The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic

Ahmed Gaballa*, Haike Antelmann†, Claudio Aguilar*, Sukhjit K. Khakh*, Kyung-Bok Song*, Gregory T. Smaldone*, and John D. Helmann*‡

*Department of Microbiology, Cornell University, Ithaca, NY 14853-8101; and †Institute for Microbiology, Ernst-Moritz-Arndt University of Greifswald, F.-L.-Jahn-Strasse 15, D-17487 Greifswald, Germany

Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved June 3, 2008 (received for review December 13, 2007)

Regulation of bacterial iron is often controlled by the (15) and are hypersensitive to H2O2, which reacts with free iron to iron-sensing ferric uptake (Fur). The Bacillus subtilis Fur generate hydroxyl radicals (16). acts as an iron-dependent repressor for siderophore bio- Despite the constitutive expression of iron uptake pathways, synthesis and iron transport proteins. Here, we demonstrate that E. coli fur mutants have significantly reduced levels of total Fur also coordinates an iron-sparing response that acts to repress cellular iron (17). This apparent paradox is now understood as the expression of iron-rich proteins when iron is limiting. When indicative of a small RNA (sRNA)-mediated repression of Fur is inactive, numerous iron-containing proteins are down- numerous iron-containing proteins (18, 19). In the absence of regulated, including succinate dehydrogenase, aconitase, cyto- Fur, the RyhB sRNA represses the synthesis of succinate dehy- chromes, and biosynthetic enzymes for heme, cysteine, and drogenase (Sdh), aconitase, fumarase, and iron superoxide dismutase (20). As a result, total cellular iron is decreased, branched chain amino acids. As a result, a fur mutant grows slowly despite a significant increase in the pool of chelatable, cytosolic in a variety of nutrient conditions. Depending on the growth iron. medium, rapid growth can be restored by mutations in one or more The repression of iron-rich enzymes when iron is limited is of the molecular effectors of the iron-sparing response. These undoubtedly adaptive and has evolved multiple times. This effectors include the products of three Fur-regulated operons that repression, which we here refer to as an iron-sparing response, encode a small RNA (FsrA) and three small, basic proteins (FbpA, allows the cell to prioritize iron usage at times of limited FbpB, and FbpC). Extensive complementarity between FsrA and the availability. Similar RyhB-mediated responses have been de- leader region of the succinate dehydrogenase operon is consistent scribed in Shigella flexneri and Vibrio cholerae (19). In Pseudo- with an RNA-mediated translational repression mechanism for this monas aeruginosa, two functionally analogous, Fur-regulated target. Thus, iron deprivation in B. subtilis activates pathways to sRNAs were described in ref. 21. In Saccharomyces cerevisiae, remodel the proteome to preserve iron for the most critical cellular iron deprivation leads to the activation of Aft1, which controls functions. expression of iron uptake functions and Cth2, an RNA-binding protein that targets for degradation the mRNAs for aconitase, ron is an essential element for nearly all cells but is often of Sdh, and perhaps dozens of additional proteins (22). Iron- sparing responses are also likely to be present in Gram-positive limited availability in natural environments (1). Bacteria have I bacteria. In Corynebacterium spp., the iron sensor DtxR re- evolved sophisticated mechanisms to scavenge iron by the elab- presses the RipA protein, which, in turn, is a repressor of oration of high-affinity iron-chelating agents, siderophores, and iron-using proteins (23). Analysis of the S. aureus proteome as their transport systems (2, 3). Expression of these iron uptake a function of iron availability also