Appl Microbiol Biotechnol (2014) 98:6933–6946 DOI 10.1007/s00253-014-5883-4

MINI-REVIEW

Oxidative stress response in Pseudomonas putida Jisun Kim & Woojun Park

Received: 15 April 2014 / Revised: 4 June 2014 / Accepted: 5 June 2014 / Published online: 24 June 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Pseudomonas putida is widely distributed in nature and is capable of degrading various organic compounds due to its high metabolic versatility. The survival capacity of P. putida stems from its frequent exposure to various endogenous and exogenous oxidative stresses. Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents existing in various niches. ROS could facilitate the evolution of bacteria by mutating genomes. Aerobic bacteria maintain defense mechanisms against oxidative stress throughout their evolution. To overcome the detrimental effects of oxidative stress, P. putida has developed defensive cellular systems involving induction of stress-sensing proteins and detoxification enzymes as well as regulation of oxidative stress response networks. Genetic responses to oxidative stress in P. putida differ markedly from those observed in Escherichia coli and Salmonella spp. Two major redox-sensing transcriptional regulators, SoxR and OxyR, are present and functional in the genome of P. putida. However, the novel regulators FinR and HexR control many genes belonging to the E. coli SoxR regulon. Oxidative stress can be generated by exposure to antibiotics, and iron homeostasis in P. putida is crucial for bacterial cell survival during treatment with antibiotics. This review highlights and summarizes current knowledge of oxidative stress in P. putida, as a model soil bacterium, together with recent studies from molecular genetics perspectives. Keywords Oxidative stress . Pseudomonas putida . Transcriptional regulation . Iron homeostasis . Antibiotics . Redox sensing J. Kim : W. Park (*) Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Anam-Dong 5Ga, Seungbuk-Ku, Seoul 136-713, Republic of Korea e-mail: [email protected]

Introduction Pseudomonas putida is a gram-negative bacterium that can live in various environmental niches because of its remarkable metabolic versatility, low nutritional requirements, and striking tolerance to diverse organic compounds (Dos Santos et al. 2004; Nelson et al. 2002; Timmis 2002). In addition, P. putida is genetically accessible, and genome-wide pathway modeling has been carried out for this organism (Dos Santos et al. 2004; Puchałka et al. 2008). These physiological characteristics and accessibility of sequencing-based bioinformatics make P. putida suitable for potential use in biotechnological and environmental applications (Nikel and De Lorenzo 2012; Poblete-Castro et al. 2012; Puchałka et al. 2008). P. putida possesses various signaling mechanisms mediating aerobic metabolism, which can degrade the hydrocarbons of organic compounds, therefore making this bacterium one of the most important microorganisms in bioremediation (Poblete-Castro et al. 2012; Yu et al. 2001). Bacteria suitable for bioremediation may go through oxidative stress due to the effects of pollutants and intermediates generated during the biodegradation process (Park et al. 2004; Schweigert et al. 2001). Our whole-cell bioreporter assay using paraquat-inducible fpr of P. putida KT2440 demonstrated that oxidative stress was generated by environmental pollutants (Lee et al. 2006a). Knowledge acquired from studies of the oxidative stress response in P. putida could contribute to advances in biotechnology. Bacterial cells exposed to endogenous or exogenous sources of oxidants have systems for detecting and removing them (Yeom et al. 2010b). The oxygen molecule can accept electrons from intracellular reductants and reactive oxygen species (ROS) (Cabiscol et al. 2000; Imlay 2013). ROS are deleterious species that react with various cellular components, thereby damaging them (Imlay 2003). Superoxide (O2•−) is generated when an oxygen molecule receives an

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electron from a donor such as a flavoprotein. Likewise, hydrogen peroxide (H2O2) is formed when two electrons are accepted by an oxygen molecule. Superoxide and hydrogen peroxide can disrupt iron–sulfur clusters such as dehydratases, thereby rendering the enzyme inactive (Dubbs and Mongkolsuk 2012; Kuo et al. 1987). They also inactivate a wide range of enzymes that utilize an iron atom as the cofactor (Anjem and Imlay 2012). Moreover, hydrogen peroxide reacts with ferric iron and forms the highly toxic hydroxyl radical (HO•) through the Fenton reaction. The hydroxyl radical is a strong oxidant that is able to react with almost every molecule within a cell. In particular, the oxidation of DNA by hydroxyl radicals causes mutations or lesions that have lethal effects on the cell (Imlay 2003, 2013). Numerous studies have been carried out to elucidate the mechanisms of the oxidative stress response by using Escherichia coli as the experimental model (Farr and Kogoma 1991). In E. coli, two redox-sensing proteins, SoxR and OxyR, are activated upon oxidation (Imlay 2013). These proteins regulate the expression of genes involved in oxidative stress defense (Imlay 2013; Pomposiello and Demple 2001). Moreover, the RpoS protein, a regulator that modulates the transcription of genes during conditions of general stress, is also involved in the oxidative stress response (Chiang and Schellhorn 2012). However, the mechanisms mediating the oxidative stress response in P. putida, a representative soil microorganism, have not yet been established. Researching the physiological and regulatory systems of P. putida will expand our knowledge on the basic mechanisms of metabolism and stress responses in soil microbes. This review focuses on the oxidative stress responses of P. putida, which differs from those of E. coli.

