Curr Microbiol DOI 10.1007/s00284-016-0998-8

Transcriptional Profiling Analysis of Bacillus subtilis in Response to High Levels of Fe3+ Wen-Bang Yu1 • Bang-Ce Ye1

Received: 29 October 2015 / Accepted: 22 December 2015  Springer Science+Business Media New York 2016

Abstract Iron is essential to microorganisms for its important biological function but could be highly toxic in excess. We have used genome-wide transcriptional analysis in Fe3?-treated (4 mM) Bacillus subtilis to reveal the effect of excess Fe3? on B. subtilis and characterized the potential pathways involved in Fe3? stress tolerance. A total of 366 and 400 genes were identified as significantly up-regulated and down-regulated, respectively. We found excess Fe3? had four major influences on B. subtilis: Fe3? resulted in oxidative stress and induced genes involved in oxidative stress resistance including the SigB-regulated genes, but the PerR regulon was not inducible in Fe3?mediated oxidative stress except zosA; Fe3? significantly disturbed homeostasis of Mn2? and Zn2?, and the mechanism was proposed in this article; the acidity of Fe3?induced genes involved in acid consuming and production of bases and shifted B. subtilis to carbon starvation state; Fe3?-induced genes related to membrane remodeling (bkd operon), which prevents Fe3?’s incorporation to membrane lipids. Moreover, Fe3? repressed the stringent control response, consistent with the induction of stringent control in iron limitation, demonstrating that iron might be a signal in stringent control of B. subtilis. This study was the first to provide a comprehensive overview of the genetic response of B. subtilis to ecxess Fe3?.

Electronic supplementary material The online version of this article (doi:10.1007/s00284-016-0998-8) contains supplementary material, which is available to authorized users. & Bang-Ce Ye [email protected] 1

Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong RD 130, Shanghai 200237, China

Introduction Iron is essential for microorganism because of its important biological function. Due to its redox activity, iron participates in numbers of important metabolic processes involving electron transfer. To some extent, the availability of iron could determine the growth efficiency of bacteria. Because of the existence of oxygen, iron was oxidized to Fe3?, which was poorly soluble in most environments. Thus to confirm iron requirement, bacteria had evolved complex and efficient pathways to acquire enough iron [38]. For pathogens, to collect iron is important for their survival in host [41]. Although it is essential, iron could be toxic in excess concentration. The well-known cellular toxicity of iron was due to its catalytic role in the Fenton reaction, in which ferrous iron (Fe2?) mediates the formation of extremely toxic hydroxyl radical leading to lethal damage [51]. Thus, to maintain iron homeostasis is important for microorganism’s cellular function. The metal ion homeostasis regulation pathways include import, distribution, and efflux. For some Gram-Positive and Gram-Negative bacteria including Bacillus subtilis, iron import and distribution were regulated by ferric uptake regulator (Fur), a dimeric DNA-binding protein with one-structural Zn2? per monomer and a regulatory Fe(II)-binding site, which represses genes involved in iron transport and siderophore synthesis in iron sufficiency environment [2, 10, 48]. Transcriptional analysis has been conducted to study the response of B. subtilis to iron starvation by Baichoo et al., and they observed that almost iron starvation inducible genes were under the control of Fur [2]. The toxicity of iron was not restricted to its catalytic role in Fenton reaction. Recently, Chamnongpol and his colleagues found that ferric iron (Fe3?) could cause damage to

