Deenah Osman

Metal Preferences and Metallation

The metal binding preferences of most metalloproteins do not match their metal requirements. Thus, metallation of an estimated 30% of metalloenzymes is aided by metal delivery systems, with ∼25% acquiring preassembled metal cofactors. The remaining ∼70% are presumed to compete for metals from buffered metal pools. Metallation is further aided by maintaining the relative concentrations of these pools as an inverse function of the stabilities of the respective metal complexes. For example, magnesium enzymes always prefer to bind zinc, and these metals dominate the metalloenzymes without metal delivery systems. Therefore, the buffered concentration of zinc is held at least a million-fold below magnesium inside most cells.

Copper Homeostasis in Salmonella Is Atypical and Copper-CueP Is a Major Periplasmic Metal Complex

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P1-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P1-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.

Copper homeostasis in bacteria.

Els ts Kin A . T he properties of copper 217 B . C opper requiring proteins 218 C . P rinciples of copper homeostasis 219 II. M echanisms of Copper Trafficking and Resistance 220 A . P 1B-type ATPases 222 B . C opper acquisition 223 C . C opper detoxification 225 D . S ensors of elevated copper levels 231 E . C opper-chaperones 233 III. C opper and Bacterial Pathogenicity 234 IV. C opper as a Biocide 237 V. C oncluding Remarks 238 Refere nces 239

Bacterial metal-sensing proteins exemplified by ArsR–SmtB family repressors

Detecting deficiency and excess of different metal ions is fundamental for every organism. Our understanding of how metals are detected by bacteria is exceptionally well advanced, and multiple families of cytoplasmic DNA-binding, metal-sensing transcriptional regulators have been characterised(ArsR-SmtB, MerR, CsoR-RcnR, CopY, DtxR, Fur, NikR). Some of the sensors regulate a single gene while others act globally controlling transcription of regulons. They not only modulate the expression of genes directly associated with metal homeostasis, but can also alter metabolism to reduce the cellular demand for metals in short supply. Different representatives of each of the sensor families can regulate gene expression in response to different metals, and the residues that form the sensory metal-binding sites have been defined in a number of these proteins. Indeed, in the case of theArsR-SmtB family, multiple distinct metal-sensing motifs (and one non-metal-sensing motif) have been identified which correlate with the detection of different metals. This review summarises the different families of bacterial metal-sensing transcriptional regulators and discusses current knowledge regarding the mechanisms of metal-regulated gene expression and the structural features of sensory metal-binding sites focusing on the ArsR-SmtB family. In addition, recent progress in understanding the principles governing the ability of the sensors to detect specific metals within a cell and the coordination of the different sensors to control cellular metal levels is discussed.

The copper supply pathway to a Salmonella Cu,Zn‐superoxide dismutase (SodCII) involves P1B‐type ATPase copper efflux and periplasmic CueP

Periplasmic Cu,Zn‐superoxide dismutases (Cu,Zn‐SODs) are implicated in bacterial virulence. It has been proposed that some bacterial P1B‐type ATPases supply copper to periplasmic cupro‐proteins and such transporters have also been implicated in virulence. Here we show that either of two P1B‐type ATPases, CopA or GolT, is needed to activate a periplasmic Cu,Zn‐SOD (SodCII) in Salmonella enterica serovar Typhimurium. A ΔcopA/ΔgolT mutant accumulates inactive Zn‐SodCII which can be activated by copper‐supplementation in vitro. In contrast, either single ATPase mutant accumulates fully active Cu,Zn‐SodCII. A contribution of GolT to copper handling is consistent with its copper‐responsive transcription mediated by DNA‐binding metal‐responsive activator GolS. The requirement for duplicate transcriptional activators CueR and GolS remains unclear since both have similar tight KCu. Mutants lacking periplasmic cupro‐protein CueP also accumulate inactive Zn‐SodCII and while CopA and GolT show functional redundancy, both require CueP to activate SodCII in vivo. Zn‐SodCII is also activated in vitro by incubation with Cu‐CueP and this coincides with copper transfer as monitored by electron paramagnetic resonance spectroscopy. These experiments establish a role for CueP within the copper supply pathway for Salmonella Cu,Zn‐SodCII. Copper binding by CueP in this pathogen may confer protection of the periplasm from copper‐mediated damage while sustaining vital cupro‐enzyme activity.

