Bacillus subtilis Regulators MntR and Zur Participate in Redox Cycling, Antibiotic Sensitivity, and Cell Wall Plasticity

Manganese and zinc are essential transition metals involved in many fundamental cellular processes, including protection against external oxidative stress. In Bacillus subtilis, Zur and MntR are key transcriptional regulators of zinc and manganese homeostasis, respectively. In this work, proteome analysis of B. subtilis wild-type, ΔmntR, and Δzur strains provided new insights into bacterial adaptation to deregulation of essential metal ions. Deletions of mntR and zur genes increased bacterial sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress and impacted the cell wall thickness. Overall, these findings highlight that Zur and MntR regulatory networks are connected to antibiotic sensitivity and cell wall plasticity.

ABSTRACT

The Bacillus subtilis MntR and Zur transcriptional regulators control homeostasis of manganese and zinc, two essential elements required in various cellular processes. In this work, we describe the global impact of mntR and zur deletions at the protein level. Using a comprehensive proteomic approach, we showed that 33 and 55 proteins are differentially abundant in ΔmntR and Δzur cells, respectively, including proteins involved in metal acquisition, translation, central metabolism, and cell wall homeostasis. In addition, both mutants showed modifications in intracellular metal ion pools, with significant Mg²⁺ accumulation in the ΔmntR mutant. Phenotypic and morphological analyses of ΔmntR and Δzur mutants revealed their high sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress. Mutant strains had a modified cell wall thickness and accumulated lower levels of intracellular reactive oxygen species (ROS) than the wild-type strain. Remarkably, our results highlight an intimate connection between MntR, Zur, antibiotic sensitivity, and cell wall structure.

IMPORTANCE Manganese and zinc are essential transition metals involved in many fundamental cellular processes, including protection against external oxidative stress. In Bacillus subtilis, Zur and MntR are key transcriptional regulators of zinc and manganese homeostasis, respectively. In this work, proteome analysis of B. subtilis wild-type, ΔmntR, and Δzur strains provided new insights into bacterial adaptation to deregulation of essential metal ions. Deletions of mntR and zur genes increased bacterial sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress and impacted the cell wall thickness. Overall, these findings highlight that Zur and MntR regulatory networks are connected to antibiotic sensitivity and cell wall plasticity.

INTRODUCTION

Metal ions such as iron, zinc, and manganese are crucial for central cellular processes, as they are structural components of many proteins and membranes while participating in the catalysis of metabolic reactions and electron transfer. However, metals are highly toxic when in excess. The mammalian innate immune system responds to infection by combining deprivation of some metals (Fe, Zn, and Mn) with a metal poisoning strategy (Cu and Zn) to prevent bacterial replication (1–3). Restrictive access to critical transition metals is a defense known as nutritional immunity (4). Examples involve lipocalin, which binds siderophores and thereby prevents Fe acquisition by bacterial pathogens (5), and calprotectin, which restricts acquisition of Zn or Mn (6). In the macrophage phagosome, NRAMP1 removes Mn and Fe to starve intracellular pathogens (7, 8). In contrast, in macrophage antimicrobial pathways, Zn and Cu toxicity is used to combat invading microbes.

Among transition metals, Mn and Zn are important in many fundamental cellular processes, including protection against reactive oxygen species (ROS). There is now evidence that the invading microbe utilizes Mn as a key micronutrient to counteract the effects of host-mediated oxidative stress. Thus, Mn plays a significant role in adaptation of pathogenic bacteria to the human host (9). It protects bacterial cells from oxidative stress, either as a cofactor for Mn-dependent catalases and superoxide dismutases or via its inherent ability to quench free-radical-mediated reactions (9–11). Similarly, Zn is important for resistance to both hydrogen peroxide (H₂O₂) and the thiol-oxidizing agent diamide (12). Zn²⁺ may protect thiols from oxidation and even displace other redox-active metals in protein thiol-containing active sites to maintain the function of proteins (13). Pathogenic bacteria deficient in maintaining proper metal homeostasis are less virulent (2, 8, 14, 15).

Intracellular metal homeostasis in bacteria is ensured by finely tuned import and efflux systems (16). Metalloregulatory proteins act as metal-sensing regulatory transcription factors. In the Gram-positive bacterium Bacillus subtilis, MntR and Zur are key regulators of manganese and zinc homeostasis, respectively. MntR is a bifunctional regulator that binds Mn²⁺ as an effector (17, 18). The MntR regulon includes 7 genes involved in Mn²⁺ uptake or efflux (18–22). The Zur metalloprotein binds Zn²⁺ as a corepressor (23–26). The Zur regulon contains 11 genes, including genes for zinc transporters (12, 23) and zinc-independent alternative ribosomal proteins (27–30). However, Zur binds 80 regions on the chromosome, indicating far broader control by this regulator (31). Zinc homeostasis is also maintained via a zinc-inducible efflux pump, CzcD, which is regulated at the transcriptional level by the metalloregulator CzrA (22). Together, Zur and CzrA sense changes in the labile Zn²⁺ pool and modify expression of genes encoding proteins that mediate zinc homeostasis (32, 33).

The roles of the MntR and Zur proteins at the transcriptional level have been studied extensively. However, a study of the global impact of mntR and zur deletions at the protein level is still missing. Here, we performed a comprehensive study on global protein changes in B. subtilis ΔmntR and Δzur mutants by quantitative proteomic analysis and validated the obtained results by physiology tests and microscopy observation. Together, our results show that Zur and MntR regulators are necessary to ensure bacterial fitness upon environmental stress.

RESULTS

Impact of mntR and zur deletions on the B. subtilis proteome.

To understand the physiological state of cells with the deleted mntR or zur gene deleted, we performed a comparative analysis of the cytosolic and membrane proteome of ΔmntR, Δzur, and wild-type (WT) cells grown in exponential phase (Table 1). Optimized analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of four technical replicates resolved more than 1,700 proteins (see Table S1 in the supplemental material). In total, 33 and 55 proteins were statistically significantly differentially abundant (P < 0.05 by the Kruskal-Wallis test and one-way analysis of variance [ANOVA]) in ΔmntR and Δzur cells, respectively (Table 2). Twelve proteins were common between ΔmntR and Δzur proteomic data sets (Fig. 1A). Among them, CarB and Spo0M changes in regulation diverged between the two mutants. Proteins involved in metal acquisition, translation, stress response, cell wall synthesis, and amino acid/nitrogen/carbon metabolism were mainly affected in ΔmntR and Δzur mutants (Fig. 1B and C).

TABLE 1.

