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Abstract Staphylococcus aureus has evolved mechanisms to cope with low iron (Fe) availability in host tissues. Staphylococcus aureus uses the ferric uptake transcriptional regulator (Fur) to sense titers of cytosolic Fe. Upon Fe depletion, apo-Fur relieves transcriptional repression of genes utilized for Fe uptake. We demonstrate that an S. aureus Δfur mutant has decreased expression of acnA, which codes for the Fe-dependent enzyme aconitase. This prevents the Δfur mutant from growing with amino acids as sole carbon and energy sources. We used a suppressor screen to exploit this phenotype and determined that a mutation that decreases the transcription of isrR, which produces a regulatory RNA, increased acnA expression, thereby enabling growth. Directed mutation of bases predicted to facilitate the interaction between the acnA transcript and IsrR, decreased the ability of IsrR to control acnA expression in vivo and IsrR bound to the acnA transcript in vitro. IsrR also bound transcripts coding the alternate tricarboxylic acid cycle proteins sdhC, mqo, citZ and citM. Whole-cell metal analyses suggest that IsrR promotes Fe uptake and increases intracellular Fe not ligated by macromolecules. Lastly, we determined that Fur and IsrR promote infection using murine skin and acute pneumonia models. 

To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed inS.aureusstrains that lacks the described Cu ion detoxification systems (ΔcopBLΔcopAZ;cop^-). Exposure of thecop^-strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. Theaptmutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production ofapt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression ofprsresulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ionsin vivoandin vitroand that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish thatS.aureusthat lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to preventS.aureusinfections. 

ABSTRACT Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The Δ bshA Δ sod and Δ bshA Δ pcat double mutant strains were as sensitive to Hg(II) as the Δ bshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δ sod and Δ pcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δ sod and Δ pcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments. IMPORTANCE Thermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus - Thermus phylum that, together with the Aquificae , constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243–249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7 ) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321–331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080 ). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes.