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The Bacillus Subtilis Iron-Sparing Response Is Mediated by a Fur-Regulated Small RNA and Three Small, Basic Proteins

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The Bacillus Subtilis Iron-Sparing Response Is Mediated by a Fur-Regulated Small RNA and Three Small, Basic Proteins The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins 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 homeostasis is often controlled by the (15) and are hypersensitive to H2O2, which reacts with free iron to iron-sensing ferric uptake repressor (Fur). The Bacillus subtilis Fur generate hydroxyl radicals (16). protein 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 repressors (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 transcription 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).
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