B. subtilis strains used in this study

Strain	Genotypea	Reference or source
BSB1	trp+	76
BSAS45	Δzur::aphA3	31
BSAS46	ΔmntR::aphA3	This work

^aaphA3, Enterococcus faecalis kanamycin resistance gene.

TABLE 2.

Total proteins differentially abundant in the cytosolic and membrane fractions of the B. subtilis ΔmntR and Δzur mutants

Mutant	No. of proteins
	Cytosolic fraction		Membrane fraction		Common in cytosolic and membrane fractions
	Upregulated	Downregulated	Upregulated	Downregulated
ΔmntR	14	14	6	1	2
Δzur	20	16	18	10	9

FIG 1.

Functional classification of proteins with altered abundance in the B. subtilis ΔmntR and Δzur mutants. (A) Venn diagram of the 76 proteins showing significant abundance changes between ΔmntR, Δzur, and WT cells. (B and C) Proteins are classified according to their involvement in biological processes.

The proteome analysis of the ΔmntR mutant indicated a greater increase of the MntA and MntB proteins, as expected since they are components of the MntABCD Mn uptake systems (Table 3). Of note, proteins of the Mn²⁺ efflux systems, MneP and MneS, were not identified in the membrane or cytosolic subfraction (Table S1), suggesting that their levels were too low to be detected under our conditions. Three proteins involved in metal ion homeostasis, ZagA, DhbB, and DhbF, were increased. The ZagA zinc metallochaperone is encoded by a Zur-regulated gene (34). DhbB and DhbF are involved in biosynthesis of the catecholate siderophore bacillibactin. The dhbABCDF operon belongs to the Fur regulon (35), whose transcriptional expression has been shown to weakly decrease in an mntR mutant (19). Under our conditions, we assumed that mismetallation of Fur occurs in ΔmntR cells, which would lead to increased expression of the dhb operon. Interestingly, the SodA Mn-dependent superoxide dismutase was less abundant in ΔmntR cells, while two increased proteins, YtsJ and RbsK, participate in metabolic pathways generating NADPH (Table 3). The malic enzyme YtsJ was proposed to be important to balance the intracellular redox pool (36, 37). The ribokinase RbsK belongs to the pentose phosphate (PP) pathway, which yields two molecules of NADPH per glucose molecule (38, 39). NADPH is the unique provider of reducing equivalents to maintain or regenerate bacterial detoxifying and antioxidative defense systems (40). One can hypothesize that ΔmntR cells need NADPH to cope with internal oxidative stress and therefore need a metabolic adaptation to maintain the NADPH/NADP⁺ ratio. However, we observed that mntR and zur deletions were accompanied by maintained levels of the NADPH/NADP⁺ ratio during the exponential phase, without significant increase in the NADPH level (data not shown).

TABLE 3.

Proteins differentially abundant in the B. subtilis ΔmntR mutant

Protein	Fold change in abundance in ΔmntR vs wild type	Gene	Function	Functional categorya
Proteins upregulated in cytosolic fraction
DhbBb	12.75	dhbB	Isochorismatase	Acquisition of iron
CitB	10.5	citB	Aconitase	TCA cycle
BglH	9.25	bglH	Phospho-beta-glucosidase	Utilization of salicin
Spo0M	9.25	spo0M	Sporulation	Sporulation
PgcA	8.25	pgcA	Alpha-phosphoglucomutase	Biosynthesis of teichoic acid
RocA	7.25	rocA	3-Hydroxy-1-pyrroline-5-carboxylate dehydrogenase	Arginine utilization/nitrogen metabolism
RbsK	7	rbsK	Ribokinase	Utilization of ribose
MntAb	6.75	mntA	Manganese ABC transporter	Acquisition of manganese
Syv	6.5	valS	Valyl-tRNA synthetase	Translation
Mao4	6.25	ytsJ	NADP-dependent malate dehydrogenase	Utilization of malate
Syh	5.75	hisS	Histidyl-tRNA synthetase	Translation
RocGb	5.25	rocG	Glutamate dehydrogenase	Arginine utilization/nitrogen metabolism
MenB	5.25	menB	Naphthoate synthase	Biosynthesis of menaquinone
ZagA	5.25	zagA	Zinc metallochaperone	Acquisition of zinc
Proteins upregulated in membrane fraction
DhbFb	29	dhbF	Involved in bacillibactin biosynthesis	Acquisition of iron
MntAb	17.5	mntA	Manganese ABC transporter	Acquisition of manganese
MtnK	8.25	mtnK	5-Methylthioribose kinase	Methionine metabolism
RocGb	7.5	rocG	Glutamate dehydrogenase	Arginine utilization/nitrogen metabolism
MntB	6.25	mntB	Manganese ABC transporter	Acquisition of manganese
DnaX	5.5	dnaX	DNA polymerase III	DNA replication
Proteins downregulated in cytosolic fraction
RplD	−25.5	rplD	Ribosomal protein L4	Translation
CarB	−22.5	carB	Carbamoyl-phosphate transferase-arginine	Biosynthesis of arginine
CspB	−20.5	cspB	RNA chaperone	Cold stress proteins
FabI	−20.5	fabI	Enoyl-acyl carrier protein reductase	Fatty acid biosynthesis
YpfD	−11.5	ypfD	Similar to ribosomal protein S1	Translation
SodM	−11	sodA	Superoxide dismutase	Resistance against oxidative stress
YkaA	−9.75	ykaA	Unknown	Proteins of unknown function
PunA	−8.75	pupG	Purine nucleoside phosphorylase	Nucleotide metabolism
RpsM	−8.25	rpsM	Ribosomal protein S13	Translation
OppA	−7.25	oppA	Oligopeptide ABC transporter	Utilization of peptides
PtsG	−6.5	ptsG	Glucose permease	Carbon core metabolism
Hbs	−6.25	hbs	Nonspecific DNA-binding protein	DNA condensation/segregation
AsnB	−6	asnB	Asparagine synthase	Control of peptidoglycan hydrolysis
PbpC	−6	pbpC	Penicillin-binding protein 3	Cell wall synthesis
Protein downregulated in membrane fraction: FlgG	−5.75	flgE	Flagellar hook protein	Motility and chemotaxis

^aAccording to the SubtiWiki database (77).

^bProtein detected in both cytosolic and membrane fractions.