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Gram-Negative bacteria’s outer membrane, which is composed of lipid bilayer, including phospholipids, lipopolysaccharides, and proteins [5, 30]. More recently, Suwalsky et al. revealed that Fe3? would incorporate into phospholipid bilayer by interacting with the phosphate of the polar head groups and disrupt its arrangement and consequently the whole of the bilayer structure. This study indicated Fe3? might interact with microorganism’s membrane phospholipids bilayer [49]. In natural environment, concentration of Fe3? might exceed 10-3 M, beyond the concentration that causes outer membrane damage or membrane structure [56]. Thus, pathways to deal with Fe3? toxicity were essential. Wosten et al. revealed that the PmrAB two-component systems in Gram-Negative bacteria could promote transcription of genes involved in modification of the outer membrane for resistance in the presence of high level Fe3? [5, 56]. However, no such pathways have been found in Gram Positive bacteria. Bacillus subtilis is a kindly characterized Gram Positive, spore-forming model bacteria in research. The genome of B. subtilis 168, the experimental strain, was sequenced in 1997 and demonstrated to encode 4106 proteins [27]. B. subtilis is a well Gram-Positive model to study the antibiotic action, stress response, and metal ion homeostasis. Moore et al. have used transcriptional profile to study the response of B. subtilis to several excess heavy metal stress, including Cd2?, Co2?, Zn2?, and Cu [35]. They have characterized that CadA is the major determinant for Cd2? resistance, while CzcD protects the cell against elevated levels of Zn2?, Cu, Co2?, and Ni2? [35]. In the present study, to reveal how excess iron affects B. subtilis and identifies the pathways that might be involved in iron detoxification, we studied the transcriptional response of B. subtilis to excess Fe3?. Transcriptional profile analysis was a useful method for research of the metal ion metabolism. Thus, we used microarrays represented 4106 protein encoding genes of B. subtilis to obtain the overview of significantly disturbed pathways under excess Fe3?. The results indicated Fe3? response involved metal ions homeostasis, general stress response (GSR), membrane remodeling, pH homeostasis, and metabolic adaptation in addition to oxidative stress response. This study provided new insights to better elucidate the effect of excess iron on B. subtilis.

Experimental Procedures Bacterial Strain and Growth Conditions Bacillus subtilis CU1065 strain was grown in Luria–Bertani (LB) medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter of distilled H2O) at 37 C with shaking

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at 200 rpm. Then the seed culture was used to inoculate in new 20 ml LB medium in a 50-ml Erlenmeyer flask. For the iron-stress experiments, excess iron was added at 4 mM (FeCl3). The FeCl3 contains Mn. Microarray Construction and Hybridization Cultures were grown to mid-log phase (OD600 of 1.3) and split into two flasks. Samples were collected after Fe3? addition at 20 min. The RNA isolation and microarray analysis was performed as previously described for amino acid additions [57]. The fold induction values were calculated by using the signal intensity values of treated samples divided by those of untreated samples. The method of microarray construction and data analysis was similar to our previous work [60]. For microarray experiments, two independent replicates were performed. Microarray Data Analysis For data extraction, normalization, and filtration, we used the methods as described in our previous work [58]. To identify differential expression genes responding to excess Fe3?, we used fold change method (twofold as a cut-off value), considering the samples of none Fe3? treatment as control. Genes with fold change bigger than two were thought significantly disturbed. The original data is deposited in GEO and the accession number is GSE71694.

Results Global Response of B. subtilis to Excess Fe31 To gain insight into B. subtilis’ response to excess Fe3?, we grew B. subtilis in LB medium to mid-logarithmic phase and added 4 mM Fe3?. Bacteria sample were harvested at 20 min after Fe3? addition to capture the initial transcriptional response elicited by Fe3?. When twofold change relative to the control was used as a cut-off value, 766 genes were identified as significantly perturbed, of which 400 genes were down-regulated and 366 genes were upregulated (Table S1). Subsequently, we used MIPS functional analysis to identify significantly perturbed pathways by Fe3?. The results demonstrated that Fe3? had a wide and prominent effect on B. subtilis’ pathways involved in functions of metabolism, energy, cell cycle and DNA processing, transcription, protein synthesis, protein fate, protein with binding function or cofactor requirement, regulation of metabolism and protein function, cellular transport, cell rescue, interaction with the environment, transposable elements, cell fate, biogenesis of cellular

W.-B. Yu, B.-C. Ye: Transcriptional Profiling Analysis of Bacillus subtilis in Response…

Fig. 1 Functional classification of significantly disturbed genes. The significantly perturbed genes in Fe3? stress were classified according to MIPS functional database (http://mips.helmholtzmuenchen.de/ funcatDB/). 01 metabolism; 02 energy; 10 cell cycle and DNA processing; 11 transcription; 12 protein synthesis; 14 protein fate (folding, modification, destination); 16 protein with binding function or cofactor requirement (structural or catalytic); 18 regulation of metabolism and protein function; 20 cellular transport, transport facilities, and transport routes; 30 cellular communication/signal transduction mechanism; 32 cell rescue, defense, and virulence; 34 interaction with the environment; 38 transposable elements, viral and plasmid proteins; 40 cell fate; 42 biogenesis of cellular components; 43 cell-type differentiation; 99 unclassified proteins (Color figure online)

component, cell-type differentiation, and unclassified proteins (Fig. 1; Table S2). Transcriptional factors play a central role to restructure the transcriptome responses to environmental signals. The microarray data were subsequently analyzed using T-profiler to identify some transcriptional factors in response to iron level change. T-profiler, a computational tool developed originally for the analysis of S.cerevisiae’s transcriptome data, has been adapted for the analysis of transcriptome data of B. subtilis [50]. T-profiler optimally uses all transcriptional data and transforms data of single genes into the behavior of gene groups, reflecting biological processes in cells. All gene groups with significant E values (\0.05) were presented in Table S3 (see Supporting information), including SigB, PerR_Negative, PurR_Negative, Strcon_Negative, SigW, and Fur_Negative. The SigB, PerR_Negative, and PurR_Negative exhibited significant positive T value, indicating these regulons were at least partially overexpressed; while the PerR_Negative, SigW, and Fur_Negative exhibited significant negative T value, indicating expression of these regulons were at least partially repressed. Oxidative Stress Response The primary toxicity of iron is ascribed to Fe2?, which mediates hydroxyl radical formation and causes oxidative stress at excess level [52]. As expected, we observed that Fe3? stress response involved a number of genes related to

oxidative stress resistance. The MIPS functional analysis revealed that Fe3? significantly induced genes in the function of oxygen and radical detoxification (Table S2). The following genes were up-regulated, including sodF (similar to Fe dependent superoxide dismutase), yqiG (probable NADH-dependent flavin oxidoreductase), and yqjM (probable NADH-dependent flavin oxidoreductase). yqiG and yqjM may involve in oxidative stress detoxification [36]. The hydroxyl radical formation mediated by Fe2? was highly toxic and destructive to DNA/proteins/ lipids [1, 25]. Following the Fe3? treatment, we expected to see the activation of DNA damage response (SOS system). As predicted, dinB, dnaE, uvrAB, xkdA, yhaZ, yneB, and yqxL that involved in SOS were up-regulated more than twofold. The GSR, regulated by the sigma factor SigB that controls at least 150 genes, can provide the B. subtilis with nonspecific protection. The SigB regulon can be induced in nutrient starvation, heat shock, ethanol, and alkaline stress [16]. Herein, the T-profiler analysis yielded a significant positive T value for SigB, indicating the SigB regulon was at least partially up-regulated (Table S3). 17 genes of the SigB gene group were up-regulated more than twofold. 11 out of the 17 genes were involved in oxidative stress tolerance, including katX, csbB, ydbD, ykgA, ydaP, yqxL, dps, yfhK, yfkJ, yfkH, and yfkI [42]. PerR is an important oxidative stress response regulator and represses expression of 15 genes in B. subtilis. However, the T-profiler analysis yielded a negative T value for PerR_Negative, indicating this regulon was at least partially down-regulated (Table S3). Ten genes of this regulon were down-regulated more than twofold (Table S3). However, the zosA gene of the PerR regulon was significantly up-regulated. zosA encodes a Zn2? uptake protein and is important for oxidative stress resistance [12]. This finding was consistent with the previous studies, which suggested PerR-mediated repression of most target genes could be elicited by either manganese or iron [11]. Effect of Fe31 on Metal Ion Homeostasis High concentration of heavy metal in environment always represses metal import transporters and induces sequestering proteins or efflux pumps so as not to excessive accumulation of metal ions [35]. The MIPS functional analysis revealed that Fe3? significantly repressed the genes in the function of metal ion transport and homeostasis of cations (Table S2). T-profiler analysis demonstrated that Fe3? significantly repressed the Fur regulon (Table S3). As presented in Fig. 2, expression of genes involved in bacillibactin synthesis (dhbABCF), bacillibactin-Fe3? uptake (feuABC), ferrichrome-Fe3? uptake (fhuBCDG), schizojinen/arthrobactin-Fe3? uptake (yfiYZ-

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W.-B. Yu, B.-C. Ye: Transcriptional Profiling Analysis of Bacillus subtilis in Response… Fig. 2 Effect of Fe3? on metal ion homeostasis. The significantly perturbed genes involved in metal homeostasis were colored. Up-regulated genes were colored in orange and down-regulated genes were colored in green (Color figure online)

yusV-yfhA), and elemental Fe3? uptake (ywbLM) were all significantly repressed ([6 fold) (Fig. 2) [38]. However, only one gene of the yfmCDEF operon encoding citrateFe3? uptake was repressed more than twofold (2.6 fold) (Fig. 2). The data of Baichoo et al. had also showed that yfmCDEF was most slightly derepressed among the iron transporters in fur mutant, consistent with our study [2]. The yfkM gene of the Fur regulon was up-regulated more than twofold (Table S1). yfkM was regulated by SigB and was not significantly repressed by Fur (Table S3) [39]. Besides the Fur regulon, the genes (mntABCD and mntH) involved in Mn2? transport and the genes (yciC and ycdH) involved in Zn2? transport were all significantly repressed (Fig. 2) [13, 40]. Expression of mntABCD and mntH was repressed by MntR, which could be activated by Mn2? and Cd2? in vivo, but not Fe2? and Zn2? (Mcguire, 2013). Thus, high level of Fe in vivo likely increased the pool of Mn. yciC and ycdH both encode Zn2? transporter and are repressed by Zur, which is also highly Zn2? specific [13]. Herein, we suggested that excess Fe3? might result high level pool of Zn2?, so as to repress the Zur regulon. This was supported by the significant up-regulation of zosA, up-regulation of which would increase Zn level in vivo [12]. Fe3?-induced genes involved in other cation transport (yqoK/yqxL/yubG/yuaA) (Fig. 1). ykoK and yqxL both encode transporters of Mg2?, which might inhibit toxic effect of Mn2?, Fe2?, Co2?, Ni2?, and Cu2? [15, 21, 44]. yubG and yuaA both encode transporters of K? [20, 35].

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A few genes encoding metal ions efflux proteins were significantly induced by Fe3?, including cadA, czcD, ydfM, yfmO, and yeaB (Fig. 2). cadA and czcD were regulated by the CrzA regulator and were strongly induced by Zn2?, followed by Cd2?, Co2?, Ni2?, and finally Cu [35]. Thus, induction of these genes might indicate accumulation of Zn2?, consistent with the repression of the Zur regulon. ydfM, yfmO, and yeaB all encode efflux pumps of unknown divalent metal ions. When NCBI Conserved Domain search tool was used to identify the conserved domain of YdfM, YfmO, and YeaB, we found that YdfM had a similar Fe2? efflux domain to the protein FieF of E. coli [14]. These results indicated that YdfM might be involved in Fe2? efflux in B. subtilis. Response to Cope with Acidic pH The Fe3? is a lewis acid and it might cause cytosolic acidification of B. subtilis. As expected, we observed a significant perturbation of the genes involved in pH homeostasis. The MIPS functional analysis revealed that Fe3? repressed a few genes in the function of pH homeostasis (Table S2). The following genes were significantly down-regulated, including mrpA (Na?/H? antiporter subunit A), mrpE (Na?/H? antiporter subunit E), mrpF (Na?/ H? antiporter subunit F), yhaUT (K?/H? antiporter), and yjbQ (similar to Na?/H? antiporter) (Fig. 3). mrpAEF and yhaUT were known to be induced in alkaline stress [23, 54]. The maeN gene encoding Na?/malate symporter was

W.-B. Yu, B.-C. Ye: Transcriptional Profiling Analysis of Bacillus subtilis in Response… Fig. 3 Effect of Fe3? on pH homeostasis. The significantly perturbed genes involved in pH homeostasis were colored. Upregulated genes were colored in orange and down-regulated genes were colored in green (Color figure online)

