Bacillus subtilis TerC Family Proteins Help Prevent Manganese Intoxication

Abstract

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.

Keywords: Bacillus subtilis; TerC family; manganese; metal resistance; metalloregulation; regulation; riboswitch.

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 complex transcriptional 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 activates expression of two cation diffusion facilitator (CDF) family Mn(II) efflux pumps. The locations of the mariner transposon (mTn) insertions in the yceC operon are shown. (b) Disk diffusion tests were performed to measure sensitivity to Mn(II). PS indicates the mneP mneS mutant strain. Mid-logarithmic cells (OD₆₀₀, ∼0.4) were plated on LB agar plates and overlaid with 10 μl of 100 mM MnCl₂ on a filter paper disk. The zone of growth inhibition was measured after overnight growth. Data represent the means ± the SD for at least three biological replicates. (c) Mn(II) sensitivity as measured using zone-of-inhibition assays. PS indicates an mneP mneS efflux defective strain. Complementation experiments indicate that the Mn(II) sensitivity of deletions in the yceC operon can be decreased to wild-type levels 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 μM. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (as determined using an unpaired two-tailed Student t test).

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 μl of 100 mM MnCl₂/1 M FeSO₄/100 mM ZnCl₂). PS indicates the mneP mneS background. Mid-logarithmic cells (OD₆₀₀, ∼0.4) were plated on LB agar plates and overlaid with 10 μl of each metal on a filter paper disk. The zone of growth inhibition was measured after overnight 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).

FIG 3

Mn(II) tolerance results from an additive effect of multiple genes. Mn(II) sensitivity was measured by using zone-of-inhibition assays. PS indicates the mneP mneS background. The del5 strain (ΔmneP ΔmneS ΔykoY ΔyceF yybP::erm) is described in Table S1 in the supplemental material. Data represent means ± the SD for three biological replicates. Multipronged significance bars indicate the statistical significance 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).

FIG 4

Growth curves highlighting the role of TerC family proteins in Mn(II) homeostasis. Strains were grown in a Bioscreen multiwell growth analyzer in LB broth amended or not with 10 μM MnCl₂, and cell growth was determined 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 as well as the wild type under these conditions. Complete results, including a range of Mn(II) concentrations, are provided in Fig. S1 in the supplemental material.

FIG 5

yceF and ykoY mutants in a mneP mneS background (PS) confer Mn(II) tolerance by reducing internal Mn(II) accumulation. ICP-MS analysis was used to measure intracellular Mn(II) accumulation after Mn(II) shock. Wild-type and mutant cells were grown to mid-logarithmic phase (OD₆₀₀, ∼0.4), 100 μM MnCl₂ was added to the LB medium, and samples were collected before shock (“0”) and 30 min after shock (“30”). Mn(II) levels were normalized against total protein. Data represent means for two biological replicates 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).

FIG 6

Expression of ykoY, yybP, mneS, and mneP and the yceC operon as a function of Mn(II) concentration. Promoter activity was monitored using β-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 were grown to mid-logarithmic phase (OD₆₀₀, ∼0.4) with Mn(II) added to the specified concentration. A proportion 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 least three 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 three biological replicates.

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. Conserved residues across species are in red. Transmembrane helices (green cylinders) are depicted as previously described (42). (b) Sequence logos showing the degree of residue conservation within the TerC superfamily of transporters. The two sequences shown here align with sections within helix 1 and helix 4 as shown in panel a. Sequence logos were obtained from Pfam. (c) Depiction of B. subtilis YceF. Six transmembrane helices are represented with the conserved DN and DS residues in helices 1 and 4.