cytochrome d but not cytochrome o rescues the toluidine blue

JOURNAL OF BACTERIOLOGY, Jan. 2010, p. 391–399 Vol. 192, No. 20021-9193/10/$12.00 doi:10.1128/JB.00881-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Cytochrome d But Not Cytochrome o Rescues the Toluidine BlueGrowth Sensitivity of arc Mutants of Escherichia coli�

Adrian F. Alvarez, Roxana Malpica, Martha Contreras, Edgardo Escamilla, and Dimitris Georgellis*Departamento de Genetica Molecular, Instituto de Fisiología Celular, Universidad Nacional Autonoma de Mexico,

04510 Mexico City, Mexico

Received 6 July 2009/Accepted 29 October 2009

The Arc (anoxic redox control) two-component signal transduction system, consisting of the ArcB sensorkinase and the ArcA response regulator, allows adaptive responses of Escherichia coli to changes of O2availability. The arcA gene was previously known as the dye gene because null mutants were growth sensitiveto the photosensitizer redox dyes toluidine blue and methylene blue, a phenotype whose molecular basis stillremains elusive. In this study we report that the toluidine blue O (TBO) effect on the arc mutants is lightindependent and observed only during aerobic growth conditions. Moreover, 16 suppressor mutants withrestored growth were generated and analyzed. Thirteen of those possessed insertion elements upstream of thecydAB operon, rendering its expression ArcA independent. Also, it was found that, in contrast to cythocromed, cythocrome o was not able to confer toluidine blue resistance to arc mutants, thereby representing anintriguing difference between the two terminal oxidases. Finally, a mechanism for TBO sensitivity and resis-tance is discussed.

The Arc (anoxic redox control) two-component system is akey element in the complex transcriptional regulatory networkthat allows facultative anaerobic bacteria, such as Escherichiacoli, to sense various respiratory growth conditions and adjusttheir gene expression accordingly (42). This system comprisesthe transmembrane sensor kinase ArcB (32) and the cytoplas-mic response regulator ArcA (34). Under reducing conditionsof growth, ArcB autophosphorylates at the expense of ATP, aprocess enhanced by various anaerobic metabolites, such aslactate and acetate (19, 49), and transphosphorylates the re-sponse regulator ArcA (22, 37). The phosphorylated form ofArcA, ArcA-P, in turn, regulates negatively the expression ofmany operons that code for enzymes involved in aerobic me-tabolism and activates the expression of genes encoding pro-teins involved in fermentative metabolism (40, 42). Under ox-idizing conditions, the kinase activity of ArcB is inhibited bythe quinone electron carriers through the oxidation of Cys 180and Cys 241, which participate in intermolecular disulfide bondformation (20, 41), allowing dephosphorylation of ArcA (18, 45).

Before the identification and characterization of Arc as atwo-component system, the arcA gene was known as the dyegene because it was observed that mutation in this gene con-ferred sensitivity to dyes such as toluidine blue O (TBO) andmethylene blue (8). Later, it was observed that mutants carry-ing mutations in arcB and in the cytochrome d-encoding operon,cydAB, exhibit a similar TBO-sensitive phenotype (15, 32).However, the causes of the dye phenotype in these mutantsremain so far unknown. It is of interest to mention that TBOand methylene blue are photosensitizers that in the presence oflight are able to instigate redox reactions producing reactive

oxygen species (ROS), which can damage nucleic acids andenzymes, leading to cell death (57). The utility of these pho-tosensitizers against a range of bacterial strains has been re-ported extensively (59). In a recent study, it was proposed thatTBO results in a significant increase of ROS in an arcA mutantand that this increase in ROS is the cause of the dye phenotype(50). Moreover, the heterologous expression of poly 3-hy-droxybutyrate was shown to be able to suppress the dye sensi-tivity in arcA mutants by diminishing O2 consumption and theproduction of ROS (50).

In this work, we examined the effect of oxygen and light andalso the effect of the antioxidants sodA, katG (hydroperoxidaseI [HPI]), katE (HPII), Hmp (flavohemoglobin), and AhpCF(alkyl hydroperoxide reductase) and the carotenoid biosyn-thetic genes of Erwinia herbicola (46) on the TBO-dependentphenotype of arc mutants. Furthermore, we generated andcharacterized several suppressor mutant strains with abolishedTBO-dependent growth defects. Our results demonstrate thatcytochrome d, but not cytochrome o, serves as a key element inthe protection of the cells against TBO by generating an anoxicintracellular environment. Finally, aerobic expression of cydABin the isolated suppressor mutants was found to be achieved bythe introduction of insertion sequences (IS) upstream of thecydAB operon, rendering its expression arc independent.

