Disruption of the OLE ribonucleoprotein complex causes magnesium toxicity in Bacillus halodurans

OLE RNAs represent an unusual class of bacterial noncoding RNAs common in Gram-positive anaerobes. The OLE RNA of the alkaliphile Bacillus halodurans is highly expressed and naturally interacts with at least two RNA-binding proteins called OapA and OapB. The phenotypes of the corresponding knockouts include growth inhibition when exposed to ethanol or other short-chain alcohols or when incubated at modestly reduced temperatures (e.g. 20°C). Intriguingly, the OapA 'PM1' mutant, which carries two amino acid changes to a highly conserved region, yields a dominant-negative phenotype that causes more severe growth defects under these same stress conditions. Herein, we report that the PM1 strain also exhibits extreme sensitivity to elevated Mg²⁺ concentrations, beginning as low as 2 mM. Suppressor mutants predominantly map to genes for aconitate hydratase and isocitrate dehydrogenase, which are expected to alter cellular citrate concentrations. Citrate reduces the severity of the Mg²⁺ toxicity phenotype, but neither the genomic mutations nor the addition of citrate to the medium overcomes ethanol toxicity or temperature sensitivity. These findings reveal that OLE RNA and its protein partners are involved in biochemical responses under several stress conditions, wherein the unusual sensitivity to Mg²⁺ can be independently suppressed by specific genomic mutations.