Functional Genomics and Biochemical Characterization of Uranium Biooxidation and Stress response in Extremely Thermoacidophilic Metallosphaera species

In this study, Metallosphaera sedula and a related archaeon isolated from a uranium mine, M. prunae, were evaluated for their ability to oxidize solid uranium (U3O8) and to resist soluble uranyl acetate. Genome sequencing results indicated that M. prunae is a spontaneous mutant of M. sedula (99.9% similarity in the genomes). Microbial physiology experiments suggested significant differences in uranium mobilization rates of the two species. M. sedula was found to oxidize solid U3O8 to soluble U(VI) while M. prunae showed passive response to U3 O8. Whole genome microarray analysis revealed that foxA', a gene encoding cytochrome c-oxidase subunit I (Cox I) and found to be mutated in M. prunae, had 30-fold lower transcript abundance in M. prunae compared to M. sedula. The CoxI enzyme has been identified as the final electron donor to oxygen in the iron oxidation pathway and its lower transcription in M. prunae could explain its inefficiency to perform uranium solubulization from solid U3O8. It was also observed that M. prunae was more tolerant to uranyl acetate (U(VI)) compared to M. sedula. In order to identify the difference in U(VI) resistance mechanisms, a U(VI) shock experiment was designed wherein the two species were challenged with 1 mM uranyl acetate at mid-exponential phase. M. sedula U (VI) trasnscriptome indicated activation of the general stress response elements (DNA repair and recombination, molecular chaperones and transcriptional regulators) while M. prunae showed a more sedate response. Interestingly, genes encoding CRISPR-cas associated proteins and VapBC loci were up-regulated for both species. In order to explore the resistance mechanisms, an early stage U(VI) culture (collected 15 min post shock) was collected and it was observed that there was extensive RNA degradation for M. prunae sample but not for M. sedula . RNA quality improved 60 min post U(VI) shock, this phenomenon was consistent with drop in U(VI) concentration in media due to complexation with inorganic sulfate and phosphate. It was proposed that M. prunae resists uranium by stalling its cellular processes and normal growth is regained when stress is alleviated. Transcriptional response analysis indicated that ribonucleases, known as VapC Toxins, could be responsible for metabolic shutdown in the cell. In order to test the hypothesis that VapC toxins could degrade cellular RNA, these proteins were recombinantly expressed in Escherichia coli and purified by affinity chromatography. VapC toxin activity was evaluated on a fluorescent tagged synthetic RNA substrate and total RNA from M. sedula and M. prunae. It was found that 4 VapC toxins (VapC4, VapC7, VapC8 and VapC9) were able to degrade ribosomal RNA in M. sedula and M. prunae at physiologically relevant toxin:RNA ratios. In order to determine the cellular targets of these rRNA degrading VapC toxins, primer extension analysis was performed with MS2 bacteriophage RNA. Several cleavage sites were determined and it was observed that VapC toxins have degenerate consensus motifs with an affinity to bind to 4 or 5 bp G-rich sequences. Bioinformatic analysis was performed with the consensus motifs to identify rRNA and messenger RNA (mRNA) targets in the cell. Heat shock response of M. prunae was performed (T=75°C to T= 85° C) for comparison with U(VI) shock. Unlike U(VI), no RNA degradation was observed for both the early and late phase heat shock samples. At late stage heat shock, several vapBC loci were up-regulated along with general heat stress responsive elements. The study points to two important conclusions - firstly, natural growth environments play a major role in dictating metal-microbe interactions for closely related Metallosphaera species, and secondly, stress-activated ribonucleases mediate the survival of these organisms under toxic metal stress.