MATERIALS AND METHODS

Bacterial strains, plasmids, and oligonucleotides. The strains and plasmidsused in this study are listed in Table 1. Strain IFC5001 was constructed by P1transduction of the h-ns::Kanr allele derived from the Keio collection (3) intostrain ECL5020 (arcA::Tetr) (21). Strains IFC5002, IFC5003, and IFC5004 wereconstructed by P1 transduction of the katG::Kanr, katE::Kanr and sodA::Kanr

alleles, respectively, derived from the Keio collection into strain MC4100. Plas-mid pMX513 was constructed by cloning a 2.3-kb SmaI fragment from pDT1.5(54), containing the sodA gene under its own promoter, into the SmaI site ofpACT3 (17). The genomic libraries of the suppressor strains were constructed bySau3A1 restriction of the genomic DNA and cloning the obtained fragments intoBamHI-digested pTZ19R (Fermentas Inc, Glen Burnie, MD).

* Corresponding author. Mailing address: Departamento de Ge-netica Molecular, Instituto de Fisiología Celular, Universidad Nacio-nal Autonoma de Mexico, 04510 Mexico D. F., Mexico. Phone: 52 555622 5738. Fax: 52 55 5622 5611. E-mail: [email protected].

� Published ahead of print on 6 November 2009.

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For the PCR amplification of the cydAB promoter region, the reverse primercydpromRev (5�-CCCGGATCCCATCATGACTCCTTGCTCATCGC-3�) wasused with the forward primers cydpromFw700, 5�-GGAATTCTGGCAAGCGTAGCGCATCAGG-3; cydpromFw1600, 5�-CGGAATTCGTGTCCTGGTCCCTACACCT-3�; cydpromFw2100, 5�-CGGAATTCGCCATGCCCGTGAGATCGA-3�; and cydpromFw4000, 5�-CGGAATTCTGCCTGGGCGCAATGGC-3�,yielding products of 700, 1,600, 2,100, and 4,000 bp, respectively, when used withgenomic DNA from a wild-type MC4100 strain (10) as a template.

The genes hmp and ahpCF were amplified by PCR using chromosomal DNAof strain MC4100 (10) as a template and the following primers: hmp-up (5�-CCCCATATGCTTGACGCTCAAAC-3�) and hmp-down (5�-CCCAAGCTTGCCGGATGTTTCCATCC-3�), yielding a 1,235-bp product; and ahpCF-up (5�-CCCCATATGTCCTTGATTAACACC-3�) and ahpCF-down (CCCAAGCTTCGGCGGCTAAGCAATTGC-3�), yielding a 2,426-bp product. Both PCR productswere digested with NdeI-HindIII and cloned between the NdeI-HindIII sites ofplasmid pMX020 (45) under the control of the arabinose promoter, generatingthe Hmp and AhpCF overexpressing plasmids pMX511 and pMX512, respec-tively.

Growth conditions. Escherichia coli strains were routinely grown in Luria-Bertani (LB) medium or tryptone broth (10 g liter�1 tryptone, 8 g liter�1 NaCl)at 37°C. Unless otherwise specified, TBO sensitivity was tested on tryptone-TBO-agar (TTA) plates (10 g liter�1 tryptone, 8 g liter�1 NaCl, 0.2 mg ml�1 TBO [8],and 15 g liter�1 of Bacto agar). When necessary, ampicillin, kanamycin, tetra-cycline, and chloramphenicol were used at final concentrations of 100, 50, 12.5,and 34 �g ml�1, respectively. To induce expression of the ara promoter-con-trolled genes, arabinose was added to TTA plates to a final concentration of0.13 mM.

Determination of carotenoid content and catalase and SOD activities. Todetermine the catalase and superoxide dismutase (SOD) activities, the strain ofinterest was grown aerobically in LB medium to an optical density at 600 nm(OD600 of �0.6). Cells were harvested and washed with 100 mM cold HEPESbuffer (pH 7.2) and disrupted by sonication, and unbroken cells and debris wereeliminated by centrifugation at 9,000 � g for 10 min at 4°C. Soluble proteins (6to 30 mg) were separated on 8% native polyacrylamide gels at 4°C and at 150 V

for 1 h or 2.5 h for the determination of SOD or catalase, respectively. Catalaseactivity was detected as described previously (12), whereas a modified photo-chemical method of Beauchamp and Fridovich (4) was used to locate SODactivities on gels. Briefly, the gel was first soaked in 25 ml of 1.23 mM nitrobluetetrazolium for 15 min and then in 25 ml of a solution containing 28 mMN,N,N�,N��-tetramethylethylenediamine and 28 �M riboflavin for another 15min. Both incubations where carried out in the dark. Subsequently, the gel wasexposed to light, to initiate the photochemical reaction, until the bands wereclearly distinguishable. Carotenoid content was determined by growing the cellsin LB broth at 37°C with constant agitation. The cells were collected by centrif-ugation at 4,000 � g for 15 min. The pellet fraction was weighed, suspended ina 1:1 (vol/vol) mixture of chloroform-methanol, and vortexed for 2 min. Thesuspension was separated by centrifugation at 4,000 � g for 5 min, and theorganic layer was used for measure of absorption spectra between 370 and 520nm. The maximum wavelength for �-carotene in chloroform-methanol solventwas 460 nm, as previously reported (5).

