCfamily proteins (42). These transporters display a conserved transmembrane archi-tecture with conserved acidic residues (Asp) in two transmembrane segments (42).This architecture is conserved for the YceF and YkoY proteins (Fig. 7). However, incontrast with this prior work, we here show that the loss of TerC homologs leads toan increase in intracellular Mn(II), suggestive of a role in Mn(II) efflux.

The YkoY TerC homolog is regulated by a Mn(II)-sensing riboswitch, and thisregulation is consistent with a role related to helping sustain optimal cytosolicMn(II) levels when Mn(II) levels are in excess. However, both YkoY and YceF areexpressed even in the absence of overt Mn(II) stress (45). We hypothesize that TerChomologs function in Mn(II) transport across the cell membrane for the purpose ofmetallating secreted or cell surface-associated metalloenzymes. This model is sug-gested, in part, from the broad conservation of TerC proteins (42, 56). For example,TerC is found in plants where it is critical for proper assembly of photosystem II, acomplex that requires Mn(II) for function. In Arabidopsis thaliana, TerC (AtTerC) islocalized to the thylakoid membrane of the chloroplast and interacts with thephotosystem II assembly factor ALB3 (57). More generally, TerC proteins are relatedto the larger UPF0016 family of proteins, which includes the plant PAM71 protein,postulated to translocate Mn(II) into the chloroplast lumen for photosystem assem-bly (58). Similarly, the yeast UPF0016 protein Gdt1p translocates Mn(II) and Ca(II)

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from the cytosol to the Golgi compartment, where it contributes to activitiesrequired for protein glycosylation (59). It is interesting to note that when expressedin the bacterium Lactococcus lactis, yeast Gdt1p mediated Mn(II) influx. A similarrole has been ascribed to the mammalian homolog, TMEM165 (60). Although wefavor a model in which bacterial TerC homologs function physiologically in Mn(II)export, perhaps coupled to metallation of secreted or membrane-localized en-zymes, they will likely function in import if inserted in the membrane in the wrongorientation. This could explain the impact of expressing a heterologous yeastprotein in L. lactis (59) and may also account for the reported increase in cytosolicMn(II) when the E. coli Alx protein was overproduced (42). Further studies will berequired to test this general hypothesis regarding the possible roles of TerCproteins in B. subtilis physiology.

In conclusion, the studies presented here expand our view of the set of proteinsaffecting cellular Mn(II) homeostasis. In addition to MntR-regulated importers (MntABCand MntH) and efflux proteins (MneP and MneS), the two TerC homologs (YceF andYkoY) appear to play overlapping functions related to Mn(II) export. For YkoY, thisexport function may provide a backup pathway for detoxifying the cytosol, as sug-gested by its regulation by a Mn(II)-inducible riboswitch. Alternatively, it may beinvolved in metallating a secreted or membrane-associated protein that is only met-allated when Mn(II) is abundant in the cell. The role of YceF may also be associated withmetallation of exoenzymes, as suggested by its ability to reduce the accumulation ofintracellular Mn(II), the lack of strong regulation by Mn(II), and by analogy with UPF0016members from Eukarya. The role of the YybP protein in Mn(II) homeostasis was notapparent under our tested conditions. Further studies will be required to define howthese and other factors help cells adapt to the changing availability of essential metalions.

MATERIALS AND METHODSBacterial strains and growth conditions. Bacterial strains were used in this study as described in

Table S1 in the supplemental material. B. subtilis was grown in lysogeny broth (LB). Ampicillin (100 mgml�1) was used for selection in Escherichia coli strains. Erythromycin (1 mg ml�1) was used for theselection of the B. subtilis strains. Techniques for B. subtilis transformation were as performed as describedpreviously (61). Clean deletions were generated using the Cre recombinase in the pDR244 plasmid (52).

To make promoter-lacZ transcriptional fusions, promoters were synthesized from CU1065 genomicDNA using the primers in Table S2, digested with EcoRI/HindIII or HindIII/BamHI, and cloned intopDG1663 (62). Constructs were then introduced into CU1065 B. subtilis. For complementation, geneproducts were amplified from CU1065 genomic DNA, digested with HindIII/BglII or HindIII/XbaI, andcloned into pPL82 (63). The new plasmids, which allow IPTG (isopropyl-�-D-thiogalactopyranoside)-inducible expression of genes, were linearized with PstI and transformed into the B. subtilis chromosomeat the amyE locus.

