interference in the balance between the environmental production of reactive oxygen species ( ros ) , including hydroxyl radicals ( oh ) and hydrogen peroxide ( h2o2 ) , and the ability of biological systems to readily detect and detoxify them , or repair the resulting damage , are defined as oxidative stress .
highly reactive radicals cause the oxidative damage of different macromolecules  proteins , dna , and lipids  leading to loss of function , an increased rate of mutagenesis , and ultimately cell death . in humans , for example , oxidative stress is involved in many diseases , such as rheumatoid arthritis , autoinflammatory diseases , neurodegenerative diseases , and cancer [ 1 , 2 ] .
however , the production of some ros ( e.g. , oh ) can also be beneficial , as they are used by the human immune system to attack and kill pathogens , such as the production of ros by macrophages .
sensing of ros - mediated signals also plays a crucial role in the biology of microorganisms .
bacteria , for example , are in continuous contact with ros generated both endogenously , as a product of aerobic metabolism , or exogenously during ionizing (  ) and nonionizing ( uv ) irradiation leading to the production of a number of radical and peroxide species through the ionization of intracellular water .
industrial contaminants that are widespread in soils and on the surfaces of plants are also sources of ros .  
iron is earth 's fourth most abundant metal , after oxygen , silicon , and aluminium .
its relevance for bacterial cells is emphasized by the fact that it is involved in a wide range of biological processes , including photosynthesis , n2 fixation , h2 production and consumption , respiration , oxygen transport , and gene regulation [ 4 , 5 ] .
however , in the presence of oxygen , ferrous ions frequently result in oxidative stress through the generation of hydroxyl radicals via the fenton reaction ( fe + h2o2fe + oh + oh ) .
therefore , bacteria have developed a variety of different mechanisms to ensure that iron is sufficiently accessible as well as being maintained in a nontoxic form .
they possess high - affinity iron transport systems ( i.e. , siderophores and membrane iron transporters ) that enable iron to be scavenged .
intracellular iron can be stored in protein complexes ( i.e. , in dps and ferritins ) .
thus , the homeostasis of these ions is tightly regulated so that their intracellular concentrations do not reach toxic levels . previously , it has also been suggested that the production of hydroxyl radicals is induced by bactericidal antibiotics to kill bacteria , in which iron ions and the fenton reaction play a role , or during a wide range of plant or human pathogen interactions .
moreover , ros can also be produced during the degradation of natural existing biopolymers ( cellulose , chitin , or xylan ) by microorganisms .
to reduce the hazardous effects of iron - based production of oh , bacteria produce proteins with an enzymatic activity to degrade ros ( i.e. , superoxide dismutases , catalases , peroxidases , and alkylhydroperoxide reductases ) , other small redox proteins ( thioredoxins and glutaredoxins ) as well as low molecular - weight thiols ( glutathione and mycothiol ) [ 8 , 9 ] .
all these cell components contribute in maintaining a reducing environment both in the cell and in controlling the extent of the oxidative burst .   in analogy to fe(ii )
ions , other transition metals ions [ i.e. , cu(i ) , co(ii ) , mn(ii ) , ti(iii ) , or cr(v ) ] are closely linked with the production of free radicals in cells .
although these metal ions can be hazardous for living organisms , they also serve as signal mediators in signalling cascades .
it is an essential trace element that is localized in the active center or in a structurally important site of many bacterial proteins .
zinc is a cofactor for more than 300  enzymes ( i.e. , superoxide dismutase and alcohol dehydrogenase ) .
it is also a structural element of at least 40 protein classes ( i.e. , rna polymerase and trna synthetases ) .
additionally , zinc can protect sulfhydryl groups from free radicals and inhibits free radical formation by competing with redox - active metals such as iron [ 12 , 13 ] .
binding of zinc as well as metal - catalyzed oxidation in proteins is closely related with the presence of redox - active cysteine residues ( e.g. , within cys - x - x - cys motifs ) and other metal - sensing amino acids . moreover
, bacterial redox sensory proteins can act either as single transcription regulators ( e.g. , furs or irr ) displaying a sensory and response domain within itself [ 14 , 15 ] , or as a part of multicomponent systems ( e.g. , hbps or chrs ) in which sensing and response are distributed among each protein component [ 1618 ] .   in bacteria , there are a high number of redox sensory proteins that show different mechanism of function . here
, we will give an overview of them and focus further on signalling pathways in which redox - active cysteines as well as iron ions are involved .