Membrane preparation and spectral analysis of cytochrome d. For the quan-tification of cytochrome d each strain was aerobically grown in 1.5 liter LBmedium to middle-exponential phase (an OD600 of approximately 0.6). Cellswere harvested and washed twice with 50 mM cold phosphate buffer (pH 7)containing 5 mM CaCl2 and 5 mM MgCl2. The cells were disrupted by sonica-tion. Unbroken cells and debris were eliminated by centrifugation at 9,000 � gfor 10 min. Membranes were prepared by centrifugation at 125,000 � g for 40min and thereafter washed twice with phosphate buffer. Membranes were sus-pended in phosphate buffer, and protein concentrations were measured by amodification of the Lowry method (16).

Conventional optical absorption difference spectra were recorded at roomtemperature in an SLM-Aminco DW 2000 spectrophotometer (SLM Instru-ments Inc.) using 1-cm light path cuvettes. Samples were reduced with solidsodium dithionite, whereas references were oxidized with air (1 min of vortexagitation). The concentration of cytochrome d in membranes was calculatedfrom the spectrum difference (dithionite-reduced samples minus air-oxidizedreferences) at room temperature, by using the wavelength pairs 630 and 650 nmand an extinction coefficient (E) of 19 mM�1 cm�1 (26).

TABLE 1. E. coli strains and plasmids

E. coli strain orplasmid Genotype or relevant characteristics Reference or

source

StrainMC4100 F araD139 (argF-lac)U169 rpsL150 relA1 flbB5301 deoC ptsF25 rbsR 10PC35 MC4100 but �arcA::Kanr 11ECL5020 MC4100 but �arcA::Tetr 21ECL5013 MC4100 but �arcB::Kanr 38ECL937 MC4100 but �cydAB::Kanr 33ECL936 MC4100 but �cyo::Kanr 33IFCS1-15 PC35, spontaneously TBO resistant This studyIFC5001 MC4100 but �arcA::Tetr, �h-ns::Kanr This studyIFC5002 MC4100 but �katG::Kanr This studyIFC5003 MC4100 but �katE::Kanr This studyIFC5004 MC4100 but �sodA::Kanr This study

PlasmidspTZ19R Cloning plasmid, Ampr Fermentas Inc.pMN2 Carries arcA gene, Ampr 34pBB25 Carries arcB gene, Ampr 32pNG2 Carries cydAB genes, Tetr 25pRG110 Carries cyoABCD genes, Ampr 2pAMkatE72 Carries katE gene, Ampr 58pBT22 Carries katG gene, Ampr 55pDT1.5 Carries sodA gene, Ampr 54pPL376 Carries carotenoid biosynthetic genes from Erwinia herbicola, Ampr 46pACT3 Expression plasmid, Ptac ori p15A, Cmr 17pMX020 Carries ArcB521–778 under the control of ara promoter, Ampr 45pMX511 Carries hmp under the control of ara promoter, Ampr This studypMX512 Carries ahpCF under the control of ara promoter, Ampr This studypMX513 pACT3 derivate carrying sodA, Cmr This studypMX515 pTZ19R derivate, IFCS1 genomic library plasmid, Ampr This studypMX515Eco EcoRI-EcoRI fragment of pMX515 cloned in pTZ19R This studypMX516 pTZ19R derivate, IFCS13 genomic library plasmid, Ampr This study

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RESULTS

ROS production is not the principal cause for the TBO-dependent growth defect of the arc and cydAB mutants. Dele-tion of the arcA, arcB, or cydAB genes has previously beenreported to result in an increased growth-sensitive phenotypein the presence of the redox dyes TBO or methylene blue (8,15, 32). In addition, it is well known that in the presence oflight, these photosensitizer dyes generate ROS that damagenucleic acids and proteins, thereby leading to cell death (57).Therefore, to test whether ROS cause the TBO-dependentgrowth-sensitive phenotype, the arcA, arcB, and cydAB mu-tants (11, 33, 38) were plated out on TTA plates and incubatedin the presence of light or in darkness. It was found that noneof the three mutants was able to grow in either condition (Fig.1A and B), indicating that either ROS do not cause the above-described phenotype or that TBO-dependent ROS productionoccurs even in darkness. To differentiate between the twopossibilities, cultures of a wild type and its isogenic arcA mu-tant strain were challenged with 0.05 mg/ml TBO for 30 min inthe presence of light or in darkness, and the activity of katG-encoded HPI, an OxyR-induced protein, was monitored. Cu-riously, a significant TBO-dependent increase of HPI activity,