suggests the presence of an pathways is usually repressed under iron-sufficient conditions by iron-sparing response, but the mechanism is not yet known (24). an iron-sensing repressor. The paradigm for iron repression in Here, we present evidence for an iron-sparing response in B. bacteria is the ferric uptake repressor (Fur) protein as originally subtilis mediated primarily by a Fur-regulated sRNA (FsrA). The described in Escherichia coli (1). Fur orthologs are found in both action of FsrA is independent of an Hfq-like protein encoded by Gram-negative and many Gram-positive bacteria, including Ba- B. subtilis but depends, in part, on three small, basic proteins cillus spp., Staphylococcus aureus, and Listeria monocytogenes (4). designated as Fur-regulated basic proteins (Fbp) A–C. These In Corynebacterium spp. and Mycobacterium spp., iron regulation basic proteins are postulated to function as RNA chaperones. is mediated instead by a functionally similar protein designated We show that together these regulators contribute to many of the DtxR (5, 6) or IdeR (7, 8), respectively. In the Rhizobiales, iron pleiotropic effects noted in fur mutant strains, including poor regulation is mediated in large part by RirA, a member of the growth on a variety of media. Rfr2 family of (9). Results and Discussion Despite differences in the nature of the regulator, there are many common themes in the bacterial responses to iron starva- Fur Regulates the Expression of a sRNA (FsrA) and Three Small, Basic tion. Notably, in most well studied systems, there is an induction Proteins. Genes repressed by Fur in B. subtilis are associated with Fur boxes (25). Classically, the Fur box is represented as a 19-bp of siderophore biosynthesis and uptake or other pathways to increase iron assimilation. Bacillus subtilis expresses uptake MICROBIOLOGY systems for its endogenous siderophore bacillibactin, ferric ci- Author contributions: A.G., C.A., and J.D.H. designed research; A.G., H.A., C.A., S.K.K., trate, and for a variety of siderophores made by other soil K.-B.S., and G.T.S. performed research; A.G., H.A., C.A., S.K.K., K.-B.S., G.T.S., and J.D.H. bacteria (10). In pathogenic bacteria, uptake pathways may also analyzed data; and A.G. and J.D.H. wrote the paper. involve hemolysins, heme-binding proteins, and transferrin- The authors declare no conflict of interest. binding proteins (2, 11, 12). B. subtilis can also use an elemental This article is a PNAS Direct Submission. Fe uptake system YwbLMN (10), which is homologous to ‡To whom correspondence should be addressed. E-mail: [email protected]. Saccharomyces cerevisiae Fet3p/Ftr1p and E. coli EfeUOB sys- This article contains supporting information online at www.pnas.org/cgi/content/full/ tems (13, 14). As a result of derepressed transport, fur mutants 0711752105/DCSupplemental. typically have elevated levels of chelatable iron within their cytosol © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711752105 PNAS ͉ August 19, 2008 ͉ vol. 105 ͉ no. 33 ͉ 11927–11932 Downloaded by guest on September 26, 2021 reveals a pattern of base substitutions consistent with a protein A ykuI FB fsrA ykuJ coding region (Fig. S3). Moreover, addition of a FLAG epitope FB fbpA ydbO fbpB ydbM allows visualization of the corresponding protein (Fig. 1D). ypbQ FB fbpC ypbR Our working hypothesis is that these three small proteins function together with FsrA to direct repression of selected B C target genes. However, as shown below, FsrA can function at some targets independent of these proteins. Moreover, the fur/fbpC fur/ fbpABC WT fur fsrA fur/fsrA fur/ fbpAB nM Fur 00.1110100 fbpAB and fbpC operons also have functions independent of fsrA FsrA and these may be mediated by the encoded peptides, the fsrA 23S RNA transcripts, or both.

