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

ABSTRACT: 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.

SUBMITTER: Harris KA

PROVIDER: S-EPMC6842403 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Harris Kimberly A KA Odzer Nicole B NB Breaker Ronald R RR

Molecular microbiology 20190922 5

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 ...[more]

PMID: 31461569

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Project description:OLE (ornate, large, extremophilic) RNAs are members of a noncoding RNA class present in many Gram-positive, extremophilic bacteria. The large size, complex structure, and extensive sequence conservation of OLE RNAs are characteristics consistent with the hypothesis that they likely function as ribozymes. The OLE RNA representative from Halalkalibacterium halodurans is known to localize to the phospholipid membrane and requires at least three essential protein partners: OapA, OapB, and OapC. However, the precise biochemical functions of this unusual ribonucleoprotein (RNP) complex remain unknown. Genetic disruption of OLE RNA or its partners revealed that the complex is beneficial under diverse stress conditions. To search for additional links between OLE RNA and other cellular components, we used phylogenetic profiling to identify proteins that are either correlated or anticorrelated with the presence of OLE RNA in various bacterial species. This analysis revealed strong correlations between the essential protein-binding partners of OLE RNA and organisms that carry the ole gene. Similarly, proteins involved in sporulation are correlated, suggesting a potential role for the OLE RNP complex in spore formation. Intriguingly, the Mg2+ transporter MpfA is strongly anticorrelated with OLE RNA. Evidence indicates that MpfA is structurally related to OapA and therefore MpfA may serve as a functional replacement for some contributions otherwise performed by the OLE RNP complex in species that lack this device. Indeed, OLE RNAs might represent an ancient RNA class that enabled primitive organisms to sense and respond to major cellular stresses.IMPORTANCEOLE (ornate, large, extremophilic) RNAs were first reported nearly 20 years ago, and they represent one of the largest and most intricately folded noncoding RNA classes whose biochemical function remains to be established. Other RNAs with similar size, structural complexity, and extent of sequence conservation have proven to catalyze chemical transformations. Therefore, we speculate that OLE RNAs likewise operate as ribozymes and that they might catalyze a fundamental reaction that has persisted since the RNA World era-a time before the emergence of proteins in evolution. To seek additional clues regarding the function of OLE RNA, we undertook a computational effort to identify potential protein components of the OLE ribonucleoprotein (RNP) complex or other proteins that have functional links to this device. This analysis revealed known protein partners and several additional proteins that might be physically or functionally linked to the OLE RNP complex. Finally, we identified a Mg2+ transporter protein, MpfA, that strongly anticorrelates with the OLE RNP complex. This latter result suggests that MpfA might perform at least some functions that are like those carried out by the OLE RNP complex.

Project description:Bacillus halodurans C-125 is an alkaliphilic microorganism that grows best at pH 10 to 10.5. B. halodurans C-125 harbors the erm (erythromycin resistance methylase) gene as well as the mphB (macrolide phosphotransferase) and putative mef (macrolide efflux) genes, which confer resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. The Erm protein expressed in B. halodurans C-125 could be classified as ErmK because it shares 66.2% and 61.2% amino acid sequence identity with the closest ErmD and Erm(34), respectively. ErmK can be regarded as a dimethylase, as evidenced by reverse transcriptase analysis and the antibiotic resistance profile exhibited by E. coli expressing ermK. Although ErmK showed one-third or less in vitro methylating activity compared to ErmC', E. coli cells expressing ErmK exhibited comparable resistance to erythromycin and tylosin, and a similar dimethylation proportion of 23S rRNA due to the higher expression rate in a T7 promoter-mediated expression system. The less efficient methylation activity of ErmK might reflect an adaption to mitigate the fitness cost caused by dimethylation through the Erm protein presumably because B. halodurans C-125 has less probability to encounter the antibiotics in its favorable growth conditions and grows retardedly in neutral environments. IMPORTANCE Erm proteins confer MLSB antibiotic resistance (minimal inhibitory concentration [MIC] value up to 4,096 μg/mL) on microorganisms ranging from antibiotic producers to pathogens, imposing one of the most pressing threats to clinics. Therefore, Erm proteins have long been speculated to be plausible targets for developing inhibitor(s). In our laboratory, it has been noticed that there are variations in enzymatic activity among the Erm proteins, Erm in antibiotic producers being better than that in pathogens. In this study, it has been observed that Erm protein in B. halodurans C-125 extremophile is a novel member of Erm protein and acts more laggardly, compared to that in pathogen. While this sluggishness of Erm protein in extremophile might be evolved to reduce the fitness cost incurred by Erm activity adapting to its environments, this feature could be exploited to develop the more potent and/or efficacious drug to combat formidably problematic antibiotic-resistant pathogens.

Dataset Information

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

Publications

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

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