P. putida is a metabolically versatile microorganism P. putida is a versatile bacterium that is capable of living in diverse environments. P. putida primarily inhabits the soil, water, plant roots, and heavy metal- or solvent-contaminated habitats (Dos Santos et al. 2004; Nelson et al. 2002; Timmis 2002). These physiological characteristics are attributed to the high metabolic flexibility of the organism and the capacity of P. putida to control cellular homeostasis (Dos Santos et al. 2004; Nelson et al. 2002; Poblete-Castro et al. 2012; Puchałka et al. 2008; Timmis 2002). The prosperity of P. putida stems from its ability to alleviate endogenous and exogenous oxidative stress. Endogenous oxidative stress is generated from aerobic metabolism, whereas exogenous oxidative stress occurs as an unavoidable consequence of interaction with various ROS-inducing agents present in different niches. To overcome the deleterious effects of oxidative stress, P. putida has developed protective cellular systems by both well-known and novel regulators such as SoxR, OxyR, HexR, and FinR

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(Chavarría et al. 2013; Hishinuma et al. 2006; Kim et al. 2008; Park et al. 2006; Yeom et al. 2009a, b, 2010a, b, c, 2012). The formation of several oxidative stress-inducing factors and the defense systems they control are summarized in Fig. 1 and Table 1.

Role of detoxifying enzymes in controlling oxidative stress in P. putida Most bacteria express several enzymes that play roles in the detoxification of ROS. Typically, superoxide dismutases (SODs), catalases, and peroxiredoxins serve to remove and detoxify ROS. SODs are metalloenzymes that catalyze the dismutation of superoxide (O2−) into oxygen and hydrogen peroxide (H2O2). Then, hydrogen peroxide is reduced to water and oxygen by catalase or alkyl hydroperoxide reductase. Thus, SODs are an important antioxidant defense in bacterial cells exposed to oxygen. SODs are categorized according to their metal ion cofactor: copper–zinc type (Cu/Zn-SOD), iron type (Fe-SOD), manganese type (Mn-SOD), and nickel type (Ni-SOD) (Fridovich 1995; Kim et al. 1996, 1998). Most P. putida strains possess two types of SODs, Mn-SOD (encoded by sodA or sodM) and Fe-SOD (encoded by sodB or sodF) (Kim et al. 1999). Interestingly, the genome of P. putida KT2440 contains two SOD genes, sodA (PP_0946) and sodB (PP_0915). It has been reported that SodA–SodB heterodimer was produced in the KT2440 cells (Heim et al. 2003). P. putida W619 has three types of SODs, including Mn-SOD, Fe-SOD, and Cu/Zn-SOD, as shown by analysis of the complete genome sequences of P. putida in the NCBI genome database. It appeared that the expression of sodA and sodB genes is differently regulated by growth phase, iron, and manganese in P. putida. Thus, iron and manganese, which are essential cofactors for SOD proteins, are important in the regulation of sodA and sodB transcription. The transcription and activity of SODs in P. putida can be also increased by the addition of methyl tert-butyl ether, phenol, toluene, cadmium, and nickel ions (Choi et al. 2013; Krayl et al. 2003; Ray et al. 2013; Shamim and Rehman 2013; Yun et al. 2006). Unlike in E. coli, paraquat does not induce transcription of the sod genes or increase SOD activity in P. putida (Kim et al. 1999). However, the absence of both sodA and sodB increases the sensitivity of the organism to paraquat as compared to the wild-type organism (Kim et al. 2000). In P. putida, the sodA sodB double mutant exhibits impaired growth on roots due to inhibition of oxidative stress-sensitive key metabolic enzymes (Kim et al. 2000). Catalases catalyze the degradation of hydrogen peroxide to water and oxygen. The catalase family is divided into monofunctional catalases, bifunctional catalase peroxidases, and manganese-containing catalases (Chelikani et al. 2004). In P. putida, all 11 strains whose genomes have been

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Fig. 1 Simplified schematic overview of oxidative stress-inducing factors and important determinants involved in the response of P. putida KT2440 to oxidative stress, which affect whole-cell physiology. P. putida lives in diverse environments and is capable of interacting with various reactive oxygen species-inducing agents existing in various niches. To overcome the deleterious effects of oxidative stress, P. putida has developed protective cellular systems, including induction of stress-sensing proteins and detoxification enzymes and regulation of oxidative stress response networks by both well-known and novel regulators such as SoxR, OxyR, HexR, and FinR. Many genes belonging to E. coli SoxR regulon (e.g., fpr, zwf-1) are controlled by other regulators. Novel FinR

regulator is required for the induction of fpr (ferredoxin-NADP+ reductase) under superoxide stress condition in P. putida. HexR is the dualsensing regulator zwf-1 induction that is able to respond to both KDPG and oxidative stress. RecG is required for the induction of the OxyR regulon by unwinding palindromic DNA for transcription in P. putida. Oxidative stress could be generated under antibiotic conditions and iron and other metals homeostasis in P. putida is crucial for bacterial cell survival under antibiotics. ED pathway regulates intracellular energy and redox status, resulting in high tolerance to oxidative stress during the P. putida lifecycle

completely sequenced have two or more genes annotated as catalases. The expression of catalase genes is known to be regulated by OxyR in E. coli (Ochsner et al. 2000; Zheng and Storz 2000; Jamet et al. 2005; Chauvatcharin et al. 2005). E. coli has two types of catalases, HPI and HPII (Schellhorn 1995). The hydrogen peroxide-inducible bifunctional catalase HPI, encoded by katG, is involved in the oxidative stress response via regulation by OxyR and RpoS (Ivanova et al. 1994; Zheng and Storz 2000). However, monofunctional HPII, encoded by katE, is independent of oxyR. P. putida mt-2 possesses two catalases (KatA and KatB) under normal incubation conditions, with the expression of KatB being dependent on RpoS (Miura et al. 1998). P. putida KT2440 chromosome has four catalase genes: PP_0015 (katE), PP_0481 (katA), PP_2887 (unknown), and PP_3668 (katB) (Nelson et al. 2002). In P. putida KT2442, which is a rifampicin-resistant mutant of P. putida KT2440, oxyR1

