VapC toxins drive cellular dormancy under uranium stress for the extreme thermoacidophile Metallosphaera prunae

Abstract

Summary When abruptly exposed to toxic levels of hexavalent uranium, the extremely thermoacidophilic archaeon Metallosphaera prunae, originally isolated from an abandoned uranium mine, ceased to grow, and concomitantly exhibited heightened levels of cytosolic ribonuclease activity that corresponded to substantial degradation of cellular RNA. The M. prunae transcriptome during ‘uranium-shock’ implicated VapC toxins as possible causative agents of the observed RNA degradation. Identifiable VapC toxins and PIN-domain proteins encoded in the M. prunae genome were produced and characterized, three of which (VapC4, VapC7, VapC8) substantially degraded M. prunae rRNA in vitro. RNA cleavage specificity for these VapCs mapped to motifs within M. prunae rRNA. Furthermore, based on frequency of cleavage sequences, putative target mRNAs for these VapCs were identified; these were closely associated with translation, transcription, and replication. It is interesting to note that Metallosphaera sedula, a member of the same genus and which has a nearly identical genome sequence but not isolated from a uranium-rich biotope, showed no evidence of dormancy when exposed to this metal. M. prunae utilizes VapC toxins for post-transcriptional regulation under uranium stress to enter a cellular dormant state, thereby providing an adaptive response to what would otherwise be a deleterious environmental perturbation.

Extremely Thermoacidophilic Metallosphaera Species Mediate Mobilization and Oxidation of Vanadium and Molybdenum Oxides

“…3). Previous work showed that sudden additions of uranium to cultures triggered a dormant phase in M. prunae (but not in M. sedula) (20) involving VapC toxin-antitoxin loci (17,20); a similar phenomenon could be a factor here. These results indicate that chemolithoautotrophic metabolism could be supported by Fe(II) oxidation in these Metallosphaera species and that the use of V and Mo as electron mediators to recycle Fe(III) to Fe(II) through their oxidation bolstered respiratory processes.…”

“…Oxygen consumption assays showed a direct reduction in the rate of oxygen consumption for iron-limited conditions, which was recovered by the addition of ferric chloride. However, the recovery was not complete for M. prunae and could indicate a VapC toxin-mediated dormant phase, previously observed during uranium "shock" for M. prunae (17,20). Solubilized molybdenum and vanadium anions could exist in more than one oxidation state, with a higher oxidation state possible, and thus serve as energetic substrates capable of being biooxidized.…”

“…The capacity for M. sedula to solubilize various metal oxides was assessed by exposing M. sedula at ϳ2.0 ϫ 10 8 cells/ml to 0.1% (wt/vol) of the oxide under chemolithoautotrophic growth conditions. M. sedula was chosen since it was previously found to be a more effective metal mobilizer than another Metallosphaera species also examined here, M. prunae (17,20). The metal oxides screened included V 2 O 3 , Cu 2 O, FeO, MnO, CoO, SnO, MoO 2 , Cr 2 O 3 , Ti 2 O 3 , and Rh 2 O 3 .…”

“…The cDNA was labeled with either Cy3 or Cy5 dye (GE Healthcare) and hybridized onto the M. sedula array, constructed as previously described (26). Slides were scanned using a Genetix 4000B scanner, and the intensity data for all of the spots on the array were normalized using an analysis of variance mixed-effects model, as used for previous transcriptomic analysis of Metallosphaera cultures (17,20). The normalized data were analyzed using JMP Genomics (SAS Institute, Cary, NC) with the criterion for significant differential response defined as a fold change of Ն2.0 and a P value of Յ1.0E-4.…”

Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis

“…S5). The VapC4 toxin is a well-known ribonuclease belonging to the VapC group of the PIN superfamily (22), indicating that the nuclease domain of Aq880 is responsible for cleavage catalysis.…”