Proteomic analysis of the Δzur mutant indicated higher levels of proteins (AdcA, AdcC, FolE2, RpmE2, YciB, ZagA, and ZinT) encoded by Zur-regulated genes, as expected (Table 4). We also observed increased amounts of the CadA efflux ATPase, which is involved in zinc and cadmium metal export (22). The Δzur strain displayed reduced levels of proteins related to stress resistance (Table 4): (i) the bacilliredoxin YqiW (renamed BrxB) promotes a redox switch in response to oxidative stress (41); (ii) the membrane protein YceD, similar to a tellurium resistance protein, is required for survival under ethanol stress (42); and (iii) LiaH is involved in resistance to envelope stress conditions (43). Therefore, the proteomic data suggest that Δzur cells could be more sensitive to environmental stresses. Interestingly, the molecular chaperone DnaK was significantly increased in Δzur cells. DnaK contributes to membrane and overall cell recovery under different stress conditions (44–46), suggesting that the Δzur strain faces internal stress conditions. Finally, the WapA tRNase toxin-WapI antitoxin system (47) was derepressed in the Δzur strain compared to the wild-type strain. Increased expression of WapA (the putative toxin) and decreased expression of WapI (the antitoxin) indicate that Δzur cells may be more susceptible to growth inhibition than wild-type cells.

TABLE 4.

Proteins differentially abundant in the B. subtilis Δzur mutant

Protein	Fold change in abundance in Δzur vs WT	Gene	Function	Functional categorya
Proteins upregulated in cytosolic fraction
ZagAb	243	zagA	Zinc metallochaperone	Acquisition of zinc
AdcAb	34	znuA	ABC transporter for zinc	Acquisition of zinc
FolE2b	22.25	folE2	GTP cyclohydrolase IB	Biosynthesis of folate
CarB	18	carB	Carbamoyl-phosphate transferase-arginine	Biosynthesis of arginine
DnaK	15.5	dnaK	Molecular chaperone	Protein quality control
RocA	14.25	rocA	3-Hydroxy-1-pyrroline-5-carboxylate dehydrogenase	Arginine utilization/nitrogen metabolism
AcoN	10.5	citB	Aconitase	TCA cycle
ZinTb	8	zinT	Zinc-binding protein	Acquisition of zinc
GlmU	7.75	glmU	Bifunctional N-acetylglucosamine-1-phosphate	Biosynthesis of peptidoglycan
RbsK	7.25	rbsK	Ribokinase	Utilization of ribose
PgcA	7.25	pgcA	Alpha-phosphoglucomutase	Biosynthesis of teichoic acid
Syi	7.25	ileS	Isoleucyl-tRNA synthetase	Translation
Fbp	7	fbp	Fructose-1,6-bisphosphatase	Gluconeogenesis
GndA	7	gndA	NADP-dependent phosphogluconate dehydrogenase	Pentose phosphate pathway
Oatb	7	rocD	Ornithine transaminase	Ornithine utilization/nitrogen metabolism
PyrGb	6.75	pyrG	CTP synthase	Nucleotide metabolism
RocGb	6.25	rocG	Glutamate dehydrogenase	Arginine utilization/nitrogen metabolism
AdcCb	5.75	znuC	ABC transporter for zinc	Acquisition of zinc
Rl31B	5.5	rpmE2	Accessory ribosomal protein	Translation
Sya	5.5	alaS	Alanine-tRNA synthetase	Translation
Proteins upregulated in membrane fraction
ZagAb	184.75	zagA	Zinc metallochaperone	Acquisition of zinc
AdcAb	100.75	znuA	ABC transporter for zinc	Acquisition of zinc
YjlD	29.25	ndh	NADH dehydrogenase	Respiration
GlpK	28.25	glpK	Glycerol kinase	Utilization of glycerol
MetK	22.75	metK	S-Adenosylmethionine synthetase	Methionine metabolism
ZinTb	19.25	zinT	Zinc-binding protein	Acquisition of zinc
AdcCb	16.75	znuC	ABC transporter for zinc	Acquisition of zinc
WapA	15.5	wapA	Cell wall-associated protein precursor	Toxins, antitoxins, and immunity against toxins
AsnB	14.75	asnB	Asparagine synthase	Control of peptidoglycan hydrolysis
FolE2b	12.25	folE2	GTP cyclohydrolase IB	Biosynthesis of folate
PyrGb	11	pyrG	CTP synthase	Nucleotide metabolism
CadA	10	cadA	Cadmium transporting ATPase	Resistance against toxic metals
YciB	8.5	yciB	Putative l,d-transpeptidase	Acquisition of zinc
Oatb	7.75	rocD	Ornithine transaminase	Ornithine utilization/nitrogen metabolism
MurG	6.75	murG	Peptidoglycan precursor biosynthesis	Biosynthesis of peptidoglycan
RocGb	6.75	rocG	Glutamate dehydrogenase	Arginine utilization/nitrogen metabolism
Smc	6.25	smc	Segregation of replication origins	DNA condensation/segregation
DnaN	5.5	dnaN	DNA polymerase III	DNA replication
Proteins downregulated in cytosolic fraction
Rl7	−46.75	rplL	Ribosomal protein L12	Translation
CspB	−37.25	cspB	RNA chaperone	Cold stress proteins
Tkt	−25.75	tkt	Transketolase	Pentose phosphate pathway
Rs6	−10.5	rpsF	Ribosomal protein S6	Translation
RpsM	−10	rpsM	Ribosomal protein S13	Translation
Hbs	−9	hbs	Nonspecific DNA-binding protein	DNA condensation/segregation
YqiW	−7	yqiW	Bacilliredoxin	Resistance against oxidative stress
YjlCb	−7	yjlC	Unknown	Protein of unknown function
YxkC	−6.5	yxkC	Unknown	Protein of unknown function
WapI	−6.25	wapI	Immunity protein	Toxins, antitoxins, and immunity against toxins
PthP	−6	ptsH	Phosphotransferase system-dependent sugar transport and carbon catabolite repression	Transporters
Rl15	−6	rplO	Ribosomal protein L15	Translation
YceD	−5.75	yceD	Similar to tellurium resistance protein	Resistance against toxic metals
Rl10	−5.5	rplJ	Ribosomal protein L10	Translation
Rs19	−5.5	rpsS	Ribosomal protein S19	Translation
YqeY	−5.25	yqeY	Unknown	Protein of unknown function
Proteins down-regulated in membrane fraction
Fla	−85	hag	Flagellin protein	Motility and chemotaxis
Rs8	−18.25	rpsH	Ribosomal protein S8	Translation
CheA	−8.5	cheA	Two-component sensor kinase	Motility and chemotaxis
YjlCb	−8	yjlC	Unknown	Protein of unknown function
AtpF	−7.5	atpF	ATP synthase	ATP synthesis
FliL	−6.5	fliL	Flagellar protein	Motility and chemotaxis
LiaH	−6	liaH	Phage shock-like protein	Resistance against oxidative stress and cell wall antibiotics
Spo0M	−6	spo0M	Unknown	Sporulation
FlgG	−5.5	flgE	Flagellar hook protein	Motility and chemotaxis
GgaB	−5	ggaB	Membrane protein	Biosynthesis of teichoic acid

^aAccording to the SubtiWiki database (77).