mutation (Phe106 to Ile in OxyR) induced KatA and KatB catalases, and KatE catalase was detected in stationary phase (Hishinuma et al. 2006). Little is known about catalase encoded by PP_2887. P. putida KT2442 KatA is similar to many monofunctional catalases, like P. aeruginosa KatA and E. coli KatE; on the other hand, P. putida KT2442 KatB is similar to E. coli KatG (Hishinuma et al. 2006). P. putida Corvallis produces three catalases (CatA, CatB, and CatC) and the stationary phase-inducible catalase encoded by catC, which has 96.7 % identity with KatE of the KT2440 strain (Hishinuma et al. 2006; Katsuwon and Anderson 1990; Miller et al. 1997). CatA activity, but not CatB activity, is inhibited by 3-amino-1,2,4-triazole, and CatA is produced during all growth phases, suggesting that CatA is a major catalase (Katsuwon and Anderson 1990). The expression of CatA increases in the presence of external H2O2 (Katsuwon and Anderson 1990). Although the three catalase isozymes (CatA,

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Table 1 Overview of oxidative stress-inducing factors and response mechanisms in P. putida Strain

Oxidative stress response trigger(s)

KT2440

Paraquat (PQ), diamide, UV light

GB-1

KT2440

Involved gene or gene product

Region from PP_4329 to PP_4397 (about 70 kb, including the flagellar gene cluster) Manganese Multicopper oxidase (cumA, prevents Mn oxidation), multicopper oxidase (mnxG and mcoA, catalyzes Mn oxidation) ß-Lactam antibiotics, heavy Polyphosphate kinase (ppk) metals (Cd, Cu)

Comments on mechanisms of oxidative stress sensing, regulation, and defense

Reference

Trade-off between motility and oxidative stress defense

Martínez-García et al. 2014

Manganese (Mn) homeostasis

Banh et al. 2013

Accumulation of inorganic polyphosphate can endure environmental stresses KT2440 Oxygen and carbon dioxide ROS-detoxification genes (ahpC, Expression of detoxifying tension glutathione metabolism genes) enzymes KT2440 Diamide, hydrogen eda and edd (comprising the Entner– Evolutionary predominance of the peroxide Doudoroff pathway) ED pathway for sugar metabolism in P. putida rather than the EMP pathway KT2440 Nickel, cobalt sodB (superoxide dismutase), trxB Nickel/cobalt homeostasis, (thioredoxin reductase), trx2 antioxidant system (thiol(thioredoxin), ohr (organic disulfide homeostasis), hydroperoxide resistance protein), expression of detoxifying tpx (thiol peroxidase) enzymes KT2440 Phenol DNA mismatch repair enzymes Homologous recombination in the (MutS or MutL) presence of phenol Cd001 (isolated from Cadmium Alkyl hydroperoxide reductase Cadmium (Cd) homeostasis, soil contaminated (AhpC), thiol peroxidase, expression of outer membrane with Cd, Zn and Pb) gamma-glutamyltransferase, porins to maintain membrane outer membrane porins integrity, expression of (OprF/OprL, OprH/OprB) detoxifying enzymes KT2440 Hydrogen peroxide OxyR, ATP-dependent RecG Cooperative action of OxyR and helicase RecG during oxidative stress response KT2440 Naphthalene Pyrroloquinoline quinone (PQQ) Expression of detoxifying coenzyme, alkylhydroperoxidaseenzymes like protein KT2440 Bipyridyl and methyl RND efflux pumps Operating RND efflux pumps viologen KT2440 Root exudation Extracellular heme-peroxidase Expression of extracellular hemeperoxidase (PP2561; essential for competitive colonization) Iron homeostasis, expression of KT2440 Antibiotics aphC (NADH peroxidase), gor detoxifying enzymes (glutathione reductase), recA (DNA repair protein), fprB (ferredoxin-NADP+ reductase) KT2440 Nitrogen availability NtrC Regulation in nitrogen metabolism via NtrC-sensing and regulation of zwf-1 gene KT2440 Superoxide (PQ) FinR (novel redox-sensing Regulation of fpr by FinR transcriptional regulator) KT2440 Biofilm formation dsbA (disulfide bond family A redox Increase EPS production and protein) biofilm formation in the absence of dsbA KT2440 PQ, MD, CHP, As zwf-1 Dual-sensing regulator HexR HexR responding to both KDPG and oxidative stress KT2440 Osmotic stress fprB (ferredoxin-NADP+reductase) Roles of FprB under osmotic stress conditions KT2440 Superoxide stress SoxR transcriptional regulator Different regulation of SoxR in P. putida

Nikel et al. 2013

Follonier et al. 2013 Chavarría et al. 2013

Ray et al. 2013

Tavita et al. 2012 Manara et al. 2012

Yeom et al. 2012

Fernández et al. 2012

Godoy et al. 2010 Matilla et al. 2010

Yeom et al. 2010a

Yeom et al. 2010c

Yeom et al. 2010b Lee et al. 2009

Kim et al. 2008

Lee et al. 2007 Park et al. 2006

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Table 1 (continued) Strain

Oxidative stress response trigger(s)

Involved gene or gene product

Comments on mechanisms of oxidative stress sensing, regulation, and defense

Reference

KT2440

Superoxide and osmotic stress

Fpr (ferredoxin-NADP+reductase)

Lee et al. 2006b

KT2440

Hydrogen peroxide

2-Cys Prx-like protein (PpPrx)

KT2440

Copper, cadmium

mt-2

Water limitation, biofilm formation

AhpC, succinate dehydrogenase, cysteine desulfurase algD (GDP-mannose dehydrogenase)

KT2440

Polyhydroxyalkanoate metabolism

rpoS (stationary sigma factor)

KT2440

Root colonization

Pmp3 family protein, GSH_peroxidase, fatty acid isomerase, glutamyltransferase

KT2442

Hydrogen peroxide

OxyR, KatA, KatB, AhpC

KT2440

Chlorophenoxy herbicides

Fumarase C, L-ornithine N5oxygenase

KT2440

Hydrogen peroxide

ChrR (soluble quinone reductase)

