Bacterial Redox Sensors

Chao Wang

Oct 5, 2005

Jeffrey Green and Mark S. PagetBacterial redox sensorsNat Rev Microbiol. 2004 Dec;2(12):954-66. Review.

Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, United Kingdom.

Redox reactions pervade living cells.

The ability to maintain redox balance is therefore vital to all organisms.

Various regulatory sensors continually monitor the redox state of the internal and external environments and control the processes that work to maintain redox homeostasis.

These sensors convert the redox signals into regulatoryoutputs, usually at the level of transcription, which allowsthe bacterium to adapt to the altered redox environment.

Some well-characterized bacterial redox sensors and their mechanisms relate to biological functions

• Thiol-based redox sensors• Fe–S cluster-based sensors• Haem-based sensors• Flavin cofactor-based redox sensors• Pyridine nucleotides• Quinone redox sensors

Thiol-based redox sensors

• Thiol-based sensor function is reviewed. Typically, these sensors use cysteine modification to sense redox alterations. Examples include OxyR in Escherichia coli, the R-RsrA system in Streptomyces coelicolor, CrtJ and the RegB−RegA in Rhodobacter sphaeroides, and OhrR from Bacillus subtilis.

• Cysteine is uniquely suited to sensing a range of redox signals because the thiol side-chain can be oxidized to several different redox states, many of which are readily reversible

OxyR — a sensor of peroxide stress

• Despite there being little doubt that OxyR uses one or more of its six cysteine thiols to directly sense oxidative stress, the precise nature of the thiol modifications and their consequences for OxyR activity are the subject of ongoing controversy.

• Storz laboratory supported the role of just one, Cys199, in the hydrogen peroxide-mediated activation of OxyR, possibly through the formation of sulphenic acid (SOH). Stamler laboratory showed that OxyR could also be activated by S-nitrosylation when treated with S-nitrosothiols (SNOs), thereby supporting the role of the same single cysteine in OxyR activation.

• Storz suggested a role for another cysteines, Cys208, following the detection in oxidized OxyR of an intramolecular disulphide bond between Cys199 and Cys208.

• Stamler’s group recently came to the conclusion that Cys208 might not have a role in OxyR activation15. Instead, they presented evidence that OxyR is modified by peroxide stress to Cys199-SOH; by nitrosative stress to Cys199-SNO; or by disulphide stress to form a mixed disulphide with glutathione(Cys199-S-SG)

σR-RsrA — a sensor of disulphide stress

Thiol-based regulators of anoxygenic photosynthesis• The facultatively photosynthetic bacterium Rhodobacter capsulatus can derive energy

from photosynthesis only when the oxygen tension falls below ~2.5%. Expression of the photosystem genes is controlled by multiple regulators, two of which, CrtJ and the RegB–RegA TWO-COMPONENT SYSTEM, seem to invoke reversible disulphide-bond formation as part of their redox-sensing mechanism.

• CrtJ aerobic conditionscontain two central PAS(PER–ARNT–SIM) domains — a motif that is implicated in sensing many environmental signals.

• RegB–RegA system anaerobic conditionsthe inhibition of RegB autophosphorylation under oxidizing conditions correlates with its accumulation in a tetrameric form, and that an intermolecular disulphide bond involving a single cysteine, Cys265, mediates this oligomerization.Mutation of Cys265 to serine led to increased photosystem gene expression during aerobiosis.

• How a ‘stable’ disulphide can form in the reducing environment of the cytoplasm.

OhrR — a sensor modulated by sulphenic acid formation

A single redox-sensing cysteine (Cys15) in the Bacillussubtilis OhrR provides perhaps the best evidence for therole of a stable sulphenic acid in modulating regulatoractivity. Biochemical studies indicate that Cys15 ofOhrR is rapidly oxidized to Cys15-SOH when treatedwith cumene hydroperoxide. This oxidation inhibitsDNA-binding, thereby inducing expression of theorganic hydroperoxidase gene ohr.

Fe–S cluster-based sensors

• Fe–S proteins have important roles as redox-responsive transcriptional and post-transcriptional regulators in many bacteria.

• The importance of Fe−S proteins in redox sensing is illustrated by the functions of several redox sensors from E. coli that use oxidation of Fe−S clusters to monitor the redox status of cell compartments and the environment to produce appropriate transcriptional responses — these include SoxR, Fnr, aconitase and IcsR.

SoxR — a sensor of superoxide and nitric-oxide stress

• The E. coli SoxR protein is a homodimer that contains one [2Fe–2S] cluster per subunit.

• When cultures of E. coli are exposed to conditions that promote the generation of superoxide (for example, in the presence of paraquat and oxygen), the [2Fe–2S]1+ clusters are oxidized to [2Fe–2S]2+ clusters. Oxidation facilitates SoxR-dependent distortion of the soxS promoter DNA to form a transcriptionally active complex with RNA polymerase. Once the source of the oxidative stress is removed, the SoxR Fe–S clusters are rapidly reduced, thereby switching off expression of the SoxRS regulon.

Fnr — a sensor of environmental oxygen

• Facultative anaerobes such as E. coli adopt different metabolic modes in response to oxygen availability. There is a hierarchy of metabolism in which aerobic respiration (in which oxygen is the terminal electron acceptor) is preferred over anaerobic respiration (in which alternative electron acceptors, such as nitrate, are used), which in turn is preferred over fermentation.

• Fnr consists of two domains, a carboxy terminal DNA-binding region, which recognizes a specific DNA sequence in target promoters, and an amino-terminal sensory domain that contains four essential cysteine residues capable of binding either a [4Fe–4S]2+ or a [2Fe–2S]2+ cluster.

During aerobic growth it is suggested that Fnr cycles between the active [4Fe–4S]2+ homodimeric form and the inactive monomeric apo-protein.

Assuming that the rate of cluster synthesis is slowerthan that of oxygen-dependent cluster degradation,Fnr will mostly be present in the inactive apo-stateunder aerobic conditions. Anaerobic conditions blockthe cycle at the [4Fe–4S]2+ to [2Fe–2S]2+ conversionstep, thereby allowing the active Fnr dimer [4Fe–4S]2+to accumulate and anaerobic gene expression to beswitched on.

Haem-based sensors

• The haem is most often bound to a PAS (PER−ARNT−SIM) domain but can reside within other protein folds. Often, the state of haem is coupled to a transmitter domain, which transduces the signal into an appropriate output.

Dos — an oxygen and redox sensor

• It has an N-terminal haem-binding PAS domain. However, the sensory domain is linked to a C-terminal phosphodiesterase domain, rather than a histidine kinase domain, which degrades cyclic AMP in a redox-dependent manner.

• Dos is a tetramer in which each subunit has two PAS domains — PAS-A and and PAS-B. The steady-state haem configuration in Dos is a six coordinate, with Met95 acting as a second axial ligand.

• When oxygen binds to the haem,Met95 is displaced, promoting conformational changes in the PAS-A domain that inhibit phosphodiesterase activity. However, phophodiesterase activity has also been shown to be regulated by the redox state of the haem iron, leading to the proposal that Dos is a redox sensor rather than an oxygen sensor.

• either oxygen binding to ferrous haem, or oxidation of the haem moiety, initiates the conformational changes that are needed to inhibit phosphodiesterase activity.

Flavin cofactor-based redox sensors

• The flavin cofactors FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) are versatile, electron-carrying coenzymes involved in both one- and two-electron transfers. The coenzymes can exist in three states: fully oxidized (for example, FAD); partially oxidized, as a semiquinone radical (for example, FADH•); and fully reduced (for example, FADH2). In the past few years, several FAD-containing primary redox sensors have been described, each of which transduces the redox signal to secondary downstream effector proteins.

Aer — a redox sensor involved in aerotaxis

• Aer regulates the motility behaviour of E. coli in gradients of oxygen (aerotaxis), redox potential and certain nutrients.Aer achieves this by interacting with the CheA–CheW complex to transmit sensory information to the flagellar motors.

Pyridine nucleotides

The pyridine nucleotides, NAD(H) and NADP(H),occupy central points in redox metabolism. The mainrole of NAD(H) is to shuttle electrons released duringsubstrate oxidation to the electron transport chain.NADP(H) is involved in reductive biosynthetic andrepair pathways.Considering the importance in maintainingredox balance of these cofactors it is perhapssurprising that sensors of their redox state have onlyrecently been described.

Quinone redox sensors

• ArcB — a regulator of respiratory gene expression

The Arc two-component system of E. coli is a globalregulator of gene expression under microaerobic andanaerobic growth conditions.When oxygen is limitingor absent, the membrane-bound sensor kinaseArcB autophosphorylates at His292 in the primarytransmitter domain, then transphosphorylates theresponse regulator ArcA at Asp54.

Conclusions

In vivo, neither redox sensors northeir signals operate in isolation. Integrated and complexregulatory networks provide an optimal responseto changeable environments. With the advent of thepost-genomic era, and the move towards more predictivebiology, understanding how these complexregulatory circuits interact to integrate transcriptionalresponses to multiple environmental cues will becrucial if meaningful predictive model systems areto be developed.