indicative of ROS production, was observed with both strainswhen incubated in the presence of light but also in darkness(Fig. 1C). Therefore, we argued that if ROS were the principalcause for the TBO-dependent growth defect of the above-described mutants, overproduction of the antioxidant enzymesSodA (54), HPI (55), and HPII (58) or the carotenoid biosyn-thetic proteins of Erwinia herbicola (46) may protect the mu-tant cells and suppress their growth defect. To test this, plas-mids carrying the above-described genes were transformedinto a �arcA::kan mutant strain (11) and plated out on TTAplates. Although all the above-mentioned plasmid-borne geneswere able to overexpress the corresponding activity (Fig. 2A toC), not one was able to suppress the dye phenotype of the arcAmutant strain (data not shown). Also, the simultaneous over-expression of katG or katE and sodA that act sequentially toinactivate ROS, did not have any effect on the growth of themutants in the presence of TBO. Moreover, deletion of katG,katE, or sodA did not result in a TBO-dependent growth-sensitive phenotype of the respective mutant strains (data notshown). Finally, the possible suppressing effect of Hmp (fla-vohemoglobin) and AhpCF (alkyl hydroperoxide reductase),

FIG. 1. Effect of light and darkness on the TBO growth defect ofthe different E. coli mutant strains and TBO-dependent katG expres-sion. A wild type and the arc and cytochrome mutant strains (1,MC4100 [wild type]; 2, PC35 [�arcA]; 3, ECL5013 [�arcB]; 4, ECL937[�cyd]; and 5, ECL936 [�cyo]) were inoculated on tryptone agar platesin the presence (left) or absence (right) of 0.1 mg ml�1 of TBO andincubated overnight at 37°C under aerobic growth environments.Plates were incubated in the dark (A), or they were incubated underincidence of white light (B). (C) Strains MC4100 (lanes 1 to 3) andPC35 (�arcA) (lanes 4 to 6) were grown on tryptone broth. At anOD600 of 0.5, the cultures were split into three aliquots; one served asa control (lane 1 and 4), whereas the others were challenged for 30 minwith 0.05 mg ml�1 of TBO in darkness (lanes 2 and 5) or under theincidence of white light (lanes 3 and 6). Protein (30 �g) were loaded ineach lane to determine HPI catalase activity.

FIG. 2. Determination of catalase and SOD activities and carote-noid content. (A) HPI and HPII catalase activities of the followingstrains: MC4100 (lane 1); PC35 (�arcA) (lane 2); PC35 carrying thekatG-expressing plasmid pBT22 (lane 3); PC35 carrying the katE-expressing plasmid pAMkatE72 (lane 4); PC35 carrying pBT22 and thesodA-expressing plasmid pMX513 (lane 5); and PC35 carrying pAMkatE72and pMX513 (lane 6). A total of 30 �g of protein was loaded in lanes1, 2, 4 and 6; whereas 6 �g were loaded in lanes 3 and 5. (B) MnSODactivity of following strains: MC4100 (lane 1); PC35 (�arcA) (lane 2);PC35 carrying pMX513 (lane 3); PC35 carrying pBT22 and pMX513(lane 4); PC35 carrying pAMkatE72 and pMX513 (lane 5). 30 �g ofprotein were loaded in each lane. (C) Carotenoid spectra of strainPC35 (continuous line) and PC35 carrying plasmid pPL376 (dottedline).

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which are suggested to be involved in the repair of oxidativedamaged lipid membranes and in the scavenge of endogenoushydrogen peroxide, respectively (7, 43), on the TBO-depen-dent growth defect of the arcA mutant was tested. The �arcA::kan mutant strain carrying plasmid pMX511 or pMX512, over-producing HMP or AhpCF, respectively, were not able to growon arabinose containing TTA plates (data not shown). Thus,ROS per se might not be the principal cause for the TBO-dependent growth defect of the arc and cydAB mutants.

The lack of oxygen cancels the toluidine blue effect on thearc mutants. It is well established that the ArcA/B system isactive under reducing growth conditions (42). Also, under suchconditions expression of the cydAB operon is activated (33,56). Therefore, we tested whether the above-described mu-tants exhibit the same TBO-dependent growth defect underanaerobic growth. Surprisingly, the mutant cells grew equallyas well as their wild-type counterparts (Fig. 3A). Moreover, itwas observed that the dye surrounding the bacterial colonies

was reduced, as judged by the bleaching of the dye, althoughit was rapidly reoxidized by exposure of the plates to air oxygen(data not shown). It therefore seems that in the presence ofoxygen the mutant cells do not have the reducing power tocope with the stress generated by TBO, which could perturbthe electron transport chain by diverting electrons to redoxcycling, thereby subverting energy production. To test this, thearcA, arcB, and cydAB mutant strains were plated on glucose-supplemented TTA plates, because in the presence of glucosethe cells do not require efficient respiration to generate suffi-cient ATP production to support cell growth. Indeed, all mu-tant strains were able to grow on the glucose-supplementedTTA plates (Fig. 3B). Hence, TBO appears to inhibit cellgrowth by impeding energy production through diverting elec-trons to futile flow.

