Báo cáo khoa học: A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB potx

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A kinetic approach to the dependence of dissimilatorymetal reduction by Shewanella oneidensis MR-1 on theouter membrane cytochromes c OmcA and OmcBJimmy Borloo*, Bjorn Vergauwen*, Lina De Smet, Ann Brige´, Bart Motte, Bart Devreeseand Jozef Van BeeumenLaboratory for Protein Biochemistry and Protein Engineering, Ghent University, BelgiumShewanella oneidensis MR-1 is a Gram-negative c-pro-teobacterium with an extremely versatile anaerobic res-piratory metabolism. Under anaerobic conditions, thisorganism reduces a variety of organic and inorganicsubstrates, including fumarate, nitrate, trimethylamineN-oxide, dimethylsulfoxide, sulfite and thiosulfate, aswell as various polyvalent metal ions and radio-nuclides, including iron(III), manganese(IV), chro-mium(VI), vanadium(V), selenium(VI), uranium(VI),and tellurium(VI) [1–7]. Bacterial dissimilatory metalKeywordskinetic enzyme parameters; metal reduction;outer membrane cytochromes c OmcA andOmcB; Shewanella oneidensis MR-1;terminal reductasesCorrespondenceJ. Borloo, Laboratory for ProteinBiochemistry and Protein Engineering,Ghent University, K.L. Ledeganckstraat 35,B-9000 Ghent, BelgiumFax: +32 9 264 52 73Tel: +32 9 264 51 26E-mail: jimmy.borloo@ugent.beWebsite: http://www.eiwitbiochemie.ugent.be/index.html*These authors contributed equally to thiswork(Received 28 April 2007, revised 25 May2007, accepted 30 May 2007)doi:10.1111/j.1742-4658.2007.05907.xThe Gram-negative bacterium Shewanella oneidensis MR-1 shows aremarkably versatile anaerobic respiratory metabolism. One of its hall-marks is its ability to grow and survive through the reduction of metalliccompounds. Among other proteins, outer membrane decaheme cyto-chromes c OmcA and OmcB have been identified as key players in metalreduction. In fact, both of these cytochromes have been proposed to be ter-minal Fe(III) and Mn(IV) reductases, although their role in the reductionof other metals is less well understood. To obtain more insight into this,we constructed and analyzed omcA, omcB and omcAomcB insertionmutants of S. oneidensis MR-1. Anaerobic growth on Fe(III), V(V), Se(VI)and U(VI) revealed a requirement for both OmcA and OmcB in Fe(III)reduction, a redundant function in V(V) reduction, and no apparentinvolvement in Se(VI) and U(VI) reduction. Growth of the omcB–mutanton Fe(III) was more affected than growth of the omcA–mutant, suggestingOmcB to be the principal Fe(III) reductase. This result was corroboratedthrough the examination of whole cell kinetics of OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction, showing that OmcB is$ 11.5 and $ 6.3 times faster than OmcA at saturating and low nonsaturat-ing concentrations of Fe(III)-nitrilotriacetic acid, respectively, whereas theomcA–omcB–double mutant was devoid of Fe(III)-nitrilotriacetic acidreduction activity. These experiments reveal, for the first time, that OmcAand OmcB are the sole terminal Fe(III) reductases present in S. oneidensisMR-1. Kinetic inhibition experiments further revealed vanadate (V2O5)tobe a competitive and mixed-type inhibitor of OmcA and OmcB, respect-ively, showing similar affinities relative to Fe(III)-nitrilotriacetic acid. Nei-ther sodium selenate nor uranyl acetate were found to inhibit OmcA- andOmcB-dependent Fe(III)-nitrilotriacetic acid reduction. Taken togetherwith our growth experiments, this suggests that proteins other than OmcAand OmcB play key roles in anaerobic Se(VI) and U(VI) respiration.AbbreviationFR, fumarate reductase.3728 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBSreduction is known to account for the majority of thevalence transitions of Fe(III) to Fe(II) in anoxic, non-sulfidogenic and low-temperature environments. Fur-thermore, microbial metal reduction represents apotential strategy for the in situ immobilization andcontainment of contaminant metals and radionuclidesin aqueous waste streams and subsurface environ-ments, as some of these metals precipitate upon reduc-tion [6,8].Although the importance of bacterial dissimilatorymetal reduction in controlling the fate