up-regulated in excess Fe3? conditions (Fig. 3). This gene was known to be induced by acids and repressed by bases. Moreover, the yxkJ gene encoding proton/malate transporter known to be induced by weak acid (sorbic acid) was also up-regulated here [50]. Polyamine is a source of base in microorganism. At high pH, polyamines could be toxic and should be exported [54]. In B. subtilis, the spermine and spermidine synthesis requires arginine/methionine and involved speA/speB/ speD/speE/metK [46]. Herein, we observed that the speD (S-adenosylmethionine decarboxylase) was up-regulated (Fig. 3). The spermine and spermidine export was initiated by acetylation [55]. We observed the genes encoding spermine/spermidine efflux pump (blt) and acetyltransferase (bltD) that induced by base stress were all downregulated [54] (Fig. 3). These results demonstrating polyamine might be helpful in acidic conditions. The putative arginine/ornithine synporter (yvsh) that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway was up-regulated (Fig. 3). Moreover, the putative arginine deiminase (ykgA) was also up-regulated (Fig. 3). In some microorganism, arginine deiminase pathway was used to resist acid stress, as it produces ammonia that could raise pH [31]. In a lot of microorganisms including B. subtilis, low pH can elicit catabolism to consume acids and pump H? out of the cell [54]. We observed a similar metabolic adaptation pattern in excess Fe3? environment. The alcohol dehydrogenase (abhA), putative formate dehydrogenase (yjgC/

yrhE), and putative NADH-dependent butanol dehydrogenase (yugK) were all up-regulated in excess Fe3? environment (Fig. 3). Moreover, the genes related to electron transport chain (ETC) including cytochrome d oxidase subunits (cydABCD), aa3 oxidase subunits (qoxABC), NADH dehydrogenase (ndh), and the menaquinone synthesis genes (menDEF) were all up-regulated (Fig. 3). Upregulation of these genes might be helpful to consume acids and export H? out of the cell. In E. coli, low pH would up-regulate genes involved in TCA cycle and glycolysis [32]. Herein, we also observed that Fe3? up-regulated a few genes involved in glycolysis and TCA cycle, including pgk (phosphoglycerate kinase), pgm (phosphoglycerate mutase), and sdhABC (succinate dehydrogenase) (Fig. 3). Although low pH elicited acids consumption, some probable negative effects were observed. The lactate dehydrogenase gene (ldh) related to lactate formation and the lactate permease (lctP) were up-regulated. However, the similar effects were also observed in E. coli, in which low pH also amplified membrane-permeant acids uptake [43]. Effect of Fe31 on Carbon–Nitrogen Balance The MIPS functional analysis revealed that Fe3? induced or repressed lots of genes in the function of metabolism, carbon metabolism including amino acids metabolism, nucleotide metabolism, and secondary metabolism. Herein, we observed a significant perturbation of genes involved in carbon metabolism. The following genes related to PTS

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components (fruA/mtlA/ydhMNO/yyzE), fructose-1-phosphate kinase (fruB), acetate kinase (ackA), and probable sugar transport system subunits (yufOP/lplAB) were all upregulated (Fig. 4), indicating that Fe3? enlarge B. subtilis’ demand on carbon sources. Besides these genes that involved in non-glucose substrate utilization, a lot of genes that repressed by carbon catabolite control protein (CcpA) were up-regulated here, including kdgRKAT, treP, ydhO, scoA, yxkJ, hutGHIMU, and araA (Fig. 4). The CcpA protein was activated by FBP in the presence of preferred carbon source (glucose, fructose and mannonse) and derepressed in carbon starvation [47]. Induction of these genes might indicate B. subtilis shift to carbon starvation state in elevated Fe3? environment. Although many CcpArepressed genes were depressed, the citM encoding citrate/divalent metal ions (Zn2?/Mg2?/Co2?/Mn2?/Ni2?) complex transporter was repressed [26] (Fig. 4). In B. subtilis, the balance between carbon and nitrogen was regulated by the metabolic flux distribution of pyruvate and 2-oxoglutarate [47]. In carbon sufficiency, pyruvate and 2-oxoglutarate were synthesized to branchedchain amino acids (BCAA) and glutamate, respectively; while in carbon starvation, synthesis of BCAA or glutamate was inhibited and the glutamate was degraded to 2-oxoglutarate [47]. Consistent with the carbon starvation response, the genes involved in BCAA synthesis (ilvBC/ leuACD) were repressed (Fig. 4). However, the gltAB (glutamate synthase) did not show significant repression.