KT2440

Phenol

AhpC, SodB, Tpx, Dsb

Corvallis

Root colonization, hydrogen peroxide

RpoS, CatB

KT2440

Methyl tert-butyl ether

AhpC, SodM, SodF

Corvallis

Root colonization, glucose MnSOD, FeSOD utilization

Removal of oxidative and osmotic stresses by FinR and the fpr gene product Chaperone to peroxidase functional switching Expression of detoxifying enzymes Effects of exopolysaccharide alginate on reduction of ROS accumulation Increases the stress tolerance of the rpoS mutant under polyhydroxyalkanoateaccumulating conditions Upregulation of stress adaptation and detoxification-related genes in rhizosphere-colonizing populations Expression of katA, katB, and ahpC by the transcription factor OxyR Expression of Fur (ferric uptake regulator)-dependent protein during treatment with herbicides Correlated with the hydrogen peroxide tolerance and levels of chrR expression Expression of detoxifying enzymes Induction of CatB during the stationary phase and increased sensitivity to oxidative stress in the RpoS mutant Expression of detoxifying enzymes Expression of detoxifying enzymes

CatB, and CatC) have been reported to play important roles in defense against oxidative stress generated by plants for which P. putida is capable of colonizing on the plant root surface, little is known about the regulation of catalase genes in the environmental strain (Katsuwon and Anderson 1989). OxyR controls the expression of peroxiredoxin AhpC, along with two major catalases (KatA and KatB) in P. putida (Hishinuma et al. 2006). Peroxiredoxin AhpC detoxifies hydroperoxide, and oxidized AhpC can be reduced by peroxiredoxin reductase (AhpF) for reactivation. AhpF is a homodimeric flavoenzyme that functions as a disulfide reductase to help reducing equivalent-dependent reduction (Poole 2005). This process requires NADH as the reducing equivalent; therefore, AhpC may be inappropriate for detoxifying large amounts of peroxide (Hishinuma et al. 2006; Poole and

An et al. 2011a Miller et al. 2009 Chang et al. 2009

Raiger-Iustman and Ruiz 2008

Matilla et al. 2007

Hishinuma et al. 2006

Benndorf et al. 2006

Gonzalez et al. 2005

Santos et al. 2004 Miller et al. 2001

Krayl et al. 2003 Kim et al. 2000

Ellis 1996). It has been suggested that AhpC removes low levels of hydrogen peroxide in E. coli. However, catalase (KatA) is the major scavenger of high levels of hydrogen peroxide (Seaver and Imlay 2001). In P. putida KT2442, the expression levels of ahpC, katA, and katB transcripts were relatively low in exponentially growing cells but were increased in the early stationary phase (Hishinuma et al. 2006). The transcription and activity of peroxiredoxin AhpC in P. putida are induced in the presence of various pollutants such as methyl tert-butyl ether, phenol, and benzoate (Cao and Loh 2008; Krayl et al. 2003; Santos et al. 2004). Interestingly, recent reports have suggested that 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) has dual functions, acting as a peroxidase or a molecular chaperone under oxidative stress in P. putida KT2440 (An et al. 2011a, b).

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Mechanisms of SoxR and OxyR regulation in P. putida are different from those in E. coli Defense mechanisms against oxidative stress are well characterized in E. coli and Salmonella enterica. Under superoxide and nitric oxide (NO) stress, the SoxR protein is activated when its redox-active clusters [2Fe–2S] are oxidized to the (Fe3+–Fe3+) form (Ding and Demple 2000; Hidalgo and Demple 1997; Hidalgo et al. 1997; Pomposiello and Demple 2001). SoxR is a transcriptional activator that induces the expression of soxS. SoxS is also a transcriptional activator that directly regulates the expression of a set of oxidative stress defense genes. These genes comprise the SoxRS regulon and include sodA (manganese superoxide dismutase), fpr (ferredoxin-NADP+ reductase), nfo (DNA repair endonuclease IV), fumC (fumarase C), and zwf (glucose-6-phosphate dehydrogenase) (Martin and Rosner 2001, 2003; Mukhopadhyay et al. 2004; Pomposiello and Demple 2001). Gene products of the SoxRS regulon engage in various activities that function to prevent and repair oxidative damage. Genomic analysis of P. putida KT2440 has shown that this strain expresses a SoxR homolog but lacks a SoxS homolog. SoxR of P. putida has 62 % amino acid identity with SoxR from E. coli, and both have four well-conserved cysteine residues that are essential for redox sensing (Park et al. 2006). The result of complementation experiments in which P. putida soxR was transformed into an E. coli soxR mutant demonstrated that P. putida SoxR is functional (Park et al. 2006). In the transformed E. coli, soxS transcription, which depends on SoxR, increases sixfold under paraquat treatment (Park et al. 2006). Moreover, SoxR is not involved in the induction of oxidative stress genes in P. putida. Exposure of P. putida to superoxide and nitric oxide stresses leads to induction of fpr, fumC-1, sodA, and zwf-1 genes (Park et al. 2006), a subset of the SoxR-dependent oxidative stress defense genes in E. coli (Pomposiello and Demple 2001). Interestingly, the expression pattern of fpr, fumC-1, sodA, and zwf-1 in the P. putida soxR mutant strain is similar to that of the wild-type organism under the same stress. Thus, these genes are induced independent of SoxR (Park et al. 2006). Additionally, compelling insights into the oxidative stress response of P. putida during exposure to oxidative stress generating agents can be obtained by transcription profiling (Yeom et al. 2010c, 2012). A study on the response of P. putida KT2440 to paraquat and cumene hydroxide (CHP) revealed that a set of genes including the SoxRS and OxyR regulons of E. coli is not uniformly induced by superoxide or hydrogen peroxide (Yeom et al. 2012). The expression of fpr is only increased in the presence of paraquat, while the other genes exhibit slight variations in expression. In the presence of CHP, katA, trxB, ahpC, ahpF, gor, and trx-2, which are involved in the OxyR regulon in E. coli, are highly expressed in P. putida (Yeom et al. 2012). Many of these upregulated