and transportof metals and their potential for remediation purposesare well recognized, the terminal reductases involvedare not yet identified, and nor are they sufficientlycharacterized, as kinetic information on metal reduc-tion is scarce. The electron transport chain involved inthe reduction of either Fe(III) or Mn(IV) in MR-1 isthought to be composed of cytochromes and a qui-none, located in both the cytoplasmic membrane(CymA and menaquinone) and the outer membrane(OmcB, and a partial role for OmcA) [4,9–11]. The21 kDa tetraheme cytochrome c CymA (SO_4591) andmenaquinone are believed to be common central com-ponents in the electron transport chain that branch toseveral reductases downstream, as cymA–or menaqui-none-deficient strains lose their ability to grow anaero-bically on Fe(III), Mn(IV), V(V), nitrate, fumarate anddimethylsulfoxide [9,10,12]. OmcA (SO_1779) andOmcB (SO_1778) are outer membrane decaheme lipo-protein cytochromes c [13,14] that are specificallyinvolved in metal reduction, although distinct func-tions have been proposed. OmcB-negative MR-1mutants are heavily affected in either Fe(III), Mn(IV)or V(V) reduction, whereas the absence of OmcAresults in metal reduction rates that are 55% and 62%of those of the MR-1 parent strain for Mn(IV) andV(V), respectively [10]. Purified and dithionite-reducedpreparations of both outer membrane proteins wererecently shown to directly transfer electrons to chelatedFe(III) at comparable rates (kcatvalues rangingbetween 1.5 and 4.1 s)1), whereas only reduced OmcBwas shown to be oxidized by uranyl acetate(kcat< 0.01 s)1) [15]. Taken together, OmcA andOmcB function as metal reductases in MR-1, albeitapparently behaving kinetically differently and display-ing a rather undefined metal specificity.To address these latter issues, we constructed omcA,omcB and omcAomcB insertion mutants of MR-1,and analyzed them in terms of dissimilatory reductionof a variety of metals, i.e. Fe(III), V(V), U(VI), andSe(VI). A ‘whole cell’ kinetics approach was used todetermine the kinetic parameters for OmcA- andOmcB-dependent chelated Fe(III) reduction, which areshown to corroborate the results of inhibition andliquid growth experiments. These results identifyOmcA and OmcB, for the first time to our knowledge,as the sole terminal Fe(III) reductases, and additionallyprovide novel insights into the dependence of dissimila-tory metal reduction by MR-1 on OmcA and OmcB.ResultsGrowth analyses of anaerobically metal-respiringomcA–, omcB–and omcA–omcB–MR-1R mutantsrelative to their MR-1R parentTo study the substrate specificities of the outermembrane decaheme cytochromes OmcA and OmcBin the process of dissimilatory metal reduction, omcA–,omcB–and omcA–omcB–MR-1R mutants were con-structed and evaluated in liquid broth growth experi-ments with lactate as electron donor and eitherFe-nitrilotriacetic acid, Fe-citrate, V2O5,Na2SeO4orUO2(CH3COO)2.2H2O as the terminal electron accep-tor. Complete growth curves were recorded for eachexperiment; those of MR-1R grown on the differentmetals are shown in Fig. 1B, whereas the increases indensity at day 3 of MR-1R and of all mutants aresummarized in Fig. 1A. For chelated forms of Fe(III)and for V2O5, culture turbidities gradually decreasedin the order MR-1R > omcA–>> omcB–> omcA–omcB–, with the greatest effect being caused by theomcB disruption. OmcA and OmcB are collectivelyessential for chelated Fe(III) dissimilatory reduction,as the omcA–omcB–double mutant cannot grow oneither Fe(III)-nitrilotriacetic acid or Fe(III)-citrate,whereas they appear to have an important, althoughredundant, function as a terminal V(V) reductase, asthe omcA–omcB–double mutant still reaches $ 50%of the MR-1R turbidity. Knocking out either omcA oromcB turned out to have no significant growth pheno-type with either U(VI) or Se(VI) as the terminal elec-tron acceptor. These results therefore provide evidencethat there are differences between the electron transferpathways towards chelated Fe(III) on the one handand either U(VI) or Se(VI) on the other. Redundancybetween these pathways may explain the growth curvesobserved for V(V) reduction.Decaheme cytochrome c quantification ofanaerobically Fe(III)-respiring omcA–, omcB–andomcA–omcB–MR-1R mutants relative to theirMR-1R parentThe major impact on Fe(III) respiration by OmcB relat-ive to OmcA can be explained by one or a combinationJ. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kineticsFEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3729of the following possibilities: (a) the steady-state OmcBconcentration is greater than that of OmcA; (b) OmcBis differentially produced (upregulated) by the omcAinsertional inactivation, but not vice versa; (c) OmcAand OmcB show different behavior patterns in termsof kinetics; and (d) OmcB is required to obtainfunctional OmcA. These possibilities are discussedbelow.A heme-staining approach was used to reveal thedecaheme cytochrome c pools present in Fe(III)-respir-ing MR-1 omcA–, omcB–and omcA–omcB–mutantsrelative to their MR-1R parent. Figure 2B shows theabsence of mature OmcA (83 kDa) and OmcB(78 kDa) in an omcA–and an omcB–background,respectively, a complete lack of both proteins in theomcA–omcB–double mutant, and approximately equalamounts of either decaheme cytochrome c in anMR-1R extract. Relative to MR-1R, Fig. 2B does notsuggest compensatory induction of either OmcB orOmcA in an omcA–or omcB–background, respect-ively.To calculate the OmcA and OmcB content inFe(III)-respiring MR-1R and single mutants, differen-tial absorption spectra for reduced-minus-oxidizedheme were recorded (Fig. 2D). As these spectra arebased on total heme content, it is imperative that allthe other heme-containing proteins in the cells are notsubjected to regulation in the respective mutants. Fig-ure 2B,C shows that, apart from OmcA and OmcB,the periplasmic fumarate reductase (FR), the cytoplas-mic CymA and other, smaller (< 20 kDa), cyto-chromes are highly abundant c-type cytochromes inMR-1R, and thus contribute substantially to the554 nm absorbance. Although not fully linear and sat-urating with increasing cytochrome content, the hemestaining experiments are indicative of the fact thatthese cytochromes are not subjected to upregulation ordownregulation in the analyzed mutants. We further-more monitored and compared FR activities in wild-type MR-1R and mutants. The enzyme assay yieldedactivity values of (in lmolÆmin)1Æmg)1) 43.8 ± 0.90,42.9 ± 0.58, 43.0 ± 0.24 and 44.3 ± 0.70 forMR-1R, omcA–, omcB–and omcA–omcB–, respect-ively, indicating no upregulation or downregulation ofFR (P ¼ 0.83). On the basis of the fact that FR is notsubjected to regulation under the applied conditions,and deducing from Fig. 2C that all other c-type cyto-chromes are also invariantly produced in the respectivemutants, we feel safe to extract OmcB and OmcAconcentrations from omcA–and omcB–mutant hemevalues minus omcA–omcB–double mutant values,respectively. The concentrations of OmcA and OmcBwere subsequently calculated on the basis on theknown stoichiometry of 10 heme groups per OmcA orOmcB molecule [16]. This approach is valid, becauseno alterations other than the expected disappearanceof either or both OmcA and OmcB in the respectivemutants are apparent from the heme-staining gels. TheomcA–background contains 4.00 pmol of OmcB per109cells, which, as to be expected from the heme stainin Fig. 2B, is similar to the OmcA concentration cal-culated for the omcB–background (3.43 pmol per109cells).By subtracting the heme concentration of theomcA–omcB–double mutant from that of MR-1Rcells, we calculated a decaheme cytochrome c content(OmcA + OmcB) in MR-1R of about 6.68 pmol per109cells. This value matches the sum of both deca-heme cytochrome c concentrations in the respectivesingle mutants, again showing that neither decahemecytochrome c is upregulated in the absence of theFig. 