^bProtein detected in both cytosolic and membrane fractions.

The proteomic analysis revealed elevated levels of proteins related to the arginine utilization pathway (RocA, RocD, and RocG) in ΔmntR and Δzur cells. The rocABC, rocDEF, and rocG operons are under the transcriptional control of RocR and are induced by the presence of arginine, ornithine, or proline in the growth medium. In contrast, expression of rocE and rocD was repressed in the transcriptomic profile of a zur mutant (12). The relationship between Zur and the RocR regulon is not clear. However, our result reinforced the idea that alteration of zinc homeostasis affects expression of the RocR regulon.

Finally, we observed changes in the levels of proteins involved in peptidoglycan synthesis or hydrolysis as well as those involved in the biosynthesis of teichoic acid (Tables 3 and 4). This set includes PgcA, PbpC, and AsnB in ΔmntR cells and AsnB, GgaB, GlmU, MurG, and PgcA in Δzur cells. This suggested a possible impact of mntR and zur deletions on the cell wall structure.

Interestingly, the global proteomics analysis brought new data and revealed some unexpected regulatory effects in the ΔmntR and Δzur mutants. It is well known that mRNA and protein expression levels may differ despite being quantified in the same bacterial cells and under similar conditions (48). Previous transcriptomic studies of zur and mntR mutants in Luria-Bertani (LB) medium identified mainly genes belonging to the Zur and MntR regulons, respectively (12, 19). Our data highlighted a broader impact of mntR and zur deletions on proteins whose levels are modified in response to perturbation of cellular metal ion pools.

Disruption of metal homeostasis in ΔmntR and Δzur mutants.

To verify whether deletion of mntR or zur modifies the cellular metal ion pool, total cell-associated metal ions were quantified in the wild-type, ΔmntR and Δzur strains. For this, mid-exponential-phase cells were cultivated at the same optical density at 600 nm (OD₆₀₀) in LB medium and analyzed by inductively coupled plasma mass spectrometry (ICP-MS).

The ΔmntR mutant displayed 2-, ∼1.5-, ∼1.5-, and ∼1.2-fold increases in the levels of Mg²⁺, Mn²⁺, Zn²⁺, and Cd²⁺, respectively, compared to the wild type (Table 5). In contrast, levels of Fe²⁺, Co²⁺, Cu²⁺, and Ni²⁺ were similar in both strains. Manganese accumulation in ΔmntR cells despite activation of efflux systems (21) may indicate that B. subtilis tolerates a mild Mn²⁺ intracellular increase without intoxication. The increased level of Cd²⁺ correlates with the higher level of the MntH transporter, which imports both Mn²⁺ and Cd²⁺ ions (18). Accumulation of Zn²⁺ and Mg²⁺ in a ΔmntR mutant was unexpected.

TABLE 5.

Metal content of B. subtilis wild type, ΔmntR and Δzur cells measured by ICP-MS

Metal ion	Concn (μM)a in:
	WT	ΔmntR mutant	Δzur mutant
Fe2+	203 ± 29.2	229.4 ± 39.1	151.1 ± 49.6
Mg2+	523.7 ± 85.5	1,155.5 ± 166.8	621.3 ± 70.1
Mn2+	26.4 ± 3.1	42.1 ± 6.4	28 ± 4.5
Zn2+	224.3 ± 25.1	302.7 ± 28.5	237.6 ± 32.7
Cd2+	2.8 ± 0.2	3.6 ± 0.4	1.4 ± 0.3
Co2+	0.25 ± 0.15	0.18 ± 0.1	0.18 ± 0.1
Cu2+	6.2 ± 0.6	6.5 ± 0.6	8.8 ± 1.4
Ni2+	6.4 ± 1.1	7.1 ± 1.4	7.5 ± 1.6

^aResults are means ± standard deviations.

The Δzur mutant displayed a 1.5-fold-increased Cu²⁺ level and a 2-fold-decreased Cd²⁺ level compared to the wild type (Table 5). The Δzur mutant had no significant differences in total Mn²⁺, Zn²⁺, Mg²⁺, Fe²⁺, Co²⁺, and Ni²⁺ levels. As the internal Zn²⁺ concentration was maintained in Δzur cells, B. subtilis appeared to tightly control the level of Zn²⁺ to avoid zinc intoxication. Copper accumulation in a Δzur mutant was intriguing. In B. subtilis, copper uptake involves a well-defined transporter, YcnJ, whose synthesis responds to copper availability (49). The lower level of Cd²⁺ detected in Δzur cells was in line with increased CadA, the major determinant for Cd²⁺ resistance (Table 4). In zinc excess, CzrA binds Zn²⁺ to trigger derepression of CadA (22, 50).

Differential Mg²⁺-dependent growth of Δzur and ΔmntR cells.

We detected an intracellular Mg²⁺ concentration of ∼0.5 to 1.0 mM in the three B. subtilis strains (Table 5). To gain insight into the physiological relevance of Mg²⁺ accumulation in the ΔmntR strain, growth assays were performed under conditions of Mg²⁺ starvation. Precultures of the wild-type, ΔmntR, and Δzur strains in LB medium were inoculated in M9 defined medium in the presence or absence of Mg²⁺. In M9 medium containing 25 mM Mg²⁺, all strains grew with similar growth kinetics (Fig. 2A). In Mg²⁺-depleted M9 medium (containing only traces of Mg²⁺), only the ΔmntR mutant showed a growth benefit (Fig. 2B). Therefore, Mg²⁺ accumulation in ΔmntR cells grown in LB medium appeared to be effective to support growth in Mg²⁺-depleted M9 medium. It is worth noting that supplementation of growth media with 5 to 25 mM Mg²⁺ may suppress morphological and vital defects of several B. subtilis mutants with mutations in cell wall-related genes (e.g., ponA, ugtP, pgcA, gtaB, or asnB) (51, 52), but the mechanisms underlying this rescuing role are still unknown. At present, we cannot explain how intracellular Mg²⁺ compensates for the absence of external Mg²⁺ ions.

FIG 2.

Effect of magnesium starvation on growth of wild-type, ΔmntR, and Δzur cells. (A) Growth curves of the wild-type (black symbols), ΔmntR (white symbols), and Δzur (gray symbols) strains grown in M9 medium in the presence of 25 mM MgCl₂. A representative assay is represented. (B) Growth curves of the wild-type (black symbols), ΔmntR (white symbols), and Δzur (gray symbols) strains grown in MgCl₂-depleted M9 medium. The doubling time of wild-type and Δzur cells is approximately 208 min. The doubling time of ΔmntR cells is approximately 122 min. A representative assay is represented. Note that the x axes are different in panels A and B for a better view of the data.