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genes (katA, trxB, ahpC, ahpF, and trx-2) are also induced during exposure to superoxide (Yeom et al. 2012). Analysis of gene sequences revealed that the soxR box in nonenteric Proteobacteria and Actinobacteria is located upstream of genes categorized as transporters, oxygenases, dehydrogenases, acetyl- or methyltransferases, and L-PSP ribonucleases (Dietrich et al. 2008). Overall, the results of these previous studies suggest that the mechanisms regulating superoxide and hydrogen peroxide stress through SoxR and OxyR activity in P. putida differ obviously from those in E. coli. OxyR is a transcriptional activator that regulates various genes induced during hydrogen peroxide-dependent oxidative stress defense (Lee et al. 2004; Imlay 2008; Zheng et al. 2001). The OxyR protein is produced constitutively and is oxidized by hydrogen peroxide. The oxidized form of OxyR binds to promoter regions of target genes and activates transcription by mediating protein–protein contact with RNA polymerase (Kullik et al. 1995; Pomposiello and Demple 2001; Wang et al. 2006). OxyR-activated genes have direct and indirect antioxidant functions. OxyR regulates the expression of two major catalases (KatA and KatB), peroxiredoxin AhpC, and thioredoxin reductase TrxB in P. putida (Fukumori and Kishii 2001; Hishinuma et al. 2006, 2008). Expression analysis on P. putida has revealed that oxyR and recG (DNA helicase) lie within the same operon (Yeom et al. 2012). Presence of oxyR-recG operon and its function for oxidative stress defense have also been reported in P. aeruginosa (Ochsner et al. 2000). Many OxyR regulon genes such as trxB, katA, aphC, hslO, trx-2, and PP0877 showed negligent levels of expression in the recG mutant under oxidative stress (Yeom et al. 2012). Purified OxyR and RecG can bind to the promoters of these genes, suggesting that RecG regulates other OxyR-controlled genes (Yeom et al. 2012). RecG participates in transcriptional regulation probably through its helicase function (Briggs et al. 2005; Yeom et al. 2012). RecG, along with single-strand binding protein (SSB), recognizes and binds to palindromic sequences located in the OxyR binding sites of the promoter regions of OxyR-regulated genes (Yeom et al. 2012). Upon binding, RecG unwinds the DNA structure using ATP as the energy source (Yeom et al. 2012). Thus, RecG activity was shown to be necessary for transcription of the OxyR regulon (Yeom et al. 2012) (Fig. 1). This finding describes a novel bacterial transcriptional mechanism by RecG helicase with OxyR in Pseudomonas.

Novel redox-sensing transcriptional regulators in P. putida Aerobic metabolism inevitably generates oxygen intermediates, called ROS, which can damage cellular components such as proteins, lipids, and nucleic acids (Imlay 2003). Redoxsensing transcriptional regulators play major roles in response to oxidative stress by regulating the expression of genes

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involved in defense against oxidative stress (Paget and Buttner 2003). SoxR and OxyR are major oxidative stress regulators in E. coli and S. typhimurium (Farr and Kogoma 1991). However, recent studies have demonstrated that the SoxR regulons in nonenteric bacteria have different roles from those of homologous proteins in the E. coli SoxRS paradigm (Dietrich et al. 2008; Park et al. 2006). The principal function of SoxR in many bacteria, including Proteobacteria and Actinobacteria, might be the control of secondary metabolite production and putative dehydrogenase expression rather than superoxide defense or detoxification (Dietrich et al. 2008). Several studies identified CyeR and QorR in Corynebacterium glutamicum (Ehira et al. 2009, 2010) and PerR and OhrR in Bacillus subtilis (Helmann et al. 2003) as novel redox-sensing transcriptional regulators involved in the response to oxidative stress. The genome of P. putida KT2440 has two important redox-sensing regulators, FinR and HexR, which are known to control the expression of the fpr and zwf-1 genes, respectively (Kim et al. 2008; Lee et al. 2006b; Yeom et al. 2010b) (Fig. 1). Interestingly, these genes are under the control of the SoxRS system in E. coli (Greenberg et al. 1990). The superoxide stress-sensing regulator FinR Many proteobacteria have FinR homologs, annotated as putative LysR-type proteins in their genomes. The P. putida KT2440 genome contains FinR, which consists of 308 amino acids and is positioned adjacent to fprA. FinR binds directly to the promoter region of fpr in P. putida KT2440 (Yeom et al. 2010b). FinR is essential for the induction of fpr (ferredoxin-NADP+ reductases) during exposure to superoxide stress in P. putida KT2440 (Fig. 1). Redox-sensing proteins harbor conserved cysteine residues to control their regulation (Green and Paget 2004). FinR in P. putida has three wellconserved cysteine residues at positions 150, 239, and 289 and two additional cysteine residues (Yeom et al. 2010b). Strains with mutations in these conserved cysteine residues, which were individually mutated to serines, showed transcription activities similar to those of the wild-type strain (Yeom et al. 2010b). Unlike other redox-sensing transcriptional regulators such as SoxR and OxyR, the five cysteines in FinR are not associated with FinR activation. The absence of ferrous iron, treatment with ferric iron, and deletion of fdxA (ferredoxin) are involved in the regulation of fpr (Yeom et al. 2010b). This experimental evidence suggests that FinR has a unique redox-sensing mechanism, different from the well-characterized mechanisms of OxyR and SoxR. Ferredoxin-NADP+ reductases (encoded by fpr) are enzymes that act as mediators in the reversible transfer of electrons between NADP(H) and one-electron carriers such as ferredoxin (Aliverti et al. 2008; Giró et al. 2006; Schröder et al. 2003). When ferredoxin is reduced through the activity of Fpr proteins, it can donate an electron to various enzymes. These enzymes participate in a number of cellular systems,