1. Anaerobic liquid growth experiments assess the role ofOmcA and OmcB in dissimilatory metal reduction. Anaerobic liquidgrowth of MR-1R, omcA–, omcB–and omcA–omcB–mutant cul-tures with either Fe(III)-nitrilotriacetic acid, Fe(III)-citrate, V(V),U(VI) or Se(VI) as terminal electron acceptor is represented asthe increase in density reached after 3 days of growth (A).Complete curves of MR-1R grown on the different metals are pro-vided in (B).Shewanella oneidensis MR-1 OmcA and OmcB kinetics J. Borloo et al.3730 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBSother. Statistical analysis (Student’s t-test) between theMR-1R values and the sum of the values of the omcA–and the omcB–mutants revealed that there is no statis-tically significant difference (P ¼ 0.43).Whole cell kinetics of OmcA- andOmcB-dependent chelated Fe(III) reductionTo establish whether differential kinetics and ⁄ or syner-gism explain the dominance of OmcB over OmcA indissimilatory chelated Fe(III) reduction, we determinedthe kinetic parameters for each decaheme cytochromec using intact actively Fe(III)-respiring cells (Table 1).Maximal activities were converted to turnover numberson the basis of either the OmcA or OmcB concentra-tions calculated in the above paragraph for the omcB–and omcA–single mutants, respectively. As explainedin Experimental procedures, Monod-based kineticmodels for whole cell kinetics simplify to Michaelis–Menten models under the conditions applied in thisstudy.Figure 3A shows Fe(III)-nitrilotriacetic acid satura-tion curves obtained using either omcA–[OmcB-dependent Fe(III) reduction], omcB–[OmcA-dependentFe(III) reduction] or MR-1R [OmcA + OmcB-dependent Fe(III) reduction] cells. In the absence ofTable 1. Enzymatic properties of OmcA- and OmcB-dependent-Fe(III)-nitrilotriacetic acid reduction. Values represent the average oftriplicate experiments ± SD.Enzymatic properties OmcA OmcBFe(III)-nitrilotriacetic acidKm(lM) 15.3 ± 2.1 28.0 ± 0.9kcat(s)1) 17.8 ± 0.4 205 ± 3.0kcat⁄ Km(M)1Æs)1) 1.17 · 1067.33 · 106V2O5Inhibition type Competitive Mixed typeKic22.5 ± 1.0 65.9 ± 0.1Kiu11.5 ± 0.6Fig. 2. Heme quantifications reveal unaltered protein productionprofiles of both OmcA and OmcB in the respective single mutantsrelative to the wild-type. (A) RT-PCR confirming the absence ofpolar effects in mutants omcA–and omcB–. Specific oligonucleo-tides were used to amplify omcA (lane 2), omcB (lane 3), mtrA(lane 4) and mtrB (lane 5) in the omcA–mutant, and omcA (lane 7),omcB (lane 8), mtrA (lane 9) and mtrB (lane 10) in the omcB–mutant. MR-1R was used as a positive control to display omcA(lane 1) and omcB (lane 6). DNA standards are indicated at the leftand right of the agarose gels. (B) Visualization and separation ofhigh molecular mass cytochromes c through heme staining of aTris ⁄ glycine SDS ⁄ PAGE gel loaded with 4 · 107whole cells fromanaerobically grown overnight cultures of MR-1R (lane 1), mutantsomcA–(lane 2), omcB–(lane 3), and omcA–omcB–(lane 4), andcomplemented strains omcA–⁄ pBAD202 ⁄ D-TOPOomcA (lane 5)and omcB–⁄ pBAD202 ⁄ D-TOPOomcB (lane 6). A molecular massstandard is indicated at the right. (C) Visualization of low molecularmass cytochromes c through heme staining of a Tricine ⁄SDS ⁄ PAGE gel loaded with 4 · 107whole cells from anaerobicallygrown overnight cultures of MR-1R (lane 1), and mutants omcA–(lane 2), omcB–(lane 3), and omcA–omcB–(lane 4). A molecularmass standard is indicated at the left. (D) Bar graph represen-tation of the cytochrome content, normalized to 109CFU, andcalculated from reduced-minus-oxidized heme absorption differ-ences at 554 nm (a peak) using the absorption coefficient of21 400M)1Æcm)1. The differences in peak height reflect theconcentrations of OmcA and OmcB in omcB–and omcA–cells,respectively.ABCDJ. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kineticsFEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3731synergism, the OmcA- and OmcB-dependent substratesaturation curves should add up to form the MR-1R(OmcA + OmcB) curve; this is a valid assumption, aswe could not identify differential protein productionprofiles as mentioned in the previous paragraph. Atfull Fe(III)-nitrilotriacetic acid saturation, the modeledsummation function corresponds well with the MR-1Rcurve, whereas it shows slightly lower than experiment-ally determined activities at nonsaturating Fe(III)-nitrilotriacetic acid concentrations. This suggests thatOmcA might synergistically enhance, albeit slightly,the affinity of OmcB for its metal substrate. However,the curves totally refute the reverse possibility, i.e. thatOmcB is needed to get functional OmcA.On the other hand, the derived kinetic