Sensitivity of ΔmntR and Δzur cells to environmental stresses.

As the proteomic analysis revealed that Δzur cells displayed reduced levels of proteins related to stress resistance (BrxB, YceD, and LiaH), we tested whether environmental stresses such as organic solvent and increased salinity could affect growth of Δzur and ΔmntR cells compared to that of the wild type. Growing bacteria were exposed to a final sublethal concentration of 4% (vol/vol) ethanol. Under the conditions used, no effect on the growth rate was observed for the wild-type cells, whereas 4% ethanol transiently affected growth of the Δzur and ΔmntR cells (Fig. 3A and B). The direct target of ethanol is the membrane bilayer. A lower resistance to ethanol exposure suggests that Δzur and ΔmntR cells are less efficient in activating an early response to stress and/or that their membrane lipids and proteins differ from those in the wild-type cells. Remarkably, we observed in ΔmntR cells a 20-fold-decreased amount of the FabI protein, which is involved in fatty acid biosynthesis (Table 3).

FIG 3.

Effects of ethanol and NaCl stresses on growth of wild-type, ΔmntR, and Δzur cells. (A) Growth curves of the wild-type (black symbols), ΔmntR (white symbols), and Δzur (gray symbols) strains grown in LB medium without ethanol addition. A representative assay is represented. (B) Growth curves of the wild-type (circles), ΔmntR (triangles), and Δzur (squares) strains grown in LB medium after addition of 4% ethanol (final concentration) at an OD₆₀₀ of 0.6. A representative assay is represented. (C) Growth curves of the wild-type (black symbols), ΔmntR (white symbols), and Δzur (gray symbols) strains grown in LB medium without NaCl addition. A representative assay is represented. (D) Growth curves of the wild-type (circles), ΔmntR (triangles), and Δzur (squares) strains grown in LB medium with addition of NaCl at 0.1 M. A representative assay is represented.

We further tested the effect of NaCl on growth. A moderate saline stress was imposed by incubation of the cells in the presence of 0.1 M NaCl. Under these conditions, we observed a slight but reproducible enhanced fitness of the Δzur and ΔmntR strains compared to the wild-type strain (Fig. 3C and D). No difference in resistance was observed with 0.5 M NaCl (data not shown). As bacterial responses to osmotic challenges are very complex, it is not obvious how the ΔmntR and Δzur strains are better suited than the wild type to cope with moderate saline stress.

Sensitivity of ΔmntR and Δzur cells to lysozyme and beta-lactam antibiotics.

The proteomic analysis revealed modified levels of proteins (AsnB, GlmU, and MurG) involved in peptidoglycan biosynthesis in ΔmntR and Δzur cells. To test whether mntR and zur deletions modified the peptidoglycan integrity, bacteria were treated with lysozyme. Both the ΔmntR and Δzur mutants were more sensitive to 0.1 mg · ml⁻¹ lysozyme than the wild-type strain (Fig. 4A and B). With 0.5 mg · ml⁻¹ lysozyme, ΔmntR, Δzur, and wild-type cells showed similar growth defects (data not shown). In addition, a disk diffusion assay was performed to compare the sensitivities of the mutant and wild-type strains to several antibiotics. The ΔmntR and Δzur mutants were more sensitive than the wild-type strain to penicillin G and oxacillin, which inhibit formation of peptidoglycan cross-links in the bacterial cell wall (Fig. 4C). In contrast, the two mutants had the same sensitivity as the wild-type strain to vancomycin and to tunicamycin, which target other steps of cell wall synthesis, and to the RNA polymerase inhibitor rifampin. The decreased resistance of the ΔmntR and Δzur strains to lysozyme and beta-lactam antibiotics strongly suggests that structural modifications take place in the cell wall of mutant cells. This is in line with the (above-described) proteomic analysis, which indicated deregulation of proteins involved in cell wall plasticity.

FIG 4.

Increased sensitivity of the ΔmntR and Δzur mutants to lysozyme and antibiotics. (A) Growth curves of the wild-type (black symbols), ΔmntR (triangles), and Δzur (gray symbols) strains grown in LB medium. A representative assay is represented. (B) Growth curves of the wild-type (circles), ΔmntR (triangles), and Δzur (squares) strains grown in LB medium in the presence of 1 mg · ml⁻¹ lysozyme. A representative assay is represented. (C) Results of disk diffusion assays with the indicated antibiotics. Bars indicate the percent increase in zone of inhibition for the ΔmntR (white bars) and Δzur (gray bars) mutants relative to the wild type. All the experiments were performed at least in three biological replicates.

Bacterial interfacial potential and cell wall thickness are altered in ΔmntR and Δzur mutants.

Modifications in cell wall composition are expected to affect bacterial surface charge (53, 54). We performed zeta potential measurements to determine bacterial surface potentials. The zeta potential indicates an electrochemical property of the bacterial cell surface which represents the transmembrane potential that maintains the cell wall/membrane architecture. Negative zeta potentials were measured for all three strains grown in LB medium, as expected for Gram-positive bacteria, which contain negatively charged teichoic acids. However, the ΔmntR mutant exhibited a lesser negative potential (−11.6 ± 0.4 mV) than the wild type (−13.5 ± 0.3 mV) and Δzur (−13.8 ± 0.7 mV) cells. This finding suggests that deletion of the mntR gene alters cell surface permeability, as a correlation between negative zeta potential and membrane integrity was previously shown (55). It should be noted that proteomic analysis indicated increased levels of PgcA, a protein involved in biosynthesis of teichoic acid that also may modify the surface charge of bacteria.

To further identify the modifications that differentiate the cell walls of ΔmntR and Δzur cells, cultures grown in LB were collected at mid-exponential phase and observed in thin cross-section by transmission electron microscopy (TEM). Bacterial cells of the 3 strains appeared as well-separated bacilli (Fig. 5A). The cell wall of the Δzur mutant was thinner (32 ± 4.5 nm) than that of the parental strain (38 ± 3.6 nm) (Fig. 5A and B). In contrast, the cell wall thickness of the ΔmntR mutant was greater (42 ± 3.5 nm) than that of the wild type. We assumed that alteration of cell wall thickness in the ΔmntR and Δzur mutants and modification of the zeta potential in ΔmntR cells resulted from changes in cell surface composition and/or structure. The interplay between manganese and zinc homeostasis and the proteins involved in cell wall plasticity identified in the proteomic approach (Tables 3 and 4) deserves future investigation.