D E FbpA MTPQSAEIRKKQLIHLLIQNGIYKKSKRHLYE fur Mutant Is Growth Impaired. Previous observations indicate that FbpB -ve FbpA FbpC LSLTELESEYKHVRRAPEHAEA PI: 9.5 fur FbpB MPKRKVKTYQQLIQENKEAIMGNPKLMNVIY a null mutation has pleiotropic effects on growth consistent DRIDRKHQKNLQEQNNT PI: 9.9 with the hypothesis that Fur mediates the iron-dependent syn- FbpC MTMLFLLGAVCTYSVLIGFVLKGISNKSV PI: 9.1 thesis of iron-using enzymes of central metabolism. Indeed, Fig. 1. Molecular components of the B. subtilis iron-sparing response. (A) transcriptome analyses revealed that a fur mutant had decreased Organization of the fsrA fbpAB, and fbpC units. FB indicates a levels of transcripts for several iron-containing enzymes and Fur box. (B) Northern blot analysis of fsrA expression in different strains. 23S cytochromes and heme biosynthesis pathways (Table S1) (25). rRNA was used as an internal loading control. (C) Binding of purified Fur to the Similarly, microarray analyses of iron-starved B. subtilis have promoter region of fsrA (0.1 nM) as detected by electrophoretic mobility shift shown significantly decreased levels of mRNAs for both gluta- assay. (D) Detection of FLAG-tagged FbpA, FbpB, and FbpC by Western blot mate synthase and amino acid biosynthetic enzymes (29). The with anti-FLAG antibodies. (E) Amino acid sequences and calculated pI of the physiological consequences of these changes were observed FbpA, B, and C proteins. directly in fluxome analyses. Fischer and Sauer (30) compared growth rates and metabolic fluxes in wild-type and 137 null mutants of B. subtilis. Most mutations led to little net change in inverted repeat, but we have noted that this sequence actually metabolic fluxes, indicating that B. subtilis has a robust central comprises two, overlapping operator sites with a 7-1-7 minimal metabolism. Of the strains tested, only one (cysP) had a slower recognition element (26). Although most Fur boxes are associ- growth rate than fur. Under the conditions used (M9 glucose ated with operons encoding siderophore biosynthesis or uptake minimal medium), the fur mutant grew at a rate one-half that of functions, or other proteins implicated in iron homeostasis, some wild-type and there was a notable perturbation in the relative sites are not closely apposed to annotated ORFs. Here, we flux through the TCA cycle (reduced by 23%), and the absolute identify three Fur-regulated transcription units that collectively flux (citrate synthase activity) was reduced nearly 3-fold (30). encode one small, noncoding RNA (sRNA) and three small, This reduced flux is likely due to down-regulation of TCA cycle basic proteins postulated to function as RNA chaperones. enzymes (see below). Motivated by these prior observations, we Anaysis of sequences downstream of one strongly predicted here initiated a detailed analysis of the phenotypes of a fur null Fur box (17/19 match to consensus) identified a Fur-regulated mutant and isogenic derivatives lacking one or more of the sRNA (fsrA) highly conserved between three closely related molecular effectors postulated to mediate this iron-sparing Bacillus strains [supporting information (SI) Fig. S1]. The fsrA response. gene is unable to encode a protein product because it lacks any potential start codons. As expected, the FsrA sRNA is strongly Iron-Sparing Response Reduces Cellular Iron Demand and Is Adaptive up-regulated in a fur mutant strain (Fig. 1B) and in iron-starved Under Severe Iron Limitation. In E. coli, cells that lack an iron- wild-type cells (data not shown). As predicted, Fur binds to the sparing response are unable to prioritize iron utilization and are fsrA regulatory region with high affinity (Fig. 1C). These results at a competitive disadvantage during growth under severe iron suggest that FsrA is a Fur-regulated sRNA. limitation (31). To determine whether the B. subtilis iron-sparing The Fur-regulated ydbN gene encodes a small, 59-aa protein response is also adaptive under iron limitation, we monitored of unknown function (25). The predicted ydbN transcript ini- growth in medium containing a concentration of the iron tiates 161 nucleotides upstream of ydbN and encodes a second chelator EDDHA sufficient to reduce yield of the wild-type small protein (53 aa) also conserved in B. subtilis, Bacillus strain by one-half. Under these conditions, a fur mutant grew amyloquefaciens, and Bacillus licheniformis (Fig. S2). The pattern significantly