cysteine residues ( cys ) in proteins are prominent targets for protein oxidation , as they easily react with h2o2 and free radicals .
oxidation of cys by h2o2 involves nucleophilic attack of the cysteinyl thiol group on the electrophilic center of h2o2 .
deprotonation of the cys thiol group to generate the thiolate anion increases its nucleophilicity , and hence reactivity towards h2o2 .
these reactions are chemically highly complex and can lead to different sulfur oxidation states , including thiols , sulfenic and sulfinic acids , thiyl radicals , disulfide s - oxides , or disulfides .
disulfides can be generated between two cys either intra- or intermolecularly ( figure 1 ) .
the sensitivity of cys thiol groups to oxidation provides them with redox sensitivity , and hence the ability to sense redox status .
for example , the thiolate anion can be stabilised by proximity to hydrogen bond donors , basic residues , and metal ions . in this review , we will focus on intramolecular disulfides that are formed within cys - x - x - cys motifs(x : any amino acid)a motif that is widespread in bacterial sensor proteins ( table 1 ) .
in addition to redox - sensing properties , these motifs are often involved in zinc binding ( i.e. , in furs , hsp33 , rsra , rsla , trx2 , and sbcc ) ( table 1 ) , and in the stabilization of protein domains that are crucial for function .
it can be expected that the molecular environment of cys is in this case also important for zinc binding . 

table 1 comprises two different types of cys - x - x - cys - containing proteins .
proteins belonging to the first group exhibit enzymatic function ( hsp33 , resa , dsba , sbcc , cytochrome c , trx2 , ahpf / ahpc , copa , and hypa ) .
for example , under reducing conditions , the chaperone hsp33 binds a single zinc ion through its c - terminal cys - x - cys - x6-cys - x - x - cys motif . upon oxidative stress , the four cys residues form intramolecular disulfide bridges resulting in release of zinc accompanied by considerable conformational changes that lead to destabilization of its c - terminus . as a consequence , hsp33 dimerises and acquires a chaperone function to prevent protein aggregation
similarly , in the dna repair protein sbcc , two monomers are linked via cys - x - x - cys motifs and a zinc ion , forming a functional zinc bridge that is important for sbcc exonuclease activity .
an extracellular low potential thiol - disulfide oxidoreductase  was shown to maintain cys residues of cytochrome c in their reduced form .
the thiolate species within the heme - binding motif ( cys - x - x - cys - his ) of apo - cytochrome c are required for covalent attachment of heme .
contrary to the other cys - x - x - cys - containing proteins described here , resa does not bind zinc through this motif .
other proteins with this signature and lacking zinc are listed in table 1 but are not within the focus of this review .
the second group of cys - x - x - cys - containing proteins is involved in transcriptional regulation and can be subdivided into two subtypes : proteins that either directly control transcription ( i.e. , whib3 , furs , catr , soxr , and surr ) or require further regulatory components ( i.e. , spx , rsra , rsla , and rsha ) .  
the redox - dependent cys - x - x - cys - containing transcriptional regulator whib3 , from mycobacterium tuberculosis , has been shown to sense the intracellular redox state in the cell , and to be required for the production of virulence polyketides , including polyacyltrehaloses ( pat ) , sulfolipids ( sl-1 ) , di - o - acyl - trehaloses ( dats ) and trehalose dimycolates ( tdms ) .
whib3 directly regulates the expression of the following genes in a redox - dependent manner : pks3 ( encoding a polyketide beta - ketoacyl synthase involved in pat and dat anabolism ) , pks2 ( encoding a polyketide synthase involved in sl-1 anabolism ) , and fbpa ( encoding a fibronectin - binding protein for tdms production ) .
whib3 contains an iron - sulfur cluster [ 4fe-4s ] that under aerobic conditions is oxidized to [ 4fe-4s ] .
further oxidative conversions result in complete loss of this cluster . unlike the superoxide stress sensor soxr
, the redox state of the whib3 iron - sulfur cluster does not modify the dna binding affinity to target gene promoters , but rather the redox state of the cysteine residues is critical for dna binding .
it was shown that , while the reduction of cys in whib3 abolishes its dna binding activity , oxidation induces it .