FIG 5.

The mntR and zur deletions are associated with altered cell wall thickness. (A) Transmission electron microscopy images of representative cells of wild-type, ΔmntR, and Δzur cells during mid-log growth phase. Bars, 100 nm. (B) Dot blot graph of the cell wall thickness. Each dot represents a measurement for a single bacillus. a, P = 1.8 · 10⁻⁴ <0.05; b, P = 4.4 · 10⁻⁶ <0.05.

Low O₂^·− and H₂O₂ accumulation in ΔmntR and Δzur mutants.

Neutralization of the surface potential of bacteria was shown to trigger the production of ROS (53, 56). Modifications in bacterial surface potential may thus result from cell wall adjustments but also from enhanced ROS production within the bacterial cells (53). However, our proteomic data were in favor of reducing ROS levels. Indeed, we observed decreased levels of BrxB and SodA in the Δzur and ΔmntR mutants, respectively. The BrxB (bacilliredoxin)- and SodA (Mn-dependent superoxide dismutase)-encoding genes can be induced by oxidative stress. Thus, a lower level of BrxB and SodA might indicate a reduction in intracellular ROS. To verify whether bacterial surface charge modifications resulted from intracellular ROS accumulation, we compared the amounts of O₂^·− and H₂O₂ production in ΔmntR and Δzur cells to those in the wild-type strain.

The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay was performed to estimate cellular production of O₂^·−. No light adsorption was observed for LB medium alone. XTT absorbs light at 470 nm only when reduced by O₂^·− and does not do so in its oxidized form. The amount of O₂^·− in cells in mid-exponential growth phase in LB medium was lower in ΔmntR and Δzur cells than in the wild type (Fig. 6A). The intracellular concentration of H₂O₂ was calculated in bacteria in mid-exponential phase based on the calibration curve obtained with pure H₂O₂ using the Amplex red assay. The generation of H₂O₂ was found to be lower in the mutants than in the wild-type strain (Fig. 6B). The ΔmntR and Δzur mutants appeared to accumulate less intracellular ROS than the wild-type strain. We thus propose that the modification in the bacterial surface potential in ΔmntR cells could result from changes in the cell wall structure rather than from intracellular ROS accumulation.

FIG 6.

ROS accumulation and oxidative stress assays in ΔmntR and Δzur mutants. (A) Quantification of O₂^·− produced in wild-type (WT), ΔmntR, and Δzur cells at an OD₆₀₀ of 1 in LB medium. (B) Quantification of H₂O₂ produced in WT, ΔmntR, and Δzur cells during 4 h of growth in LB medium. (C) Plates from a disk diffusion assay with a drop of 3 M hydrogen peroxide (H₂O₂). (D) The histograms represent the percentage of survival of WT, ΔmntR, and Δzur cells at 15 min after addition of 0.01, 0.05, or 0.1 mM H₂O₂. The survival rate was determined as the ratio of the number of CFU per milliliter after 15 min to the number of CFU per milliliter before addition of H₂O₂.

Effect of external H₂O₂ on ΔmntR and Δzur cells.

To estimate bacterial sensitivity to external oxidative stress, we compared the survival of wild-type, ΔmntR, and Δzur cells when challenged with external H₂O₂. Disk diffusion assays indicated an increase in the zone of growth inhibition for the ΔmntR and Δzur mutants compared to that for the wild-type strain (Fig. 6C). Similarly, both the ΔmntR and Δzur mutants showed a growth defect compared to the wild-type strain when exposed to 50 or 100 μM H₂O₂ for 15 min in LB medium (Fig. 6D). Increased sensitivity of the ΔmntR mutant to H₂O₂ correlates with the role of Mn²⁺ as corepressor of the PerR regulator in response to peroxide stress (57–60). Increased susceptibility of the Δzur mutant to H₂O₂ suggests that Zur is required for expression of oxidative stress defenses, as observed in Corynebacterium diphtheriae (61).

DISCUSSION

Our results provide a new view on B. subtilis mntR and zur mutants under stress-related conditions. Proteomic analysis revealed 33 and 55 proteins whose abundance was affected in ΔmntR and Δzur mutants, respectively. The identified proteins are involved in various cellular processes, notably in translation, amino acid/nitrogen/carbon metabolism, and cell wall homeostasis. Commonly affected proteins suggest coordination between the MntR and Zur regulatory networks.

The ΔmntR and Δzur mutants displayed growth kinetics similar to those of the parental strain, although intracellular pools of some essential metal ions differed (Table 5). The 1.5-fold-greater accumulation of Mn²⁺ in the ΔmntR mutant versus the WT indicates that B. subtilis can tolerate an intracellular increase in Mn²⁺ without bacterial growth being affected. In contrast, maintenance of the Zn²⁺ content in a Δzur mutant provides evidence that alternative pathways can maintain intracellular Zn²⁺ homeostasis. Surprisingly, Mg²⁺ and Zn²⁺ metal ions also accumulate in the ΔmntR mutant (2.2-fold and 1.4-fold increased, respectively), while Cu²⁺ accumulates 1.5-fold in Δzur cells. It is possible that metabolic changes in ΔmntR and Δzur cells provoke secondary demands for other metal ions, leading to their increased levels. How bacteria simultaneously regulate the content of multiple metal ions merits further studies.

Metal ions catalyze numerous metabolic processes by their roles in electron transfer. Essential pathways such as the tricarboxylic acid (TCA) cycle (CitB) and the stringent response are impacted by bacterial metabolic status (62–65). Our proteomic analyses revealed higher levels of RocA, RocD, and RocG proteins in both ΔmntR and Δzur mutants. These enzymes are involved in arginine and ornithine utilization to generate ammonium and may contribute to maintaining intracellular pH homeostasis (66).

We previously performed a genome-wide identification of Zur-binding sites by chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip) (31). We were initially surprised that promoters identified by ChIP-on-chip did not match proteins identified in the Δzur mutant. However, the differences may be explained by the importance of posttranscriptional control of translation, as well as the effects of transcriptional regulators other than Zur in controlling gene expression.

We showed that the ΔmntR and Δzur mutants accumulate less ROS (e.g., O₂^·− and H₂O₂) than the wild-type counterpart (Fig. 6). Mn²⁺ and Zn²⁺ metal ions are intimately linked to the ability to withstand ROS. Mn²⁺ itself or in a complex with a cellular component is involved in cellular defense against O₂^·− stress and can therefore substitute for SodA (67). An elevated extracellular Zn²⁺ amount protects B. subtilis from peroxide stress (59). Hence, slight increases in Mn²⁺ and Zn²⁺ pools in ΔmntR cells (Table 5) may sufficiently promote the antioxidative defense mechanism to diminish the need for Mn-dependent superoxide dismutase SodA (Table 3). It is intriguing that the Δzur mutant shows a similar decrease of ROS, whereas the Mn²⁺ and Zn²⁺ levels are not modified (Table 5 and Fig. 6). This raises questions about the regulatory role of Zur in maintaining redox potential.

