Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

Abstract

Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of K -dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3 untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.Aconitase catalyzes the reversible isomerization of citrate and isocitrate in the second step of the tricarboxylic acid branch of the Krebs citric acid cycle. In Bacillus subtilis, the Krebs cycle is induced during late exponential phase in nutrient-exhausted medium and is responsible for the production of biosynthetic intermediates and ATP and for reducing power during stationary phase and sporulation. When induced, aconitase becomes one of the most abundant proteins in the cell (8). Induction of the aconitase gene, citB, is dependent on inactivation of two DNA binding proteins, CcpC (22) and CodY (25). Repression by CcpC is antagonized by citrate (22), whereas CodY loses repressing activity when the intracellular pools of GTP (32) and branched-chain amino acids (38) decrease. A citB null mutant is severely defective in spore formation and has a relative sporulation efficiency of 1.4 ϫ 10 Ϫ4 % compared to its wild-type parent (6). Previous work by Craig et al. (6) showed that the defect in sporulation of a citB null mutant is caused, in part, by the accumulation of citrate, a chelator of Mn 2ϩ and Fe 2ϩ . Chelation of divalent cations disrupts phosphorylation (activation) of Spo0A, the major transcription factor for early sporulation-specific genes (19). However, addition of excess Mn 2ϩ and Fe 2ϩ only partially overcomes the sporulation defect of an aconitase null mutant (6).When a constitutively active allele of spo0A is introduced into the citB null mutant, cells proceed past the stage 0 block in sporulation but are still blocked at a later stage (6). A similar result is seen when an aconitase null mutant is genetically modified to prevent synthesis of citrate (6); such cells proceed past stage 0 but do not complete sporulation efficiently. Taken together, these results suggest that citrate accumulation may be responsible for the stage 0 block, but aconitase may have an additional function unrelated to citrate metabolism that is important during the later stages of sporulation. The only other known activity of B. su...

Phosphinothricin-tripeptide biosynthesis: An original version of bacterial secondary metabolism?

“…The apo-protein is able to bind human IRE as well as IRE-similar sequences within its own genome. The regulatory role of aconitase on the sporulation of B. subtilis was shown by Serio et al (2006). Here, the authors could demonstrate the binding of CitB to the 3' UTR of the gerE transcript.…”

The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen

“…Bacillus subtilis was shown to contain aconitase that could bind to rabbit ferritin IRE and to IRE-like sequences that were located in B. subtilis operons encoding the major cytochrome oxidase and an iron uptake system [65]. Moreover, an analogous mRNA/aconitase interaction was linked to efficient sporulation of B. subtilis [66]. Finally, Escherichia coli aconitases A and B bind to stem-loop structures in the cognate mRNAs, stabilizing them during oxidative stress [67,68] .…”

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR–DNA complexes

“…This regulation can occur by a variety of different mechanisms. For instance, aconitase and uracil phosphoribosyltransferase are regulatory enzymes that bind RNA (29,30). Proline oxidase and biotin synthetase contain separate DNAbinding domains that allow these proteins to function as transcriptional repressors (31,32).…”