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including oxidative stress defense (Wackett 2003). P. putida possesses two different types of ferredoxin reductases: FprA and FprB (Yeom et al. 2009a, b). The exposure of P. putida to oxidative and osmotic stresses induces fprA expression (Lee et al. 2006b; Lee et al. 2007; Yeom et al. 2009a, b). Therefore, fprA mutant strains show high sensitivity towards both of these stresses (Lee et al. 2006b). Induction of fprA requires FinR, a transcriptional factor located near fprA (Yeom et al. 2010b). In contrast to fprA, fprB is induced only by osmotic stress, and fprB mutant strains are exceptionally susceptible to highly osmotic conditions (Lee et al. 2007). Induction of the fprB gene does not require FinR or LuxR, a transcriptional factor adjacent to fprB, indicating the existence of unknown regulators. The dual-sensing regulator HexR In P. putida, there are three homologs of zwf: zwf-1, zwf-2, and zwf-3. Among these three homologs, zwf-1 plays a major role in glucose metabolism by converting glucose 6-phosphate to 6-phosphogluconate (6PG) (Kim et al. 2008). In the presence of glucose or gluconate, zwf1 is highly expressed, while zwf-2 and zwf-3 expression levels remain low (Kim et al. 2008). In addition, zwf-1 is involved in the oxidative stress response (Giró et al. 2006; Li and Demple 1994; Park et al. 2006). Treatment with ROS-producing agents such as paraquat, menadione, arsenic, CHP, and nitric oxide dramatically elevates the expression of zwf-1 but does not cause substantial changes in the expression of zwf-2 and zwf-3 (Park et al. 2006). Expression of zwf-1 is regulated by the transcriptional factor HexR in P. putida (Kim et al. 2008) (Fig. 1). HexR regulates the gene encoding glyceraldehyde-3phosphate dehydrogenase (gap, encoded by PP_1009) and two operons, including the zwf, pgl (6phosphogluconolactonase), and eda operon and the edd (6phosphogluconate dehydratase), glk (glucokinase), and gltR (DNA-binding response regulator) operon (Del Castillo et al. 2008; Hager et al. 2000; Petruschka et al. 2002; Temple et al. 1994). HexR directly binds to the zwf-1 promoter and acts as a repressor (Kim et al. 2008). Experimental results suggest that HexR is a dual-sensing protein that responds to both 2-keto-3deoxy-6-phosphogluconate (KDPG) and oxidative stress (Kim et al. 2008). Previously, 6PG was shown to serve as an inducer of the HexR operon (Petruschka et al. 2002; Wu and Weiss 1992). However, KDPG, not 6PG, seems to be the real inducer that hinders HexR from binding to the zwf-1 promoter (Kim et al. 2008). In an experiment with edd mutants, which do not have the ability to synthesize KDPG because of a mutation in 6PG dehydratase, and eda mutants, which exhibit accumulation of KDPG in cells because of a mutation in KDPG aldolase, edd mutants showed low expression of zwf1, whereas eda mutants exhibited significantly increased zwf-1 expression (Kim et al. 2008). Interestingly, KDPG is not required for the induction of zwf-1 during conditions of oxidative stress. edd mutant strains exposed to oxidative stress

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induce the expression of zwf-1 without KDPG (Kim et al. 2008). In addition, in vitro studies have shown that HexR binding to the zwf-1 promoter region is weakened by menadione and CHP (Kim et al. 2008). These findings imply that HexR can directly sense oxidative stress through an unidentified pathway.

Effects of carbon and nitrogen metabolism on oxidative stress Various xenobiotic compounds, such as toluene, xylenes, styrene, phenol, and naphthalene, are present in environments due to increasing industrial activities (Ju and Parales 2010). When bacteria degrade xenobiotic compounds, endogenous oxidative stress could be generated owing to defective oxidation reactions, membrane damage, and catalytic uncoupling (Domínguez-Cuevas et al. 2006; Lee 1999; Sikkema et al. 1995). When xenobiotic compounds across the cell membranes, cells undergo increasing loss of ions, ATP, other cellular metabolites, and energetic perturbation (Sikkema et al. 1995). Many oxygenases play a critical role in biodegradation of aromatic and xenobiotic compounds (Lee 1999; Díaz et al. 2001). Oxygenases can actually produce ROS by uncoupling of their catalytic mechanism (Lee 1999). Exposure to ROS by toxic chemicals can result in disrupting the cell membrane, proteins, and lipids, which leads to cell death (De Smet et al. 1978; Ramos et al. 2002). Generation of high levels of endogenous ROS may induce DNA damages and increasing mutagenesis during the process of biodegradation (Gibson and Parales 2000; Pérez-Pantoja et al. 2013). Thus, it is important to understand the effects of carbon and nitrogen metabolism on oxidative stress when P. putida consumes xenobiotic compounds as carbon or nitrogen sources. The Entner–Doudoroff pathway is required to cope with oxidative stress Glycolysis is the metabolic pathway that converts glucose into pyruvate. The production of ATP and reducing power [NAD(P)H] are involved in the glycolytic pathway (Romano and Conway 1996). The Embden–Meyerhof– Parnas (EMP) pathway is the universal glycolytic pathway in both eukaryotes and prokaryotes (Romano and Conway 1996). However, prokaryotes have diverse glucose metabolism, such as Entner–Doudoroff (ED) pathway and phosphoketolase pathway (Conway 1992; Romano and Conway 1996). The EMP and ED pathways are the most archetypal bacterial glycolytic pathways (Conway 1992; Flamholz et al. 2013; Fuhrer et al. 2005). The EMP and ED routes yield different levels of ATP and NAD(P)H (Conway 1992; Romano and Conway 1996). This energetic difference can affect to select pathway in evolutionary process (Bar-Even et al. 2010). The prevalence of the ED pathway in environmental bacteria might be due to less enzymatic protein