Fig. 4 Effect of Fe3? on carbon–nitrogen balance. The significantly perturbed genes involved in carbon–nitrogen balance were colored. Upregulated genes were colored in orange and down-regulated genes were colored in green (Color figure online)

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Moreover, several genes that related to threonine synthesis (thrBC/hom) were also repressed (Fig. 4). In B. subtilis, threonine was synthesized indirectly from oxaloacetate of the TCA cycle [47]. In B. subtilis, the enzymes of de novo purine and pyrimidine synthesis were encoded by the pur and pyr operon, including 25 genes. Herein, 12 genes (purBCDEFHKLMNQS) involved in purine synthesis and five genes (pyrAACDFG) involved in pyrimidine synthesis were up-regulated (Fig. 4). The pur and pyr operon were repressed by PurR and PyrR, respectively, both of which were inactivated by PRPP [29]. Up-regulation of these genes might indicate Fe3? result in an increased pool of PRPP. Effect of Fe31 on Cell Membrane Remodeling As suggested by Suwalsky, Fe3? would incorporate into phospholipid bilayer by interacting with the phosphate of the polar head groups and disrupt its arrangement and consequently the whole of the bilayer structure [49]. Thus, we expected to observe that high level of Fe3? induces membrane adjustment response to prevent Fe3?’s incorporation. Genes involved in the functions of cell surface are regulated by ECF sigma factors, like SigW/SigX [18]. Indeed, expression of many SigW-regulated genes was significantly altered (Table S3). The T-profiler analysis yields a significant negative T value for SigW, indicating

W.-B. Yu, B.-C. Ye: Transcriptional Profiling Analysis of Bacillus subtilis in Response…

this regulon was at least partially repressed. 36 genes out of 62 SigW-regulated genes were repressed more than twofold (Table S3). Repression of these genes suggested that the SigW regulon was expressed in our control conditions. This has also been observed in other studies [61]. In B. subtilis, most membrane lipids synthesis genes are regulated by FapR, which regulates initiation genes (fabHA/fabHB), fabD, and elongation genes (fabF/fabG/ fabI) [45]. Neither of these genes showed significant expression alteration. However, most of the genes of BkdR regulon were significantly down-regulated, including bcd, bkdAA, bkdAB, bkdB, ptb, and buk (Fig. 5). The BkdR regulon is to provide precursors for branched-chain fatty acids (BCFA) synthesis [8]. Down-regulation of these genes would reduce the number of BCFA in membrane lipids and increase percentage of straight-chain fatty acids (SCFA) in lipids, leading to a more rigid lipids bilayer, which might limit Fe3?’s binding to cell membrane [24, 50]. The effect of Fe3? on membrane structure remodeling has also been noticed in our previous work [58]. Effect of Fe31 on Stringent Control The T-profiler analysis revealed that the Strcon_Negative gene group produced a significant Positive T value, indicating an induction pattern of this group. 33 out of 87 Strcon_Negative genes were induced more than twofold (Table S3). While 28 up-regulated genes were involved in protein synthesis, murE was involved in cell wall synthesis

and yjlD encoded NADH dehydrogenase. Strcon_Negative refers to the gene group repressed in stringent response, which coordinates a global transcriptional pattern in response to nutrient starvation [6, 9]. Researches have revealed that the stringent response could be initiated by iron limitation in B. subtilis [17, 34, 37, 53]. Thus, these results suggested that iron might be a signal of stringent control in B. subtilis.

Discussion In this work, we used microarrays to study how B. subtilis responds to excess Fe3?. Our results revealed that a lot of pathways, including iron homeostasis, oxidative stress response, membrane remodeling, and pH homeostasis, were involved in Fe3? response. These data suggested Fe3? very likely had multiple effects on B. subtilis in addition to oxidative damage. Herein, we proposed a model how Fe3? affects B. subtilis’’s growth. High concentration of Fe3? would likely cause cytosolic acidification and induce acid response. Then, when Fe3? binds to the cell surface, it would disturb membrane structure. Finally, high concentration of Fe3? resulted in elevated Fe2? pool in vivo and caused oxidative damage. To maintain iron homeostasis is essential for microorganism to ensure enough iron import and prevent iron toxicity [56]. Thus, it is reasonable to observe that a set of genes involved in iron homeostasis was perturbed.