Effect of TBO on the survival of the arc mutants. In anattempt to test whether TBO exerts a bactericidal or bacteri-ostatic effect on the arc mutants, the �arcA::kan mutant strainwas grown in LB, and �103 cells were plated out either ontryptone-agar plates or on several TTA plates. As expected,the arcA mutant strain grew on the plates without TBO but noton the ones with TBO. Subsequently, every 24 h the cells of aTTA plate were transferred to a plate without TBO by replicaplating in order to estimate the number of living cells. A twofolddecrease in the number of CFU, indicative of cell death, wasobserved after day 4 (Fig. 2C). Thus, the presence of TBO ap-pears to lead to the death of the arcA mutant cells, although witha rather slow death rate.

Curiously, various colonies start appearing on the TTAplates after 4 days of incubation. These colonies (IFCS1 to -16)were picked and restreaked on TTA plates. All of them wereresistant to kanamycin, indicating that they were not contam-inants, and were able to produce colonies the size of those ofa wild-type strain after overnight incubation (data not shown),indicative of a suppressor mutation event(s).

Characterization of the arcA suppressor mutants. To char-acterize the suppression event(s), pTZ19R-based genomiclibraries of the suppressor mutants were constructed andscreened by transformation of the plasmid libraries into thearcA::kan mother cells, which in turn were plated out on TTAplates. Two plasmids, pMX515 and pMX516, derived from thelibraries of strains IFCS1 and IFCS13 were found to suppressthe TBO-sensitive phenotype of the arcA mutant (data notshown). Subsequent sequencing analysis of the two plasmidsrevealed the same 4,654-bp fragment containing the cydABstructural genes with 170 bp upstream of the cydA start codonand 1,758 bp downstream of the cydB stop codon. To find outwhether cydAB or the downstream genes (ybgTEC and tolQ)were responsible for the suppression effect, the EcoRI-EcoRIfragment of plasmid pMX515 containing the cydAB operonwas subcloned into the same vector, resulting in plasmidpMX515Eco, and transformed into the arcA::kan mutant cells.The transformed cells grew equally as well as the wild-typecells on TTA plates (data not shown). Thus, cydAB appears tobe able to suppress the TBO-dependent growth defect of thearcA mutants.

Expression of cydAB suppresses the TBO-growth defect ofthe arc mutants. To confirm that the increased expression ofcydAB suppresses the TBO-dependent growth defect of arcmutants, the effect of ectopic expression of wild-type cydAB,

FIG. 3. Effect of anoxia and glucose on the TBO growth defect ofthe different E. coli mutant strains. A wild type and the arc andcytochrome mutant strains (1, MC4100 [wild type]; 2, PC35 [�arcA]; 3,ECL5013 [�arcB]; 4, ECL937 [�cyd]; and 5, ECL936 [�cyo]) wereinoculated on tryptone agar plates in the presence (left) or absence(right) of 0.1 mg ml�1 of TBO. (A) Plates were incubated overnight at37°C under an anaerobic growth environment. (B) Plates were supple-mented with 10 mM glucose and incubated overnight at 37°C under anaerobic growth environment. (C) Survival of strain PC35 on TBO agarplates. Approximately 103 cells of strain PC35 were plated out onseveral TTA plates. Every 24 h the cells of a TTA plate were trans-ferred to a plate without TBO by replica plating, and the number ofliving cells was measured and plotted against time. The data are theaverage of three independent experiments (the variation was less than10% of the means).



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using plasmid pNG2 (25), on the TBO growth-sensitive phe-notype of the mutants was tested. In agreement with the above-described result, overexpression of cydAB allowed the arc mu-tants to grow on TTA plates (Fig. 4A and B), indicating that an

increase in the expression of the cydAB operon in the suppres-sor strain is responsible for the suppression effect.

E. coli is able to use two different terminal oxidases: cyto-chrome o, which is encoded by the cyoABCD operon and isused during oxygen-rich conditions, and cytochrome d, en-coded by cydAB, which is preferably expressed in microaerobicgrowth conditions. Since expression of cytochrome d appearsto suppress the TBO-dependent phenotype of the arc mutants,we tested whether the ectopic expression of cyoABCD, usingplasmid pRG110 (2), also suppresses the growth defect of thearc mutants. No suppression was observed (Fig. 4A and B),indicating that in contrast to cytochrome d, cytochrome o isnot able to protect the arc mutants from the deleterious effectof TBO.