Metal Sensing in Salmonella: Implications for Pathogenesis

Both the essentiality and toxicity of transition metals are exploited as part of mammalian immune defenses against bacterial infection. Salmonella serovars continue to cause serious medical and veterinary problems worldwide and detecting deficiency and excess of different metal ions (such as copper, iron, zinc, manganese, nickel, and cobalt) is fundamental to their virulence. This involves multiple DNA-binding metal-responsive transcription factors that discriminate between elements and trigger expression of genes that mediate appropriate responses to metal fluxes. This review focuses on the metal stresses encountered by Salmonella during infection and the roles of the different metal-sensing regulatory proteins and their target genes in adapting to these changing metal levels. Current knowledge regarding the mechanisms of metal-regulated gene expression and the structural features of sensory metal binding sites are described. In addition, the principles governing the ability of the different sensors to detect specific metals within a cell to control cytosolic metal levels are also discussed. These proteins represent potential targets for the development of new therapeutic approaches.

Differential expression from two iron-responsive promoters in Salmonella enterica serovar Typhimurium reveals the presence of iron in macrophage-phagosomes.

The metal status of macrophage-phagosomes during Salmonella infection is largely unknown. In this study, we have precisely calibrated the metal-specificities of two metal-responsive promoters, P(iroBCDE) and P(sodB), from Salmonella enterica serovar Typhimurium and used these to directly monitor iron-levels in Salmonella-containing macrophage-phagosomes. Expression from the P(iroBCDE) promoter is highly elevated in metal-depleted media but low in media supplemented with iron or cobalt, and to a lesser extent manganese. In contrast, P(sodB) shows low levels of expression in metal-depleted media but is induced in media supplemented with iron but no other metals at maximum permissive concentrations. In both cases, iron-responsive expression corresponds to changes in the number of iron atoms per bacterial cell and is unaffected by pH or the presence of reactive oxygen species (hydrogen peroxide and superoxide). Importantly, expression from P(iroBCDE) remained low while expression from P(sodB) was elevated during infection of both Nramp1(+/+) and Nramp1(-/-) macrophages. Expression from a control promoter, P(polA), unaffected by metal ions, remained unchanged. These findings are therefore consistent with the presence of iron within Salmonella-containing macrophage-phagosomes and support a model in which the toxic potential of iron may be exploited as a component of the respiratory burst killing mechanism.

The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo. Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro. Sensitivity to formaldehyde requires a cysteine (Cys35 in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmRE64H reveals that an FrmR-specific amino-terminal Pro2 is proximal to Cys35, and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmRP2S and RcnRS2P, respectively, impair and enhance formaldehyde reactivity in vitro. Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro. Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

Fine control of metal concentrations is necessary for cells to discern zinc from cobalt

Bacteria possess transcription factors whose DNA-binding activity is altered upon binding to specific metals, but metal binding is not specific in vitro. Here we show that tight regulation of buffered intracellular metal concentrations is a prerequisite for metal specificity of Zur, ZntR, RcnR and FrmR in Salmonella Typhimurium. In cells, at non-inhibitory elevated concentrations, Zur and ZntR, only respond to Zn(II), RcnR to cobalt and FrmR to formaldehyde. However, in vitro all these sensors bind non-cognate metals, which alters DNA binding. We model the responses of these sensors to intracellular-buffered concentrations of Co(II) and Zn(II) based upon determined abundances, metal affinities and DNA affinities of each apo- and metalated sensor. The cognate sensors are modelled to respond at the lowest concentrations of their cognate metal, explaining specificity. However, other sensors are modelled to respond at concentrations only slightly higher, and cobalt or Zn(II) shock triggers mal-responses that match these predictions. Thus, perfect metal specificity is fine-tuned to a narrow range of buffered intracellular metal concentrations.Bacteria possess transcription factors whose DNA-binding activity is altered upon binding to specific metals, but the binding of metals is not specific in vitro. Here, Osman et al. show that tight regulation of buffered intracellular metal concentrations is a prerequisite for metal specificity.