better than wild-type (Fig. 2A, lane 2 vs. lane 1), of base substitutions argues that these are protein-coding re- presumably because of the constitutive expression of the iron- gions: many substitutions are in the degenerate third codon sparing response in the precultures. Note that, under these position. Both proteins are detected as FLAG-epitope tagged conditions, even the wild-type strain derepresses the Fur regu- products in B. subtilis (Fig. 1D), and after expression in E. coli lon, so both strains should have a similar complement of iron (data not shown). Here, we rename these proteins as Fur- uptake pathways. However, these strains are sfp0 (32) and are regulated basic proteins (FbpA) and FbpB (formerly YdbN) therefore unable to synthesize bacillibactin, which is necessary to (Fig. 1 A and E). access iron chelated by EDDHA (10). The third Fur-regulated basic protein, FbpC, is encoded by the To determine whether the fsrA, fbpAB,orfbpC operons were first identified member of the Fur regulon, metal-regulated gene important for these growth effects, we extended this analysis to C(mrgC) (27). An mrgC-lacZ transcriptional fusion is induced a panel of mutant strains. In contrast with the fur mutant, a by the iron chelator EDDHA or by growth under iron-limiting double fur fsrA mutant strain is severely growth impaired in this conditions (27). With the completion of the B. subtilis genome medium (Fig. 2A, lane 3). We infer that the inability of these cells sequence in 1997 (28), we noted that the mrgC ORF terminates to prevent the synthesis of iron-consuming proteins hinders after only 29 codons (Fig. 1E). The nearest annotated ORF adaptation to these iron-limiting conditions. A severe growth (ypbR) lies 296 base pairs downstream of the mrgC transcript defect was also noted in a fur mutant strain that still contains initiation site and is not regulated by Fur or by iron (25). The FsrA but lacks all three basic proteins (the fur fbpABC quadruple presence of a Rho-independent terminator predicts a Ϸ188-nt mutant; Fig. 2A, lane 6). These results suggest that the iron- RNA product from this region. As noted for fbpA and fbpB, sparing response requires both FsrA and one or more of these comparison of this region between sequenced Bacillus genomes small proteins postulated to function as RNA chaperones. The

11928 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711752105 Gaballa et al. Downloaded by guest on September 26, 2021 0.3 1 2 3 4 5 6 7 8 9 fsrA C CA A A G U U C 0.25 5’ GAG AGAA ACUCUCUGUUCCC ACCCC CUAUCAGAU AAGAU 3’

0.2 3’CUUUU UUGAGAGACAAGGG UGGGGGACAAACUAUUCA 600 A 5’ U U A 0.15 U SD sdhC OD C A 0.1 U A Start G G 0.05 U U AA B 0.35 0.3 200 400 0 10 100 150 0.25 B fsrA 20 40 80 0.2 nM 0.15 0.1 g Fe/mg proteins 0.05 sdhC* CU1065 fur fbpAB fur fbpC fbpABC fur fur fsrA fur fbpC fur fsrA fbpAB fur fsrA fbpABC fur fsrA

1.8 C 1.6 1.4 Fig. 2. Growth effects of mutants altered in the iron-sparing response. (A) 1.2 The iron-sparing response is adaptive under severe iron limitation. Cell density 1 after overnight growth under severe iron limitation (Chelex-treated glucose 0.8 minimal medium with 1 ␮M EDDHA). Under these conditions, the fur mutant 0.6

grows better than wild-type, but a fur mutant additionally lacking either fsrA Ratio (relative to WT) 0.4 or all three protein chaperones grew poorly. (B) Cell iron content (in micro- 0.2 grams of Fe per milligram of soluble protein) as determined by using spec- trophotometric analysis of ferrozine-iron complexes. 1.4 D 1.2 600 1

OD 0.8 fbpAB and fbpC operons, however, appear to be at least partially 0.6 0.4 redundant in function. There appears to be a sufficient degree 0.2 CU1065 fur fbpABC fur fbpAB fbpC fur fsrA fbpAB fur fsrA fur fsrA fur fbpC fbpABC fur fsrA of iron-sparing in strains containing either FbpC alone (Fig. 2A, fur lane 4) or FbpAB (Fig. 2A, lane 5), to allow reasonably good growth (relative to wild-type cells) under these conditions. We hypothesized that these effects of the FsrA sRNA and the FbpABC proteins might be explained by their role in reducing cellular iron demand. To measure the amount of iron needed to Fig. 3. FsrA regulation of succinate dehydrogenase expression. (A) Predicted support cell growth we monitored iron content in wild-type and pairing between FsrA and the 5Ј-leader region of sdhC, the first gene of the mutant cells grown under iron sufficient conditions (Fig. 2B). sdhCAB operon. (B) Binding of the FsrA to the 5Ј leader region of sdhC (200 The amount of cellular iron (after washing to remove surface- nM) as detected by EMSA, using radioactively labeled sdhC (200 nM) and cold fsrA transcripts. (C) Level of SdhA protein as detected by proteomics (see Fig. associated ions) as measured by using ferrozine (33) was reduced Ϸ S4 and S5). (D) Growth (OD600 after overnight growth at 37°C) in minimal 30–40% in the fur mutant (Fig. 2B, lane 2). Significantly, this medium containing succinate as carbon source. effect was largely reversed in the fur fsrA and fur fbpAB strains (Fig. 2B, lanes 3 and 4). Surprisingly, this effect was exacerbated in the fur fbpC double mutant (Fig. 2B, lane 5). These results To identify candidate target operons for FsrA, we focused on suggest that FsrA and the FbpAB proteins may function in the the Ϸ240 genes that were decreased in mRNA level at least same pathway in determining iron content and that FbpC has an 4-fold in the fur mutant (25). Many of these genes (e.g., encoding opposing effect. The imperfect correlation between the effects ribosomal proteins and translation factors) are likely expressed of these mutations on growth and on iron content hints at the at a lower level as a result of the slower growth rate of the complex nature of this stress response: FsrA clearly plays a major mutant. Others encode iron-containing enzymes important for role in helping prioritize iron utilization but the effects of the key metabolic pathways including enzymes of the TCA cycle, FbpABC proteins are complicated and vary among the different respiratory cytochromes, and amino acid biosynthesis (ILV, Cys, protein targets. and Glu) (Table S1). Many of these same mRNAs are also down-regulated in cells starved for iron as measured 15 min after Transcriptome Analyses Identify Genes Positively Regulated by Fur. In treatment with the iron chelator 2,2Ј-dipyridyl (DP) (Table S1). E. coli, RyhB inhibits expression of Ϸ18 operons by RNA-RNA interaction (19). Like other sRNAs, RyhB affects steady state FsrA sRNA Represses Expression of Succinate Dehydrogenase. By mRNA levels because of degradation of the resulting RNA-RNA analogy with RyhB, we hypothesized that FsrA might act as an duplexes, or simply because of the decreased stability associated antisense RNA for some or all of these target genes. Indeed, we with the lack of translation (34, 35). Regardless of mechanism, identified several possible targets for FsrA by searching the B. the effects of transacting sRNAs can be visualized, to a first subtilis genome, using TargetRNA (36) (Table S1). Among the approximation, by monitoring their effects on the transcriptome. strongest matches was the leader region of the operon encoding We hypothesized that the FsrA-mediated iron-sparing re- Sdh (Fig. 3A), a common target of iron-sparing responses in MICROBIOLOGY sponse should lead to specific changes in the transcriptome. In several organisms. In our previous microarray analyses, we noted a previous microarray analysis, we demonstrated that, under that the sdhA, B, and C transcripts were down-regulated Ϸ3-fold iron-replete conditions, Fur negatively regulates the expression in the fur mutant. This decrease is largely reversed in the fur fsrA of Ϸ20 operons (by direct binding to associated operators) and double mutant (data not shown). This suggests that the decrease functions as a positive regulator for numerous operons, including in transcript level is due to increased degradation subsequent to several encoding iron-containing enzymes and cytochromes formation of the FsrA:sdhC duplex. In vitro, FsrA and the sdhC (25). Down-regulation of some of these same genes was noted in leader region form an RNA-RNA complex as judged by RNA a recent transcriptome analysis of B. subtilis under conditions of mobility shift (Fig. 3B). iron deprivation (29). The sequence complementarity between FsrA and sdhC pre-