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requirements for converting the same amount of glucose than the EMP pathway (Flamholz et al. 2013). Glucose catabolism in P. putida occurs through the ED pathway because of the absence of the critical glycolytic enzyme 6phosphofructokinase (PFK) (Del Castillo et al. 2007; Chavarría et al. 2012). From an evolutionary perspective, the ED pathway confers a selective advantage for P. putida over organisms possessing a functional EMP pathway (Chavarría et al. 2013). The ED pathway is more efficient for producing NADPH, which could improve cellular reducing power to repair oxidative stress damages (Kim et al. 2008). In P. aeruginosa, closely related to P. putida, the ED pathway is also essential in the presence of a steady level of hydrogen peroxide (Deng et al. 2014). It was proven that inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, encoded by the gap gene) by ROS through the oxidation of catalytic active cysteine residue induces glycolysis and pentose phosphate pathway in P. aeruginosa and Staphylococcus aureus (Deng et al. 2013, 2014). Elevated intracellular levels of KDPG under the same condition cause dissociation of glycolytic repressors HexR from zwf promoter, which enhances production of NADPH in P. aeruginosa (Deng et al. 2014). This observation is consistent with the fact that release of HexR repressor is important for increasing NADPH production in P. putida under oxidative stress (Kim et al. 2008). Taken together, these data suggest that the ED pathway regulates intracellular energy and redox status, resulting in high tolerance to oxidative stress in the Pseudomonas species. NtrC-dependent sensing of nitrogen availability is essential for the cellular response against oxidative stress Bacteria can activate nitrogen assimilation pathways when grown in nitrogen-limited environments (Leigh and Dodsworth 2007; Merrick and Edwards 1995). Under nitrogen-limited conditions, NtrC is a global activator of nitrogen-controlled genes and allows cells to uptake ammonia (Gyaneshwar et al. 2005; Ninfa et al. 2000; Zimmer et al. 2000). NtrC is a twocomponent signal transduction system that is controlled by NtrB via phosphorylation (Arcondéguy et al. 2001; Ninfa and Jiang 2005). The zwf-1 gene product, glucose-6-phosphate dehydrogenase, acts as a defensive response against oxidative stress (Lundberg et al. 1999; Pomposiello and Demple 2001; Kim et al. 2008). The putative NtrC binding site in the promoter region of zwf-1 has been established and was shown to be located upstream of the HexR binding site in P. putida KT2440 (Yeom et al. 2010c). Nitrogen availability is often limited in soil ecosystems. Interestingly, under nitrogenlimited oxidative stress conditions, NtrC represses zwf-1 expression, causing P. putida to become more sensitive to oxidative stress (Hervás et al. 2008; Kim et al. 2008). Moreover, deletion of NtrC from P. putida results in increased resistance to oxidative stress (induced by menadione and CHP) under conditions of poor nitrogen supply, nevertheless leading to

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significantly reduced growth in the ntrC mutant (Yeom et al. 2010c). The derepression of several sets of genes [oxidative stress genes (katE, catalase; radC, DNA repair protein), cell wall biosynthesis genes (murI, glutamate racemase), and regulatory genes (PP2070, PP1307, PP3603, and PP4734)] as well as zwf-1 may facilitate the acquisition of high resistance to oxidative stress in the ntrC mutant, as demonstrated by transcriptional profiling comparing nitrogen-limited and nitrogen-rich oxidative stress conditions (Yeom et al. 2010c). Consequentially, NtrC-dependent sensing of nitrogen availability is another requirement for cellular defense mechanisms to oxidative stress.

Antibiotic-mediated oxidative stress and iron homeostasis In aerobic habitats, bactericidal antibiotics cause oxidative stress, which leads to cell death (Dwyer et al. 2009; Kohanski et al. 2007, 2008). Antibiotics generate superoxide and hydrogen peroxide, leading to inactivation of various enzymes and subsequently inhibiting cellular growth (Imlay and Linn 1988). Moreover, these two oxidants form the hydroxyl radical through the Fenton reaction (Touati 2000). Unincorporated intracellular iron transfers an electron to hydrogen peroxide, and intracellular reductants, including cysteine and reduced flavins, then reduce the oxidized iron back to its ferrous form (Touati 2000). The Fenton reaction is cyclic within cells because cellular reductants reduce ferric iron to ferrous iron (Touati 2000). The hydroxyl radical is a destructive oxidant that causes DNA damage and leads to cell death (Imlay and Linn 1988). Thus, factors that can promote the Fenton reaction stimulate the cytotoxic activity of antibiotics (Touati 2000). Antibiotics increase the level of ROS within cells, allowing accumulation of oxidative stress-inducing agents (Dwyer et al. 2009; Kohanski et al. 2007, 2008). Exposure to ampicillin, norfloxacin, and gentamycin induces the expression of the oxidative stress genes aphC, gor, and recA. The aphC (NADH peroxidase) and gor (glutathione reductase) genes are members of the OxyR regulon (Yeom et al. 2010a). recA is involved in repairing DNA damage caused by hydrogen peroxide (Carlsson and Carpenter 1980; Park et al. 2005). In addition, antibiotic treatment facilitates the activities of catalase and peroxidase. These findings indicate that antibiotics generate oxidative stress in P. putida (Yeom et al. 2010a) (Fig. 2). Iron availability is a critical factor in the mechanism of antibiotic-mediated cell death (Yeom et al. 2010a). In P. putida, oxidative stress caused by antibiotics is dependent on iron concentration. Ampicillin treatment in P. putida causes a dramatic increase in the expression of oxidative stress genes when iron is added to the growth medium (Yeom et al. 2010a). Previously, FprB was shown to function as a ferric reductase in P. putida (Yeom et al. 2009a, b). Therefore, FprB facilitates