Considering that the two oxidases differ, in that cytochromed has high affinity to O2, whereas cytochrome o has low affinityto O2 (13, 14, 48), we argued that this higher affinity to O2

might provide the basis for the TBO growth defect suppressionof the arc mutant. To test this, we examined whether theheterologous expression of recombinant hemoglobin 1 (rHb1)and leghemoglobin a (Lba), two plant-derived hemoglobinswith high affinity to O2, in arcA and cydAB mutants is sufficientto suppress their TBO sensitivity. To this end, the arcA andcydAB mutants were transformed with plasmid prHb1 (1) orpLba1 (29), carrying rHb1 of Oryza sativa and Lba of Glycinemax, respectively, and the transformants were plated out onTTA plates. Interestingly, the ectopic expression of both he-moglobins suppressed the TBO growth defect of both the arcAand cydAB mutants (Fig. 4C), suggesting that the high affinityof cytochrome d to O2 provides the key element for the TBO-dependent growth phenotype of the arcA mutant. This, incombination with the fact that oxygen convection on agarplates is too slow to compensate for respiration, raises thepossibility that cytochrome d provides TBO resistance by pro-moting an anoxic intracellular environment. If true, cytochrome dshould fail to protect the cells when grown in air-saturatedliquid cultures in the presence of TBO, because local oxygenconcentrations cannot be depleted. To test this, the wild type,the arcA mutant, and the arcA mutant strain complementedwith cydAB-carrying plasmid pNG2 were inoculated in tryp-tone broth in the presence or absence of TBO, and theirgrowth was monitored by counting the number of CFU of eachculture. It was found that neither strain was able to grow inair-saturated liquid cultures in the presence of TBO (Fig. 4D).Therefore, it can be concluded that cytochrome d protects thecells against TBO on TTA plates by creating an anaerobicintracellular environment.

Quantification of cytochrome d expression in the wild-type,arcA mutant, and mutant suppressor strains. Because theectopic expression of cytochrome d plays a central role inconferring TBO resistance to arc mutants, we tested whetheraerobic cydAB expression is elevated in the suppressor mutantscompared to the arcA::kan mother cells. To this end, the wild-type strain, the arcA mutant, and the mutant suppressor strainswere grown aerobically to mid-exponential growth phase, andthe amount of cytochrome d was measured by spectroscopicanalysis of the membrane fractions (Fig. 5). It was found thatmembranes of the wild-type strain contained circa 60 pmol ofcytochrome d per mg of protein, whereas no cytochrome d inthe membranes of the arcA mutants was detected. As expected,

FIG. 4. Expression of cydAB and also heterologous hemoglobins sup-press the TBO growth defect of arc and cydAB mutants. The arcA (A) andarcB (B) mutant strains were transformed with the following plasmids: pMN2(arcA), 3; pBB25 (arcB), 4; pNG2 (cydAB), 5; and pRG110 (cyoABCD), 6.Transformed and untransformed cells (indicated as 2) were inoculated ontryptone agar plates in the presence (left) or absence (right) of 0.1 mg ml�1

of TBO. In panels A and B, 1 represents the wild-type strains (MC4100).Growth was scored after overnight incubation at 37°C. (C) Strains PC35(�arcA) and ECL937 (�cyd) were transformed with plasmids prHb1 (recom-binant rice hemoglobin) and pLba (soybean leghemoglobin a), and theirgrowth on tryptone agar plates in the presence (left) or absence (right) of 0.1mg ml�1 of TBO was scored after overnight incubation at 37°C. 1, PC35; 2,PC35 carrying prHb1; 3, PC35 carrying pLba; 4, ECL937; 5, ECL937 carryingprHb1; 6, ECL937 carrying pLba. (D) Effect of TBO on the growth of strainsMC4100 (square), PC35 (triangle), and PC35 carrying plasmid pNG2 (dia-mond) in air-saturated liquid cultures. Cells were grown in tryptone brothcontaining 0.1 mg ml�1 of TBO (filled symbols) or without TBO (opensymbols), and growth was monitored by counting the number of CFU. Thedata are the average of three independent experiments (the variation was lessthan 10% of the means).



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membranes of the arcA mutant harboring either pNG2 orpMX515Eco contained amounts of cytochrome d �2.5-foldlarger than the ones of the wild-type strain. Finally, a consid-erable amount of cytochrome d, ranging from 5 to 120 pmol/mgof protein, was detected in all suppressor strains (Fig. 5). It thusappears that the suppression event(s) leads to an ArcA-indepen-dent expression of cydAB.

Analysis of the cydAB promoter region in the suppressorstrains. It has been demonstrated previously that the expres-sion of cydAB is negatively regulated by the histone-like pro-tein H-NS (24) and the transcriptional regulator Fnr (56).Therefore, the ArcA-independent expression of cydAB in thesuppressor mutants might be a result of its derepression, due toa possible inactivation of H-NS. The transcriptional regulatorFnr was discarded as a candidate because its activity is practi-cally null in the presence of molecular oxygen (36, 53). To testwhether inactivation of H-NS suppresses the �arcA growthphenotype by derepressing expression of the cydAB operon, wecreated a �arcA �h-ns double mutant strain and tested itsability to grow on TTA plates. No growth was observed (datanot shown), thereby eliminating the possibility of H-NS inac-tivation as an explanation for the ArcA-independent expres-sion of cydAB.