it is noteworthy to mention that reduction and oxidation in whib3 are reversible processes . in the case of the zinc - containing transcriptional repressor furs from streptomyces
reticuli , it was biochemically and spectrophotometrically shown that h2o2-mediated disulfide bond formation between cys93 and cys96 in furs is accompanied by the release of the bound zinc ion .
this leads to conformational changes in furs , resulting in a loss of furs binding to its own dna operator sequence within the regulatory region of the target operon , furs - cpeb . as a result ,
the expression of this operon is no longer repressed , leading to an increase in production of furs and the mycelium - associated catalase - peroxidase cpeb .
this enhanced expression in turn detoxifies the surrounding environment of hazardous h2o2 [ 9 , 14 ] .  
proteins with cys - x - x - cys motifs are also indirectly involved in transcriptional regulation , by interacting with additional components .
reversible disulfide formation can regulate the activity of the sigma factor r (  ) of streptomyces coelicolor a3(2 ) . here
, the thiol - specific oxidant diamide induces -mediated transcription of > 30 genes , including those encoding thioredoxins and thioredoxin reductases that are involved in antioxidative stress response .
however ,  does not contain any cys and in vitro studies have demonstrated that its transcriptional activity is not dependent on oxidative stress .
indeed , researchers have characterised the protein rsra , which interacts with  and regulates its transcriptional activity in a oxidative stress - dependet manner .
rsra is a zinc - containing and redox - sensitive antisigma factor that possesses seven cys , two of which ( cys41 and cys44 ) are located within a cys - x - x - cys motif that is important for activity . under oxidising conditions ,
a disulfide bond is formed in rsra , and this oxidized state can not bind  or inhibit its transcriptional activity .
oxidised rsra can be re - reduced by thioredoxin and in the thiol - reduced state rsra reassociates with  and blocks its transcriptional activity .
the redox sensing cys in rsra also coordinate zinc ions that provide the protein with a higher structural stability , strengthening its interaction with . upon redox stress and induction of disulfide bond formation , zinc is released and rsra undergoes conformational changes , generating a structure that does not bind . structural studies of an rsra homolog , rsla ( table 1 ) from mycobacterium tuberculosis revealed that the redox sensing and zinc binding cys ( cys54 and cys57 ) are closely located ( ~3 ,   ; figure 2 ) to each other and are solvent exposed in the complex , thus , providing a structural basis for the redox sensitivity of rsla .
the solvent exposed state of cys within the cys - x - x - cys motif is also an essential feature in redox sensing by the heat - shock protein hsp33 ( table 1 ; pdb : 1vq0 ) from escherichia coli .
to assure that iron is sufficiently accessible as well as being maintained in a nontoxic form , bacteria have developed a variety of protein - based protection mechanism .
they employ iron - sensing regulatory proteins ( i.e. , perr and hbps - sens - senr ; see also table 2 ) that control the expression of proteins ( i.e. , h2o2-degrading enzymes such as catalases ) blocking iron - dependent damage . upon sensing of iron - based stress ,
these proteins undergo different oxidative modifications , including oxidation of histidines ( figure 3 ) , dityrosine formation , or carbonylation .  

table 2 lists different iron - sensing proteins that mostly control redox stress resistance systems .
they can directly regulate the transcription of target genes by dna binding to regulatory regions ( i.e. , fur , dtxr , rira , irr , perr , dmdr1 , ider , sirr , fnr , and tror ) or utilize further regulatory elements ( hbps - sens - senr ; pmra - pmrb , chrs - chra , and feca - fecr - feci ) that mediate the adaptive response .  
iron - dependent gene regulation has been demonstrated for the diphtheria toxin repressor ( dtxr ) from corynebacterium diphtheriae and corynebacterium glutamicum .
binding of divalent iron ions to the global regulator dtxr results in the transcriptional repression of a number of genes required for iron uptake ( i.e. , htaa coding for a secreted iron acquisition and transport protein ) , as well as for usage and storage ( i.e. , hmuo coding for a heme oxygenase and ftn coding for a ferritin - like protein ) .
mutational analyses have additionally demonstrated that dtxr is also involved in the iron - dependent expression of dna - protecting proteins , including dps - like proteins that bind iron and dna .   in bacillus subtilis ,
the peroxide resistance regulator perr has been shown to sense metal dependent as well as h2o2-based oxidative stress , and to regulate the corresponding adaptive response .
the metalloprotein perr is a small dimeric protein that coordinates two metal ions per monomer .