Mariner transposon mutagenesis. Mariner transposon mutagenesis procedure was carried out inthe mneP mneS mutant strain (PS strain) using the pMarA mariner (mTn) delivery plasmid as describedpreviously (64). Cells were grown at 30°C until stationary phase to maintain pMarA and were thenselected for growth on LB plates containing elevated levels of Mn(II) (100 and 150 �M) at 42°C. Coloniesthat grew overnight were inoculated in LB supplemented with kanamycin (15 �g ml�1) at 37°C to ensurethe integration of the transposon. Genomic DNA was isolated and then subjected to Taq�� restrictionenzyme digestion, followed by a ligation of sticky ends to generate circular DNA. An inverse PCR usingprimers annealing to the ends of the mTn was performed, followed by sequence analysis to identify theorientation and location of the mTn insertions.

Zone of inhibition assays. Cells were grown in LB with shaking at 37°C to an optical density at 600nm (OD600) of �0.4. A 100-�l aliquot of cultures was mixed with 4 ml of prewarmed LB soft agar (0.75%agar), poured onto LB agar plates (containing 15 ml of 1.5% LB agar), and allowed to solidify at roomtemperature for 10 min. Filter paper disks (7 mm) were placed on top of the soft agar, 10 �l of 100 mMMnCl2 was added to the disks, and the plates were incubated at 37°C overnight. For IPTG induction, IPTGwas added to both the soft agar and the plates to a final concentration of 0.1 mM. The data shownrepresent the averages and standard deviations (SD) of three biological replicates.

Bioscreen assay. Strains were grown at 37°C to an OD600 of �0.4, from which 2 �l of cells wasinoculated to 200 �l in a Bioscreen 100-well microtiter plate. Growth was monitored spectrophotometri-cally (OD600) every 15 min for 25 h using a Bioscreen C growth analyzer (Growth Curves USA, Piscataway,NJ) at 37°C with continuous shaking. Data shown are representative growth curves (or cell density at afixed time), and experiments were repeated at least three times.

ICP-MS. Cells were grown in LB medium to an OD600 of �0.4 and then shocked with 100 �M MnCl2.Samples (4 ml) were collected immediately before shock and at 30 min after shock. Samples were

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prepared and analyzed by ICP-MS as previously described (37). Cells were washed twice with phosphate-buffered saline (PBS) buffer containing 0.1 M EDTA, followed by two Chelex-treated PBS-buffer-onlywashes. Cells were resuspended in 500 �l of Chelex-treated PBS buffer with 1% Triton X-100 andcontaining 75 mM NaN3, followed by incubation at 37°C for 90 min. The samples were centrifuged, and10 �l of the supernatant was used to measure the total cell protein concentration by Bradford assay.Then, 750 �l of 5% HNO3 with 0.1% (vol/vol) Triton X-100 was added to the rest of the supernatant, whichwas boiled at 95°C for 30 min. Samples were centrifuged, and then the supernatant was diluted with 1%HNO3. Metal levels were measured by ICP-MS (Perkin-Elmer ELAN DRC II using ammonia as the reactiongas and gallium as an internal standard) and normalized against the total cell protein concentration. Datarepresent means � ranges of two separate experiments.

�-Galactosidase assays. Strains containing promoter-lacZ fusions were grown in LB to an OD600 of�0.4 with a gradient of concentrations of MnCl2 added to the media. �-Galactosidase assays wereperformed as described previously (65), except that cells were lysed with lysozyme at 0.1 mg ml�1 for30 min at 37°C instead of chloroform.

SUPPLEMENTAL MATERIALSupplemental material is available online only.SUPPLEMENTAL FILE 1, PDF file, 4.3 MB.

ACKNOWLEDGMENTSThis study was supported by a grant from the National Institutes of Health

(R35GM122461) to J.D.H. S.P. was supported by undergraduate grants from the CornellUniversity College of Agriculture and Life Sciences and the Cornell Institute of Host-Microbe Interactions and Disease.

We thank members of the Helmann lab for helpful comments. We also thank ChrisFurman for the construction of yybP and ykoY overexpression strains and Daniel Kim forpreliminary ICP-MS data.

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