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the Fenton reaction and enhances antibiotic activity. Additionally, the frpB mutant exhibits higher survival and less DNA damage in response to ampicillin treatment as compared to the wild-type strain (Yeom et al. 2010a). On the other hand, FprB overexpressing strains show heightened sensitivity toward antibiotic stress (Yeom et al. 2010a). Correspondingly, cyanide, another molecule that promotes the Fenton reaction, boosts the effects of antibiotics (Woodmansee and Imlay 2002; Yeom et al. 2010a). However, cells treated with desferrioxamine, an iron chelator that impedes the Fenton reaction, show enhanced survival under antibiotics (Yeom et al. 2010a) (Fig. 2). Microarrays conducted in the presence of ampicillin in both P. putida and P. aeruginosa have shown that antibiotic treatment induces the upregulation of genes involved in oxidative defense, including ahpC and DNA repair genes such as uracil DNA glycosylase (ung); these genes are not induced in the ferric reductase mutant (Yeom et al. 2010a). In Pseudomonas, NADH levels and iron concentrations are important factors modulating the activity of antibiotics, and iron homeostasis has significant roles in bacteria cell survival following treatment with antibiotics (Yeom et al. 2010a).

Conclusions and future perspectives Defense against oxidative stress is an important feature of bacterial cell survival in habitats where exogenous and endogenous ROS are generated. Through several recently conducted studies (Chavarría et al. 2013; Hishinuma et al. 2006; Kim et al. 2008; Park et al. 2006; Yeom et al. 2009a, b, 2010a, b, c, 2012), our understanding of the oxidative stress system in P. putida KT2440 has been expanded. The oxidative stress response in P. putida is unique, differing from E. coli. In P. putida, the E. coli SoxRS regulon genes fpr and zwf are modulated by other regulators, FinR and HexR, respectively (Kim et al. 2008; Yeom et al. 2010b). OxyR is another regulator of P. putida that induces oxidative stress genes in cooperation with the helicase RecG (Yeom et al. 2012). In addition to OxyR, the well-studied peroxide-responsive regulators PerR and OhrR exist in E. coli (Dubbs and Mongkolsuk 2012). OxyR and PerR primarily function to sense hydrogen peroxide; however, OhrR is able to respond to organic peroxide and sodium hypochlorite as well (Dubbs and Mongkolsuk 2012). PerR plays a role as peroxideresponsive repressor and is considered a member of the Fur family of meta-responsive transcriptional regulators (Antelmann and Helmann 2011). The LysR family transcriptional regulator (PputW619_2615, 294aa) in the P. putida W619 strain has 61 % amino acids identity with E. coli PerR, but no studies describing the role of this regulator have been published. Instead of PerR as a transcriptional regulator, several studies have investigated the role of Fur, a ferric

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Fig. 2 Proposed model of antibiotic-mediated oxidative stress and iron homeostasis. Oxidative stress can be generated during treatment with antibiotics. Additionally, iron homeostasis in Pseudomonas is crucial for bacterial cell survival during treatment with antibiotics

uptake regulator, in maintaining iron homeostasis (Heim et al. 2003; Venturi et al. 1995). Under conditions of iron deprivation, iron starvation sigma factor (PvdS) and fumarate hydratase (FumC), which are regulated by Fur protein, are upregulated in P. putida KT2440. Additionally, the promoter region of pfrl (pseudomonas ferric regulator) has a Fur-box consensus sequence and is regulated by Fur in P. putida WCS358 (Heim et al. 2003; Venturi et al. 1995). OhrR is a transcriptional repressor of the MarR family of proteins (Dubbs and Mongkolsuk 2012). Most P. putida strains harbor OhrR-like protein (a MarR family transcriptional regulator) in their genome sequences; this protein exhibits high amino acid sequence similarity (over 70 %) compared to well-known OhrR proteins in other bacteria. To date, no studies have investigated the function or regulation of OhrR in P. putida. Along with OhrR, OspR in P. aeruginosa and MgrA and SarZ in S. aureus have been shown to act as oxidant-responsive MarR family regulator (Kaito et al. 2006; Lan et al. 2010; Luong et al. 2003). Therefore, a detailed understanding of the mechanisms through which these regulators function in P. putida will further our understanding of adaption and evolution to various environmental stresses. An overview of oxidative stress and cellular response in P. putida is illustrated in Fig. 1. We are able to understand the basic process how P. putida defends itself against ROS generated by various conditions. P. putida possesses multidirectional mechanisms of oxidative stress sensing, regulation, and defense. However, these mechanisms of oxidative stress response are not fully understood. Identification of target genes of SoxR and their physiological functions in P. putida has not

yet been conducted. To improve the mechanisms of sensing and regulation of oxidative stress, SoxR and OxyR regulon in P. putida must be fully established. Other oxidative stress response regulators, such as the well-known PerR and OhrR and their regulon, are not characterized in P. putida. It is also worth investigating the interacting regulatory network between oxidative stress and other stress response systems, such as desiccation, cold or heat shock, and starvation. These further studies will enhance the understanding of the physiology and ecology of P. putida during its life cycle. New findings on oxidative stress mechanisms of P. putida, as a model soil bacterium, will broaden our knowledge of bacterial regulatory mechanisms and potentiate the biotechnological application of P. putida in the future.

Acknowledgments This work was supported by the Mid-career Researcher Program through an NRF grant (2014R1A2A2A05007010) funded by the Ministry of Science, ICT, & Future Planning (MSIP).

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