one binding site binds a zinc ion , whereas the second one a regulatory metal , either iron or manganese [ 37 , 38 ] . in vivo
, the regulatory metal is required for repression of the transcription of target genes , including those ones encoding a catalase , alkylhydroperoxide reductase , and the dps - like dna - binding protein mrga . under h2o2-based oxidative stress conditions , fe catalyzes the oxidation of histidines ( either his37 or his91 ) in the regulatory binding site of perr .
analysis of electron maps density in the crystal structure of the oxidised perr protein ( perr - zn - ox ) showed the presence of a 2-oxo - histidine residue at position 37 .
further , maldi - tof and tandem esi - ms studies additionally revealed the oxidation of his91 within per - zn - ox [ 36 , 38 , 39 ] .
it was concluded that the structural conformation of perr is dependent on the oxidation state of the regulatory site .  
oxidative modifications have also been shown to regulate the activity of the redox sensor and heme - binding protein hbps from streptomyces reticuli .
hbps is extracellularly located and interacts with the membrane - embedded histidine autokinase sens from the two - component system sens senr [ 40 , 41 ] .
analyses of the crystal structure of hbps combined with size exclusion chromatography and static light scattering [ 4244 ] allowed the identification of hbps as an octamer .
further studies demonstrated that the octameric assembly in hbps is required for an efficient interaction with sens .
phosphorylation analyses also revealed that under nonoxidative stress conditions hbps inhibits the autophosphorylation of sens , whereas oxidative stress induces the hbps - mediated activation of sens .
after autophosphorylation at a conserved histidine , sens transfers the phosphate group to its cognate response regulator senr , resulting in senr~p .
the unphosphorylated form of senr binds to specific sites upstream of the furs - cpeb operon ( encoding for the redox regulator furs and the mycelia - associated catalase - peroxidase cpeb ) , leading to its transcriptional repression .
once senr has been phosphorylated , it loses the ability to bind to this operator , leading to a de - repression of the furs - cpeb transcription .
comparative physiological analyses have demonstrated that the presence of hbps and sens - senr provides s. reticuli with a defence system against redox - stressing conditions [ 40 , 41 ] .
it was proposed that the switching of hbps from its inhibitor to activator state of sens autophosphorylation under oxidative stress conditions is controlled by conformational changes in hbps .
indeed , cd spectroscopy as well as fret analyses revealed that after iron - mediated oxidative stress hbps undergoes secondary structure and overall intrinsic conformational changes , which are accompanied by oxidative modifications ( i.e. , carbonylation and dityrosine formation ) .
while the sites of carbonylation have not yet been determined , the tyrosine residues participating in dityrosine formation have been identified as tyr77 .
these residues are localized in the interface of hbps subunits and are located in close proximity to each other , over the monomer - monomer interface .
interestingly , tyr77 is situated near to a postulated iron - binding site , containing glu78 and glu81 within an e - x - x - e motif ( figure 4 ) that has been previously characterized as an iron - binding motif [ 4547 ] .
the reported oxidative modifications in hbps result in the degradation , either autonomously or protease dependent , of the oxidized protein .
a metal - catalyzed protein oxidation accompanied by cross - linking and degradation has been reported for the bradyrhizobium japonicum iron response regulator ( irr ) protein , which is involved in the regulation of iron transport as well as of heme biosynthesis genes .
it is proposed that the iron - catalyzed oxidation with subsequent degradation of irr is the molecular basis for the modulation of the activity of this regulator .
the membrane - embedded sensor kinase pmrb from the two - component system pmra - pmrb of the pathogen salmonella enterica serovar typhimurium exhibits two copies of the exxe iron - binding motif .
these are periplasmically located and have been proposed to be involved in sensing of extracellular ferric iron . upon iron sensing and autophosphorylation , pmrb transfers the signal to its cognate response regulator pmra , which in turn regulates fe resistance genes : pbgp and ugd both coding for enzymes that modify the lipid a region of lipopolysaccharide ( lps ) with 4-aminoarabinose .
the gene product of pmrc catalyzes the addition of phosphoethanolamine to lipid a , whereas the phosphatase encoded by pmrg targets the phosphate located in the core region of the lps .
these modifications result in a less - charged cell surface and diminished binding of ferric iron to the membrane .