Remarkably, deletion of mntR or zur enhanced sensitivity to lysozyme and beta-lactam antibiotics, both of which target the cell wall (Fig. 4). Our results and previous studies suggest mechanisms that may explain these findings. First, increased sensitivity to peptidoglycan synthesis inhibitors can be due to modification of the cell wall structure and/or composition in the mutants. Our findings are in line with this, since the cell wall thickness and bacterial surface zeta potential were modified in the ΔmntR and Δzur mutants compared to the wild-type strain (Fig. 5). Second, beta-lactam antibiotics were proposed to generate ROS that contribute to bacterial death (68). We observed a greater sensitivity to external ROS of ΔmntR and Δzur cells than of the wild-type strain (Fig. 6). Third, there is an intimate connection between metal ions and antibiotic activity Some antibiotics are known to require metal ions for their activity (69). When not required to mediate target binding, interaction of metal ions with antibiotics can provide an additional mode of antibacterial action, as seen, for example, with Zn²⁺, which potentiates the antibiotic activity of vancomycin (70), and with ZnMgO, which increases ciprofloxacin activity (71). We showed here that ΔmntR and Δzur cells accumulate some metal ions (Table 5), which may potentiate antibiotic activity. Further investigations are needed to determine the exact role(s) of Zur and MntR in bacterial sensitivity to antibiotics.

Altogether, this work might uncover new targets for intervention to successfully combat emerging bacterial multiresistance against conventional antibiotics.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The B. subtilis strains used in this work are listed in Table 1. B. subtilis cells were grown in Luria-Bertani (LB) medium or in M9 minimal medium (72). In media containing NaCl, the concentrations are indicated. Antibiotics were added at the following concentrations when required: 5 μg kanamycin ml⁻¹ and 60 μg spectinomycin ml⁻¹. Solid media were prepared by addition of 20 g Noble agar (Difco) liter⁻¹. Standard procedures were used to transform B. subtilis (73).

DNA manipulations.

In PCR, the Pfu DNA polymerase was used as recommended by the manufacturer (Biolabs). DNA fragments were purified from agarose gels using the QIAquick kit (Qiagen).

Construction of strains.

The mntR mutant BSAS46 was constructed by homologous replacement of the mntR coding sequence with the kanamycin resistance gene aphA3 using a joining PCR technique. The aphA3 gene was first amplified. The region upstream of the mntR gene (nucleotides 2542520 to 2543556) was amplified by PCR with a 24-bp aphA3 fragment at its 3′ end. The region downstream of mntR (nucleotides 2543796 to 2544739) was amplified with a 24-bp aphA3 fragment at its 5′ end. The three DNA fragments were combined, and then a PCR was performed with the two external oligonucleotides. The final product, corresponding to the two regions flanking mntR with the inserted aphA3 cassette in between, was purified from a gel and used to transform B. subtilis. Integration and deletion were confirmed by PCR and verified by DNA sequencing.

Intracellular metal concentration measurement by ICP-MS.

Overnight cultures of wild-type, ΔmntR, and Δzur B. subtilis strains were diluted to an optical density at 600 nm (OD₆₀₀) of 0.05 in 15 ml of fresh LB medium cultured in 50-ml Falcon tubes. Bacteria were incubated at 37°C until exponential phase (OD₆₀₀ of around 0.8). Cell cultures were centrifuged at 4,000 × g at 4°C for 10 min and washed three times in 5 ml of ultrapure water (Millipore) with EDTA added to 1 mM. Samples were dried overnight at 80°C and then acidified twice with Suprapur 65% nitric acid (Merck Millipore) until mineralization. Samples were further analyzed at the University of Montpellier II (Laboratoire ICP-MS, UMR5543 Géosciences). Samples were dissolved with 250 ml of nitric acid (65%) for 1 h. They were diluted 1,000-fold in double-distilled water and analyzed using an Agilent 7700x quadrupole inductively coupled plasma mass spectrometer. Concentrations were determined by analyzing standard solutions. To obtain the number of atoms of metal ions per cell, the raw data were normalized to the washing solution. The measurement for each strain was performed in three replicates.

Quantification of NADPH and NADP⁺.

Detection and quantification of NADPH and NADP⁺ content were done using the NADP/NADPH microplate assay kit (Cohesion Biosciences). Assays were performed on cells grown in LB medium until the OD₆₀₀ was around 0.8. Each experiment was performed in three independent replicates.

Effects of ethanol and NaCl.

Overnight cultures were diluted in fresh LB medium to an initial OD₆₀₀ of 0.1 and incubated in flasks with shaking (200 rpm) at 37°C. Bacterial growth was measured by following the optical density at 600 nm. In the ethanol stress assay, ethanol at a final concentration of 4% was added when the OD₆₀₀ reached 0.6. In the saline assay, NaCl was added at final concentration of 0.1 M at the beginning of the cultures.

Effects of lysozyme and MgCl₂.

Overnight cultures were diluted at least 100-fold in fresh medium. Growth curves were done in 96-well plates in a Tecan plate reader (Tecan Infinite M200PRO) with continuous agitation at 37°C. Growth conditions were as indicated elsewhere in the text. At least three independent biological replicates were performed.

Assay of sensitivity to antibiotics.

The assay of antibiotic sensitivity was performed on solid media. B. subtilis strains were grown in LB medium to an OD₆₀₀ of 1. One milliliter of growing culture was spread onto petri plates containing LB medium. The plates were dried briefly in a laminar flow hood before 5-mm paper disks (Whatman) containing 25 μl of antibiotic solution were placed on the plates (penicillin G, 8 mg · ml⁻¹; oxacillin10, μg · ml⁻¹; tunicamycin, 375 μg · ml⁻¹; vancomycin, 0.5 μg · ml⁻¹). The plates were incubated at 37°C overnight. The zones of inhibition were measured by using ImageJ after scanning the plates. Standard deviations were calculated from three independent assays.

Peroxide stress assays.

To perform disk diffusion assays, the cell number was determined in exponential-phase cultures by evaluating the CFU per milliliter. One milliliter of each microbial suspension containing 1 × 10⁸ bacterial cells ml⁻¹ (corresponding to a 0.5 McFarland standard) was spread over the surface of the agar plates. The plates were dried briefly in a laminar flow hood. A sterile 5-mm paper disk was placed on the agar surface, and 10 μl of 3 M hydrogen peroxide (H₂O₂) was added to the disk. For the stress assay in liquid medium, cultures were inoculated at an OD₆₀₀ of 0.05 in LB medium and incubated at 37°C with agitation at 200 rpm. At an OD₆₀₀ of 0.5, H₂O₂ was added at 0.1 mM. After 15 min of incubation, serial dilutions were plated on LB agar. The CFU per milliliter were counted. The number of CFU per milliliter before the stress was used as a reference to measure the rate of survival.

Quantification of O₂^·− and H₂O₂ free radicals.

The bacterial production of superoxide radical ion (O₂^·−) was evaluated by measuring the adsorption of XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] (Sigma). XTT was dissolved in PBS (pH 7) and added to the bacterial culture to a final concentration of 0.4 mM. When reduced by O₂^·−, XTT forms water-soluble XTT-formazan, which adsorbs light at 470 nm. The changes in absorbance at 470 nm were monitored using an Infinite M200 luminescence reader (Tecan, Germany). XTT absorbs light at 470 nm only when reduced by O₂^·−.

Intracellular production of H₂O₂ was quantified using the Amplex red assay. B. subtilis strains were grown in LB medium to mid-exponential phase. After centrifugation, pellets were resuspended in phosphate-buffered saline (PBS) and transferred to a 96-well plate that contained 20 μl of enzymatic mix (1 μl 10-acetyl-3,7-dihydroxyphenoxazine [ADHP] reagent, 1 μl horseradish peroxidase, and 18 μl assay buffer) in each well. Resorufin fluorescence was measured using a spectrofluorometer (Tecan Infinite M200PRO) with excitation and emission wavelengths of 530 and 590 nm, respectively. H₂O₂ calibration was done using H₂O₂ standard solutions ranging from 100 to 1,500 nM. Each experiment was performed in duplicate and repeated at least twice.

Whole-cell surface potential.

The zeta potential of the bacterial cell growth in exponential phase in LB medium was measured using a Zetasizer Nano ZS90 instrument (Malvern, UK). The results for zeta potential are presented as the average value from three independent cultures (10 measurements per culture).

TEM.

For transmission electron microscopy (TEM), bacterial pellets were collected at an OD₆₀₀ of 1 and fixed with 2% glutaraldehyde in 0.1 m sodium cacodylate buffer, pH 7.2, at room temperature for 3 h. Samples were then contrasted with 0.5% oolong tea extract in sodium cacodylate buffer (74), postfixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate, gradually dehydrated in ethanol (30% to 100%), substituted gradually in a mixture of propylene oxide and Epon, and embedded in Epon (Delta Microscopy, Labège, France). Thin (70-nm) sections were collected onto 200-mesh copper grids and counterstained with lead citrate. Grids were examined using a Hitachi HT7700 electron microscope operated at 80 kV (Elexience, France). Images were acquired with a charge-coupled device camera (Advanced Microscopy Techniques Corp., Japan). The thickness of the cell wall was measured on TEM micrographs of at least five cells at a magnification of ×70,000, taking at least six measurements on each cell. Statistical analysis was performed with the unpaired Student t test, and a P value of <0.05 was considered significant. Analyses were performed using Prism 7 (GraphPad Software, San Diego, CA).

Sample preparation for proteomic analysis.

To analyze the proteomic profiles of the wild-type, ΔmntR, and Δzur cells, protein extraction and tryptic digestion in gel were performed as described in detail previously (75). Briefly, four independent cultures of each B. subtilis strain were grown in LB medium at an OD₆₀₀ of 0.8. Cells were harvested by centrifugation and disrupted by a passage through a One Shot cell disrupter (Constant Systems Ltd., Warwickshire, UK). After centrifugation, the resulting supernatants were ultracentrifuged (100,000 × g, 1 h, 4°C). Cytoplasmic fractions were designated the soluble parts after a single ultracentrifugation step. The remaining pellets were considered the crude membrane fraction. The total protein concentration was measured using a NanoDrop instrument. All of the samples were loaded on 10% NuPAGE Bis-Tris gels (Invitrogen). In-gel digestion of the proteins was performed on bands excised from one-dimensional SDS-PAGE gel. The quantity of modified trypsin (Promega, sequencing grade) was 0.5 μg per sample. In the final step, tryptic peptides were resuspended in 25 μl of precolumn loading buffer containing 0.05% (vol/vol) trifluoroacetic acid (TFA) and 5% (vol/vol) acetonitrile prior to LC-MS/MS analysis.

LC-MS.

Mass spectrometry was performed on the Plateforme d'Analyse Protéomique de Paris Sud Ouest (PAPPSO) platform as described in detail previously (75). A NanoLC-Ultra Eksigent (Sciex) system connected to a Q-Exactive mass spectrometer (Thermo Fisher) by a nanoelectrospray ion source was used. The protein identification was performed with X!TandemPipeline (open-source software developed by PAPPSO, version 3.3.1) against a Bacillus subtilis 168 protein database (4,253 entries). The X!Tandem search parameters were as follows: trypsin specificity with two missed cleavages, fixed alkylation of cysteine (+57.0215), and variable oxidation of methionine (+15.9949). The protein identification was run with a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da. For all proteins identified with a protein E value of <0.01 in the first step, we searched for additional peptides to reinforce identification using similar parameters except that semitryptic peptides and protein N-terminal acetylations were accepted. The final search results were filtered as follows: (i) peptide E value of <0.01 with a minimum of 2 peptides per protein and (ii) protein E value of <0.05.

Relative quantification of peptides and proteins.

Peptide were analyzed by spectral counting (SC). SC takes into account the number of assigned spectra for each protein and is correlated to relative protein abundance. For control quality of data, normalization, filtration, and statistical analysis, MassChroqR (http://pappso.inra.fr/bioinfo/masschroqr/) was used. Proteins with fewer than two peptides were removed. Peptides with a variation ratio of <1.5 were eliminated, as were peptides with a standard deviation from the retention time of 20 s and higher. Repeatable peptides were those which were present in at least 7 of 8 samples. The data set was normalized based on the median retention time, and missing peptide intensities and protein abundances were imputed. The proteins whose numbers of peaks were significantly different (with a minimum difference of 5 peaks between the mutant and the wild type) were determined by using the Kruskal-Wallis test. A one-way ANOVA model was used to analyze changes, with the genotype as a fixed effect. A protein was considered significantly variable when the P value was <0.05 (see Table S1 in the supplemental material).

Availability of data.

All supporting data are included in the main article and in the supplemental material.