We then examined whether the ArcA-independent expressionof cydAB in the suppressor mutants is a result of mutations inthe complex promoter region of the cydAB operon. To thisend, the cydAB promoter region of each suppressor strain wasPCR amplified using the forward primers cydpromFw700,cydpromFw1600, cydpromFw2100, or cydpromFw4000 and thereverse primer cydpromRev. Curiously, only the reactions withDNA from strains IFCS7, -8, and -13 yielded a PCR product ofthe expected size, whereas the reactions with DNA from all

other suppressor strains yielded specific but variably sized PCRproducts. Sequencing analyses of the obtained PCR productsrevealed that suppressor strains IFCS7, -8, and -13 possessed awild-type promoter sequence, whereas IS elements (IS1, IS2,and IS5) were integrated into the regulatory promoter regionof cydAB of all other suppressor strains (Table 2 and Fig. 6A).Noteworthy, strains IFCS7, -8, and -13, which possess a wild-type promoter sequence together with IFCS16, which has anIS5 integrated at approximately position �500 relative to thestart codon of cydAB, were the ones with the smallest amountof spectroscopically detectable cytochrome d (5 to 20 pmol/mgof protein). Moreover, it was observed that the promoter ofcydAB in strains IFCS1 and IFCS9 to -12, in addition to theinsertion of IS1, suffered deletions of 800 to 2,850 bp upstreamof the positions of insertion (Table 2 and Fig. 6A).

Subsequent analysis of the new generated sequences, usingthe Neural Network Promoter Prediction software (47), re-vealed that potential chimeric promoters were generated forsuppressor strain IFCS1, -2 to -6, -9 to -12, and -14 to -16 (Fig.6B). These hybrid promoters combine a �35 region providedby the IS with an endogenous �10 region, thereby enabling theArcA-independent expression of cytochrome d. However, noexplanation can be provided for strains IFCS7, -8, and -13,which possess a wild-type promoter. One possibility might bethat a transcriptional factor has been altered in such a way thatit now is able to activate expression of cytochrome d. Alterna-tively, a cytochrome d-independent mechanism might be re-sponsible for the suppression of the dye sensitivity in thesemutant suppressor strains.

DISCUSSION

Almost 30 years ago, arcA was discovered as a gene withdifferent phenotypic properties, and therefore, it was desig-nated dye (8), fexA (39), msp (9), seg (30), or sfrA (6). Thedesignation dye described the growth sensitivity of these mu-tants to redox dyes, such as toluidine blue and methylene blue.Later on, a similar phenotype was described for mutants ofarcB (32) and cydAB (15). Redox dyes, such as TBO andmethylene blue, in the presence of light are able to produceROS that were suggested to be the cause for the dye sensitivityof the arcA mutants (50). However, the results presented inthis study indicate that the dye phenotype of arc mutants butalso the TBO-dependent production of ROS is light indepen-dent. Also, they demonstrate that the overproduction of the

TABLE 2. Genotypic characteristics of cydAB promoters of arcAmutant suppressors

Suppressorstrain(s)

IS (size�bp)

Position ofinsertion Deletions

IFCS1 IS1 (768) �376 �376–�3226IFCS2 IS1 (768) �447 NoIFCS3, IFCS4 IS2 (1,327) �101 NoIFCS5, IFCS6 IS1 (768) �376 NoIFCS9 IS1 (768) �410 �410–�1210IFCS10, IFCS11 IS1 (768) �410 �410–�2008IFCS12 IS1 (768) �410 �410–�2510IFCS14 IS5 (1,195) �323 NoIFCS15 IS1 (768) �467 NoIFCS16 IS5 (1,195) �517 No

FIG. 5. Spectroscopic quantification of cytochrome d. Membranefractions were prepared from cells grown in Luria broth to an OD600of 0.6. Reduced-minus-oxidized difference spectra were recorded, andthe levels of cytochrome d quantified from A630 to 650 and an absorptioncoefficient of 19 mM�1 cm�1, as described in Materials and Methods.The data are the averages of two independent experiments (the vari-ations were less than 10% of the means).



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antioxidant enzymes HPI, HPII, SodA, HMP, and AhpCF orthe carotenoid biosynthetic enzymes from Erwinia herbicola,which are known to provide protection to oxidative stress givenby H2O2 and near-UV in recombinant E. coli strains (5, 51),are not able to suppress the TBO-sensitive growth of arc andcydAB mutants. Although, these results do not support thesuggestion that ROS formation is the main cause for the dyephenotype, the possibility of the participation of ROS in thedye phenotype cannot be discarded. Nevertheless, the cellularresponses against ROS do not appear sufficient to cope withthis stress.