Gaballa et al. PNAS ͉ August 19, 2008 ͉ vol. 105 ͉ no. 33 ͉ 11929 Downloaded by guest on September 26, 2021 dicts that FsrA will down-regulate translation of Sdh. Consistent A fur fur fur fur fsrA with this expectation, the level of SdhA protein detected by 2D WT fur fsrA fbpAB fbpC fbpABC fbpABC PAGE was significantly decreased in the fur mutant strain, but CitB CitB CitB CitB CitB CitB it was restored to wild-type levels in the fur fsrA mutant (Fig. 3C and Fig. S4). In addition, measurement of Sdh activity indicates Ϸ an 8-fold decrease in the fur mutant strain but normal levels in LeuC LeuC LeuC LeuC LeuC LeuC the fur fsrA double mutant (data not shown). In general, there YvfW YvfW YvfW YvfW YvfW YvfW is a correlation between the level of expression of SdhA and the ability of the cells to grow on succinate as sole carbon source (Fig. 3D). fur B 0.3 fur fsrA Many small regulatory RNAs require a chaperone, such as fur fbpAB 0.2 fur fbpC Hfq, which is important for the iron-sparing response in E. coli fur fbpABC fur fsrA fbpABC (18). Although the B. subtilis ymaH gene encodes a protein 0.1 homologous to Hfq, this protein is not required for FsrA- 0 mediated inhibition of Sdh expression because a fur ymaH -0.1 double mutant does not regain the ability to grow on succinate. -0.2 To date, the function of Hfq remains unclear in B. subtilis and -0.3 log fold repression it is apparently not needed for the function of other known -0.4 CitB LeuC YvfW sRNAs (37, 38). Perhaps because of the extensive sequence fur fur fsrA fur fbpC fur fbpAB fur fbpABC complementarity between FsrA and the sdhC mRNA (Fig. 3A), C 6 this binding interaction also does not require the postulated 3 FbpAB and FpbC protein chaperones either in vitro (Fig. 3B)or 0 in vivo (growth on succinate is not restored in a fur fbpABC mutant; Fig. 3D). -3 -6 FsrA sRNA Represses Expression of Aconitase. To futher explore the -9 effects of FsrA and these Fur-regulated proteins on gene regu- -12 lation, we have used 2D PAGE to monitor both cytosolic cydA -15 cysH proteins and membrane-associated lipoproteins in our collection gltA of mutant strains (Figs. S4-S6 and Table S2). As expected, those FoldWT changerelatedto -18 ilvC proteins directed repressed by Fur are up-regulated in all of the -21 resA yvfW fur mutant strains. Among those proteins that are down- -24 ctaO regulated in the fur mutant, many are increased in level in strains additionally carrying mutations in fsrA and/or the fbpAB or fbpC Fig. 4. Remodeling of the proteome functions to reduce cellular iron operons. Three examples of proteins with unique patterns of utilization. (A) Selected examples of proteins positively regulated by Fur (CitB, regulation are illustrated in Fig. 4A and quantified in Fig. 4B. LeuC, and YvfW) as visualized by using 2D PAGE (see Figs. S4–S6 for complete Aconitase (CitB) is decreased in intensity Ϸ2-fold in the fur datasets). (B) Quantitation of protein expression levels for CitB, LeuC, and YvfW from A.(C) RT-PCR analysis of mRNA levels for representative FsrA target mutant and this effect is reversed only in those strains lacking genes. Fold-change in mRNA expression levels (relative to wild type) was FsrA. Thus, like Sdh, the repression of aconitase requires FsrA measured by using quantitative RT-PCR. Results shown are representative of but is independent of the fbpAB and fbpC operons. Aconitase experiments performed at least three times. contains a 4Fe–4S cluster and is a known target of iron-sparing responses in C. glutamicum (mediated by the RipA protein, ref. 23) and in S. cerevisiae (mediated by Cth2p; 22). It is interesting Table S1). However, in this case repression is alleviated by either to note that B. subtilis aconitase, like its mammalian counter- mutation of fsrA or the fbpAB operon (Fig. 4). Regulation of parts that function as iron-regulatory element binding proteins YvfW by both FsrA and the fbpAB operon is confirmed by (IRE-BP), has an iron-regulated RNA-binding activity (39). RT-PCR (Fig. 4C) and has also been seen in transcriptome Thus, under conditions of severe iron deprivation FsrA mediates comparisons of these same strains (G.T.S. and J.D.H., unpub- repression of continued synthesis of aconitase and loss of iron lished results). from existing aconitase molecules may further reduce enzyme activity and activate its RNA-binding function. Although the Repression of Isopropyl Malate Dehydratase (LeuCD) Requires Prod- roles of aconitase as an RNA-binding protein are not well ucts of the fsrA and fbpC Operons. The expression of enzymes understood in B. subtilis, is has been reported to bind internally involved in branched chain amino acid biosynthesis was also to the feuABC mRNA (39), which encodes the major uptake strongly regulated by iron. This likely reflects the fact that system for the endogenous siderophore bacillibactin (10). isopropyl malate dehydratase, the product of the leuC and leuD In addition to