A Global Investigation of the Bacillus subtilis Iron-Sparing Response Identifies Major Changes in Metabolism

Abstract

The Bacillus subtilis ferric uptake regulator (Fur) protein is the major sensor of cellular iron status. When iron is limiting for growth, derepression of the Fur regulon increases the cellular capacity for iron uptake and mobilizes an iron-sparing response mediated in large part by a small noncoding RNA named FsrA. FsrA functions, in collaboration with three small basic proteins (FbpABC), to repress many "low-priority" iron-containing enzymes. We have used transcriptome analyses to gain insights into the scope of the iron-sparing response and to define subsets of genes dependent for their repression on FsrA, FbpAB, and/or FbpC. Enzymes of the tricarboxylic acid (TCA) cycle, including aconitase and succinate dehydrogenase (SDH), are major targets of FsrA-mediated repression, and as a consequence, flux through this pathway is significantly decreased in a fur mutant. FsrA also represses the DctP dicarboxylate permease and the iron-sulfur-containing enzyme glutamate synthase (GltAB), which serves as a central link between carbon and nitrogen metabolism. Allele-specific suppression analysis was used to document a direct RNA-RNA interaction between the FsrA small RNA (sRNA) and the gltAB leader region. We further demonstrated that distinct regions of FsrA are required for the translational repression of the GltAB and SDH enzyme complexes.T he tricarboxylic acid (TCA) cycle is a central pathway of Bacillus subtilis metabolism. The flux of carbon through the TCA cycle generates ATP through substrate-level phosphorylation and by the generation of reducing equivalents that feed the electron transport chain. TCA cycle intermediates also serve as biosynthetic precursors for numerous amino acids, heme, and other key metabolites (4,9,18,21,43). Of particular note, ␣-ketoglutarate is transaminated by glutamate synthase using glutamine as a donor to generate two glutamate molecules. Thus, glutamate synthase serves as a direct link between central carbon metabolism and nitrogen metabolism. Citric acid, another key intermediate in the TCA cycle, can also play a role in metal ion homeostasis by facilitating the uptake of cations, including Fe(III), Mg(II), and Mn(II) (25,30).Since the TCA cycle is central to many biosynthetic and metabolic processes, several regulators exact tight control over the expression of TCA cycle enzymes, including both global (CcpA, CodY, and TnrA) and pathway-specific (CcpC, RocR, and GltC) regulators (39). To this list, we can now add the ferric uptake regulator (Fur). Fur has dual roles in iron homeostasis. Under iron-limiting growth conditions, derepression of numerous Furregulated operons (2) allows expression of siderophore biosynthesis and uptake pathways (30). Many other genes are downregulated in the fur mutant (2), including several with roles in central metabolism. Many of the genes downregulated in the fur mutant may be the targets of a Fur-regulated sRNA A (FsrA) and three small, Fur-regulated basic proteins (FbpABC) that can act as coregulators with FsrA. These effectors repress the transla...

Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

“…Three previously reported sRNAs are also part of the top-10 list, namely, FsrA/S512 (22/62 genomes, MFE Z-score Ϫ4.8), RnaC/ S1022 (19/62 genomes, MFE Z-score Ϫ5.2), and CsfG/S547 (61/62 genomes, MFE Z-score Ϫ4.7). As discussed above, FsrA/ S512 is an important regulator of the iron-sparing response of B. subtilis (43,102). Interestingly, in the tiling array study by Nicolas et al (15), FsrA/S512 was found to be expressed also under conditions with plentiful iron.…”

“…The individual mutations in either gltA or FsrA/S512 abolished the interaction while the presence of the respective complementary mutations restored the interaction (102). It is likely that the FsrA/S512 regulon extends to the expression of aconitase (encoded by citB), the lactate oxidases (encoded by lutABC), the C 4 -dicarboxylate permease (encoded by dctP), the exocytoplasmic thioredoxin ResA, and the cytochrome bc 1 component QcrA (102). These targets can all be rationally linked to the iron-sparing response (102), making this the only documented sRNA regulon in B. subtilis.…”

“…Second, compensatory mutations were made in both gltA, encoding glutamate synthetase, and FsrA/S512. The individual mutations in either gltA or FsrA/S512 abolished the interaction while the presence of the respective complementary mutations restored the interaction (102). It is likely that the FsrA/S512 regulon extends to the expression of aconitase (encoded by citB), the lactate oxidases (encoded by lutABC), the C 4 -dicarboxylate permease (encoded by dctP), the exocytoplasmic thioredoxin ResA, and the cytochrome bc 1 component QcrA (102).…”

“…It is likely that the FsrA/S512 regulon extends to the expression of aconitase (encoded by citB), the lactate oxidases (encoded by lutABC), the C 4 -dicarboxylate permease (encoded by dctP), the exocytoplasmic thioredoxin ResA, and the cytochrome bc 1 component QcrA (102). These targets can all be rationally linked to the iron-sparing response (102), making this the only documented sRNA regulon in B. subtilis. This implies that, as in Gram-negative bacteria (103), RNA regulons with multiple targets from the same or functionally related processes are also present in B. subtilis and thus in Gram-positive bacteria.…”

α-fur, an antisense RNA gene to fur in the extreme acidophile Acidithiobacillus ferrooxidans

“…Genes regulated in this manner include those involved in the biosynthesis of siderophores and iron uptake (Friedman & O'Brian, 2003; Quatrini et al, 2007;Gao et al, 2008;Butcher et al, 2012), genes encoding small RNAs such as fsrA and ryhB (Smaldone et al, 2012;Massé & Gottesman, 2002;Salvail & Massé, 2012), and many other genes involved in iron homeostasis (reviewed by Salvail & Massé, 2012). Under iron-limiting conditions, Fur no longer binds Fe 2+ , reducing its affinity for Fur boxes and thus allowing transcription to proceed (Baichoo et al, 2002;Ochsner et al, 2002;Butcher et al, 2012).…”