Yet, our results demonstrate that cytochrome d plays a vitalrole in the TBO resistance of E. coli. Cytochrome d, usedmainly under oxygen-limiting growth, and cytochrome o, pre-dominating under highly aerated growth, are the two terminalquinol oxidases in the respiratory chain of E. coli (31). Despitethat both these enzymes catalyze the same reaction, that is, thereduction of oxygen to water, they differ in that cytochrome dhas high affinity to O2 and a low Vmax, whereas cytochrome ohas low affinity to O2 and a high Vmax (13, 14, 48). Now, ourresults demonstrate that expression of cytochrome d but notcytochrome o suppresses the TBO growth-sensitive phenotypeof the arc mutants. Moreover, they clearly demonstrate thatthe high affinity to O2 is the key characteristic of cytochrome dthat explains the dye phenotype of the arc and cydAB mutants.Therefore, it is tempting to speculate that cytochrome d servesas a potent O2 scavenger able to generate an anoxic intracel-lular environment, thereby restricting TBO-generated stress by

not permitting TBO reoxidation. In such a scenario, the reduc-ing power of the cell would be sufficient to reduce TBO andabolish its detrimental effects on cell growth. Moreover, thefact that the dye phenotype is observed only in the presence ofoxygen raises the possibility that TBO perturbs the electrontransport chain by diverting electrons to redox cycling, therebyrestricting energy production. This is supported by the previ-ous observation demonstrating that cyanide-treated E. colimembranes that are unable to respire recover oxygen con-sumption in the presence of TBO (50) and also by the fact thatthe presence of glucose reverts the TBO-dependent growthdefect of the arc mutants. Interestingly, a protective role forcytochrome d in generating an intracellular anaerobic environ-ment has previously been observed with Azotobacter vinelandii,which uses the terminal oxidase to protect nitrogenase, anoxygen-sensitive enzyme, enabling nitrogen fixation even dur-ing aerobic growth (35).

The expression of the cydAB operon is negatively regulatedby the histone-like protein H-NS and the transcriptional reg-ulator Fnr and positively regulated by the ArcA/ArcB two-component system (23, 24, 40, 56). In agreement, we observedsignificant amounts of spectroscopically detectable cytochromed with a wild-type strain but not with the arcA mutant. Thus,the TBO-dependent growth defect of the arc mutants might beattributed to the lack of cytochrome d. This is supported by thefact that ectopic expression of cydAB suppresses the growthdefect of the arc mutants and further supported by the fact thatconsiderable amounts of cytochrome d were expressed in 13

FIG. 6. (A) Schematic representation of the cydAB promoter. Gray arrows represent the mng and cydA open reading frames. In the mng-cydAintergenic region are shown the ArcA (black blocks) and Fnr (gray blocks) regulatory binding sites and also the five proposed promoters (P1 toP5) of the cydAB operon (23). Arrows indicate the IS insertion site for each �arcA suppressor strain. Position numbers are relative to the startcodon (1) of cydA. (B) Predicted new promoters created after IS integration in the cydAB promoter region of suppressor strains. IS are shownin bold italic characters. Predicted �35 and �10 boxes are underlined.



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out of 16 arcA suppressor mutants. ArcA-independent cydABexpression in the suppressor strains appears to be achieved bythe integration of IS in different locations of the cydAB pro-moter region. The activation of expression of bacterial silentgenes (cryptic genes) by insertion of IS elements is reportedwidely. In general, transposable elements contribute positivelyto the fitness of the cells by activating catabolic operons thatare otherwise silent, and their transposition rates increase un-der such conditions, such that the activation of those operonsis beneficial (27). Examples include the E. coli operons bgl, cel,and asc, which code for enzymes involved in the utilization of�-glucoside sugars (28). Noteworthy, similarly to the case re-ported here, the insertion position of the IS in the crypticoperons bgl and cel is not at a single site but confined to aregion of 223 (52) and 108 bp (44), respectively. However,whether the presence of TBO itself enhances the occurrence ofIS transposition remains unknown.

It would be interesting to identify the suppressor gene(s) instrains IFCS7, IFCS8, and IFCS13, as they may provide furtherinsights into the mechanism(s) by which bacteria copes withstress generated by redox dyes, such as TBO and methyleneblue.

ACKNOWLEDGMENTS

We thank Raul Arredondo-Peter for plasmids prHb1 and pLba1,NBRP (NIG, Japan) for h-ns, katG, katE, and sodA E. coli mutants,Claudia Rodriguez for technical assistance, Diego Gonzalez Halphenand Bertha Michel for helpful discussions and for critically reading themanuscript, and the Unidad de Biología Molecular from the Institutode Fisiología Celular, Universidad Nacional Autonoma de Mexico foroligonucleotide synthesis and sequencing.

This work was supported by grants 37342-N from the Consejo Na-cional de Ciencia y Tecnología (CONACyT) and IN221106/17 fromDGAPA-PAPIIT, UNAM.

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cytochrome d but not cytochrome o rescues the toluidine blue

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