its role in mediating repression of Sdh and genes, is an iron-sulfur containing enzyme. In B. subtilis, the aconitase, FsrA appears to be the major factor determining ilv-leu operon encodes seven proteins that participate in several phenotypes characteristic of the fur mutant. In addition to an inability to grow on succinate, a fur mutant is unable to branched chain amino acid biosynthesis and expression of this grow on fumarate as sole carbon source or on ammonium as sole operon is subject to complex control (40). At least three different nitrogen source (possibly reflecting regulation of the gltAB transcription factors regulate transcription of this operon, and operon). In both cases, growth is restored in the fur fsrA double the primary transcript is processed to yield several discrete mutant, but not in the fur fbpAB fbpC mutant. Therefore, these mRNA species with differing stabilities (41). LeuC protein levels growth phenotypes depend on FsrA, but not on the three were reduced in a fur mutant, but, in this case, repression was protein-coding genes. largely alleviated by mutation of either fsrA alone or fbpC alone (Fig. 4) and completely relieved in multiply mutant strains. The Repression of YvfW Requires Products of Both the fsrA and fbpAB leuCD genes were shown to be derepressed in a ripA mutant in Operons. Expression of YvfW, an unknown function ferredoxin- C. glutamicum, indicating that this enzyme is also subject to the like protein, is also repressed in a fur mutant strain (Fig. 4A and iron-sparing response in this organism.

11930 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711752105 Gaballa et al. Downloaded by guest on September 26, 2021 fsrA, fbpAB, and fbpC Operons Function in Combination to Regulate operon (Fig. 3A). In some, but not all, cases, fbpC is also Protein Synthesis. To extend this analysis and determine whether involved. As monitored by proteomics, the effects are also these changes in protein levels were correlated with changes in complex and FsrA appears to be the most important effector for mRNA levels, we used quantitative RT-PCR to monitor the many of the protein targets. transcript levels for several presumed target operons of the The fbpAB and fbpC genes encode small, basic proteins that iron-sparing response (Fig. 4C). The fur mutant had a decreased we propose function as dedicated Fur-regulated RNA chaper- level of mRNA corresponding to biosynthetic enzymes for ones (either alone or in heterooligomeric complexes). We do not branched chain amino acids (ilvC), cytochromes (cydA, resA, and exclude the possibility that the fbpAB and fbpC transcripts may ctaO), cysteine (cysH), glutamate (gltA), and the ferredoxin-like also function as sRNAs to affect . Indeed, E. coli protein (yvfW). The levels of many of these RNAs were restored sgrS has recently been shown to regulate sugar transport by to near wild-type levels in the fur fsrA, fur fbpAB,orfur fbpAB acting both as a sRNA and by encoding a protein product (43). fbpC mutant strains. As noted in the examples above, FsrA Ongoing studies are directed at defining the complete suite of appears to have the strongest effect on expression and RNA targets for FsrA-mediated regulation and the precise roles of the levels were restored to near wild-type levels for each of these fbpAB and fbpC genes in what appears to be a complex combi- targets in the fur fsrA double mutant strain. Strong effects were natorial mechanism for regulation of gene expression. also noted for the fur fbpAB mutant strain, whereas the fur fbpC Materials and Methods strain displayed a full derepression for only one tested target gene, gltA (Fig. 4C). These results highlight the complexity of this Strain Construction and Growth Conditions. B. subtilis strains were constructed by using long-flanking homology PCR as described in ref. 44. For selection, regulatory system and suggest a model in which the FbpAB and antibiotics were added as follows: 1 ␮g/ml erythromycin and 25 ␮g/ml linco- FpbC proteins may function to modulate the activity or the mycin (for selecting for macrolide-lincosamide-streptogramin B resistance), targeting of the FsrA sRNA. 100 ␮g/ml spectinomycin, 10 ␮g/ml chloramphenicol, 15 ␮g/ml kanamycin, and 10 ␮g/ml neomycin. B. subtilis was grown in LB or in a Mops-based glucose Constitutive Expression of the Iron-Sparing Response Is Maladaptive minimal medium suitable for iron starvation studies (27). Unless