Fig. 5 Membrane remodeling response. The significantly perturbed genes involved in membrane remodeling were colored. Up-regulated genes were colored in orange and down-regulated genes were colored in green (Color figure online)

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However, the repression of Zur and MntR regulon was surprising to us. Repression of Zur could be explained by the induction of zosA. Up-regulation of this gene would increase Zn pool in vivo. For the repression of MntR regulon, we explained as follows. Although Fe in vivo could not activate MntR, Fe could compete with Mn to bind MntR [33]. Thus, a relative high level of Fe would restrain Mn to bind MntR, likely leading to malfunction of MntR. Then malfunction of MntR might result in increased Mn transport. But when Mn in vivo was increased to high enough, Mn might displace Fe from MntR, thus leading to repression of MntR regulon. Elevated Fe3? in the environment must increase Fe in vivo and result oxidative stress. However, most of the PerR-regulated genes were repressed except zosA. The repression of PerR regulon might impair B. subtilis’ oxidative stress resistance, since the PerR-regulated genes (like katA/ahpCF) were important in H2O2 detoxification [3]. In B. subtilis, PerR could be in two active form, PerR:Zn,Fe and PerR:Zn,Mn [19]. Activity of PerR:Zn,Fe was easily eliminated by H2O2, whereas PerR:Zn,Mn was comparatively resistant to H2O2 [19]. Excess Mn could displace Fe from PerR and enhanced its repression [19]. Thus, we proposed that repression of the PerR regulon due to increased Mn in vivo in excess Fe3? environment, since high level of Fe probably increased transport of Mn as we discussed. Down-regulation of the SigW regulon was somehow surprising, as this regulon was induced in envelope stress environment. Note that the medium we used had no stress factors, but the microorganism was cultured in the shaker with a shaking speed at 200 r/min. Since the study of Butler et al. had revealed that shearing force could enhance fluidity of cell membrane, it was possible that the shearing force produced in the shaking process could enhance B. subtilis’s membrane structure fluidity, thus the SigW was activated, since Kingston et al. suggested that SigW was activated by compounds that increased membrane fluidity [4, 24]. SigW was a regulator of B. subtilis’s homeoviscous adaptation, thus excess Fe3? might somehow block homeoviscous adaptation of B. subtilis [24]. After exposure to Fe3?, a significant repression of the bkd operon that involved in membrane lipids synthesis was observed. Repression of bkd operon would reduce BCFA synthesis and membrane fluidity [7, 24]. This remodeling might effectively prevent Fe3?’s incorporation to phospholipids, since Suwalsky suggested that a tighter membrane lipids bilayer could prevent Fe3?’s incorporation to lipids phosphate head group [49]. To maintain pH homeostasis is essential for microorganism to survive in pH stress. In general, B. subtilis showed metabolic pattern that favored to consume acids and pump H? out of the cell in excess Fe3? environment. The expulsion of protons to maintain pH homeostasis by the respiratory chain could lead to a higher demand on

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energy resources. Thus, enhancement of respiration might be the reason that induced carbon starvation response in excess Fe3? environment. Moreover, the enhancement of protein synthesis, purine synthesis, and cation export all need more energy. All of these might contribute to enlargement of energy demand and cause carbon starvation response. The carbon starvation response was also observed in sorbic acid stress and Fusaricidins stress in B. subtilis [50, 59]. Fe3? also enhanced expression of genes involved in the polyamine and ammonia production. Production of ammonia was important for low pH tolerance [31]. Polyamines are small aliphatic amines found in all living organisms except some Archaea [28]. In plants, polyamines are major components that are not only involved in fundamental cellular processes but also in adaptive responses to environmental stress [28]. Moreover, polyamine and its modulon were important for E. coli’s growth in acid pH [22]. Thus, we suggested polyamine synthesis might be helpful in high level of Fe3?. Acknowledgments This study was supported by the China NSF (21276079, 21335003), SRFDP (No. 20120074110009), the Key Grant Project (No. 313019) of the Chinese Ministry of Education, and the Fundamental Research Funds for the Central Universities. Compliance with Ethical Standards Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, ‘‘Transcriptional profiling analysis of Bacillus subtilis in response to high levels of Fe3?.’’

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Transcriptional Profiling Analysis of Bacillus subtilis in Response to High Levels of Fe(3.).

Iron is essential to microorganisms for its important biological function but could be highly toxic in excess. We have used genome-wide transcriptiona...
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