in contrast to other iron - sensing systems ( i.e. , perr and hbps - sens - senr ) , pmra - pmrb seems to protect the cell against iron toxicity independent of the simultaneous presence of oxygen . beside the posttranslational modifications ( i.e. , oxidation of histidines , carbonylation , dityrosine formation , or s  s bonding ) that regulate iron - sensing proteins , posttranscriptional regulation of gene expression in response to oxidative stress
has been reported for the aconitases acna and acnb of escherichia coli [ 49 , 50 ] . in their holo form ,
both acna and acnb contain an iron - sulfur cluster , [ 4fe-4s ] , and exhibit enzymatic function . under reducing conditions ,
they catalyze the isomerisation of citrate to isocitrate . upon oxidative stress and iron depletion ,
the resulting apo - acna and apo - acnb proteins act as rna - binding proteins that stabilize acna- and acnb - mrna transcripts , leading to increased amount of the respective aconitases that subsequently complex iron - sulfur clusters .
moreover , mutational analyses have revealed that the presence of acna provides e. coli with enhanced resistance against superoxide - mediated oxidative stress .
additionally , proteomics studies demonstrated that the expression of anti - oxidative stress - working proteins ( i.e. , soda and trxb ) is dependent on acna .
as in humans , the exposure of bacteria to ros causes damage to a variety of macromolecules , resulting in mutations and often in cell death .
however , ros may also be considered to be beneficial compounds , as they function as signalling molecules that lead to a coordinated response of bacteria under redox - stress conditions .
these signals can be sensed by redox - active and zinc - coordinating cys - x - x - cys centres in proteins . under reducing conditions , zinc stabilizes protein structure , but
the presence of h2o2 provokes the release of zinc and the formation of s  s bridges that significantly alter the conformation and structure of the protein ( i.e. , furs or rsra ) . as a result , transcriptional activity is altered ( by furs ) , or the ability to interact with the partner dna - binding protein is lost ( by rsra ) . in both cases , the transcription of genes involved in the anti - oxidative stress response is ultimately activated . the structural basis for the redox sensitivity
is given by the location of s  s - forming cysteines in the protein , namely , their solvent exposition within the three - dimensional structure .
importantly , reduction and oxidation processes within cys - x - x - cys redox centres are reversible and provide bacteria with an elegant switch on / off mechanism .
iron - dependent oxidative modifications ( i.e. , 2-oxo - histidine or dityrosine formation ) are also involved in ros - based signalling . under h2o2-based stress , iron catalyzes the oxidation of histidines in the peroxide resistance regulator perr , leading to release of iron and subsequently to de - repression , and expression of target genes . in analogy ,
iron - based stress activates the hbps - sens - senr - mediated signalling cascade .
hbps is a redox sensor that upon oxidative modifications undergoes conformational and structural changes , inducing the phosphorylation cascade between the sensor kinase sens and the response regulator senr . as known for other regulators , oxidative modifications
therefore , de novo protein biosynthesis is required to switch off the corresponding signal cascade .  
the diversity of responsive protein elements among bacteria correlates with the diversity in : ecological niches , life cycles , pathogenic , and nonpathogenic character .
 bacteria are permanently in contact with reactive oxygen species ( ros ) , both over the course of their life cycle as well that present in their environment . 
 these species cause damage to proteins , lipids , and nucleotides , negatively impacting the organism . to detect these ros molecules and to stimulate the expression of proteins involved in antioxidative stress response , bacteria use a number of different protein - based regulatory and sensory systems . 
 ros - based stress detection mechanisms induce posttranslational modifications , resulting in overall conformational and structural changes within sensory proteins . 
 the subsequent structural rearrangements result in changes of protein activity , which lead to regulated and appropriate response on the transcriptional level . 
 many bacterial enzymes and regulatory proteins possess a conserved signature , the zinc - containing redox centre cys - x - x - cys in which a disulfide bridge is formed upon oxidative stress . 
 other metal - dependent oxidative modifications of amino acid side - chains ( dityrosines , 2-oxo - histidines , or carbonylation ) also modulate the activity of redox - sensitive proteins . using molecular biology , biochemistry , biophysical , and structure biology tools , molecular mechanisms involved in sensing and response to oxidative stress 
 have been elucidated in detail . in this review , 
 we analyze some examples of bacterial redox - sensing proteins involved in antioxidative stress response and focus further on the currently known molecular mechanism of function . 
1. Introduction
2. Zinc-Containing Cys-X-X-Cys Motifs as Sensor Centres
3. Iron-Based Oxidative Stress
4. Summary