otherwise Under a Variety of Growth Conditions. To explore the physiological indicated, liquid media were inoculated from an overnight preculture and role of this iron-sparing response, we have used an automated incubated at 37°C with shaking at 200 rpm. E. coli DH5␣ was used for routine growth (optical density) monitoring system (BioScreen) to mon- DNA cloning as described in ref. 45. Restriction enzymes, DNA ligase and DNA polymerases were used according to the manufacturer’s instructions (New itor our panel of mutant strains in LB and glucose minimal England Biolabs). Northern blot analysis and EMSA experiments were done by medium with and without supplementation with various amino using standard techniques as described in SI Methods. acids (Table S3). Consistent with previous results, the fur mutant displayed both a reduction in the maximal rate of growth and a Overexpression and Detection of FbpA, B, and C Proteins. Expression constructs prolonged lag phase in a variety of conditions and was unable to for C-terminal FLAG-tagged fusions were constructed by PCR (46). The result- grow with succinate or fumarate as sole carbon source. These ing products were cloned in pET16B and pDGG1664 (47) and expressed in E. effects were partially suppressed either by mutation of fsrA or, coli and B. subtilis respectively. The fusions were detected by Western blot in some cases, by mutation of one or both of the fbpAB and fbpC analysis, using anti-FLAG antibodies (Sigma). operons. When the tabulated growth data were analyzed by using a numerical taxonomy approach (42), it became clear that the fur, Quantification of Target Genes Expression by Real-Time PCR. RNA was extracted from strains grown in LB to mid-log phase, using an RNA extraction kit fur fbpAB, and fur fbpC mutants all cluster together, indicating (Qiagen). cDNA was synthesized by using random hexamer primers and that the individual mutation of either the fbpAB or the fbpC Taqman reverse transcription kit (Roche), and real-time PCR was carried out by operons did little to suppress the poor growth phenotypes under using SYBR Green (Bio-Rad) and gene-specific primers according to the man- most conditions (Table S3). Conversely, the fur fsrA and the fur ufacturers’ instructions. fbpAB fbpC mutants clustered with the wild-type strain indicat- ing that mutation of either the sRNA or all three peptide- Analysis of RNA–RNA Complex Formation. Both target RNA (5Ј 166 sdhC mRNA) encoding genes suppressed many of the growth phenotypes. and fsrA were synthesized in vitro from PCR-generated template fragments, These results are consistent with the general notion that the using a T7 RNA polymerase kit (Ambion) and purified from 6% denaturing fbpAB and fbpC loci are at least partially redundant in function, polyacrylamide gels according to the manufacturer’s instructions. RNA-RNA com- plex formation was measured according to (38), using 6% native polyacrylamide as noted for growth in medium supplemented with EDDHA gels containing 1ϫ TBE. Dried gels were analyzed by PhosphoImaging. (Fig. 2A). Proteome Analysis. B. subtilis strains were grown in Belitsky minimal medium Conclusion (48) to an OD500 of 0.4 and harvested for the proteome analysis, using dual The results presented here establish the broad outlines of the channel imaging of 2D PAGE gels as described in ref. 49 (Figs. S4 and S5 and iron-sparing response in B. subtilis. Like the enterobacteria, this SI Methods). To define the membrane-attached lipoproteome (Fig. S6), strains response involves at least one sRNA (FsrA) that acts to down- were grown in LB and harvested 1 h after entry into the stationary phase at ϭ regulate the synthesis of numerous iron-containing enzymes and OD540 3.0. Sdh activity was measured in cells grown in minimal succinate iron-consuming biosynthetic pathways. However, FsrA- medium according to ref. 50. mediated regulation also involves the fbpAB and fbpC genes. As ACKNOWLEDGMENTS. We thank E. Masse´ for helpful comments on this monitored by RT-PCR, down-regulation many RNA targets in manuscript. This work was supported by National Institutes of Health Grant the fur mutant requires both the FsrA sRNA and the fbpAB GM059323. MICROBIOLOGY 1. Andrews SC, Robinson AK, Rodriguez-Quinones F (2003) Bacterial iron homeostasis. 6. Wennerhold J, Bott M (2006) The DtxR regulon of Corynebacterium glutamicum. 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