parametersfor OmcA- and OmcB-dependent chelated Fe(III)reduction summarized in Table 1 do rationalize thedominance of OmcB in dissimilatory Fe(III) reduction:under physiologically relevant low micromolar concen-trations of Fe(III), OmcA should outnumber OmcBsix-fold to catalyze electron transfer at a similar rate.Complementation of the omcA–and omcB–mutantsrestored Fe(III)-nitrilotriacetic acid reduction activityto MR-1R levels (Fig. 3B).Inhibition assays of OmcA- and OmcB-dependentchelated Fe(III) reduction as a measure of enzymespecificityTo determine whether the lack of phenotype ofomcA–omcB–strains observed during anaerobicgrowth on either of the electron acceptors U(VI) andSe(VI) is due to the decaheme cytochromes c notrecognizing either of these electron acceptors, weprobed the relative affinities via competition assays.Figure 4 shows the IC50plots of the inhibition data ofwhole cell OmcA- and OmcB-dependent Fe(III)-nitrilo-triacetic acid reduction by either V(V), U(VI), orSe(VI). Only V(V) appears to significantly inhibitFe(III) reduction, as characterized by IC50s of 10.7 lmand 81.4 lm for inhibition of OmcA and OmcB,respectively.Modes of inhibition of either OmcA or OmcBby V(V)The modes of inhibition of either OmcA- or OmcB-dependent Fe(III)-nitrilotriacetic acid reduction byV(V) were investigated for the two following reasons:(a) to derive the relevant inhibition constants; and (b)to establish whether both decaheme cytochromes cmay differ mechanistically. Fe(III)-nitrilotriacetic acidsaturation curves in the absence and in the presence oftwo different concentrations of V(V) were plotted andmodeled to obtain the apparent Vmaxand Kmvalues(Fig. 5A,B). These parameters were subsequently usedto generate double-reciprocal Lineweaver–Burk plotsto easily determine inhibitor modality (Fig. 5C,D;Table 1).OmcA inhibition by V(V) is characterized by anincrease in apparent Kmand no change in apparentFig. 3. Kinetic characterization of OmcA- and OmcB-dependentFe(III)-nitrilotriacetic acid reduction rationalizes the dominance ofOmcB in anaerobic ferric iron respiration. (A) Monod-based kineticmodel curves [34] for Fe(III)-nitrilotriacetic acid reduction by MR-1Rcells (inverted triangles), omcA–cells (squares), and omcB–cells(triangles). As explained in Experimental procedures, the two lattercurves simplify to the Michaelis–Menten formulation under the con-ditions applied. Adding up these curves generates the dotted-linecurve, which, as explained in Experimental procedures, shouldresemble the MR-1R curve. Because this assumption is only validat saturating Fe(III)-nitrilotriacetic acid concentrations, slight synergymay modulate activity when both OmcA and OmcB are present inthe outer membrane. (B) In trans complementation of omcA–andomcB–cells restores Fe(III) reductase activity to MR-1R levels. SeeExperimental procedures for details.Shewanella oneidensis MR-1 OmcA and OmcB kinetics J. Borloo et al.3732 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBSVmax, generating Lineweaver–Burk lines with intersect-ing y-axis intercepts, which is the characteristic signa-ture of competitive inhibition. We calculated a Kivalue of 22.5 lm, suggesting that the kinetics of V2O5binding to OmcA are similar to those for binding ofFe(III)-nitrilotriacetic acid.Fig. 4. Competition assays of OmcA- (left panel) and OmcB-dependent (right panel) Fe(III)-nitrilotriacetic acid reduction with other metalsshow that only V(V) may represent an alternative substrate for both cytochromes. Fe(III)-nitrilotriacetic acid reductase activity in the absenceof a competing metal substrate is set to 100%. Relative activities are plotted as a function of increasing concentrations of either V(V) (asvanadate; red), U(VI) (as uranyl acetate; green), or Se(VI) (sodium selenate; purple). Inhibition curves were fitted to the standard hyperbolicinhibition equation (see Experimental procedures).Fig. 5. Analysis of the modes of inhibition of OmcA- and OmcB-dependent Fe(III) reduction by V(V) reveals mechanistic differences betweenthe two cytochromes. (A, B) Direct plots of the steady-state velocities of OmcA-dependent (A) and OmcB-dependent (B) Fe(III)-nitrilotriaceticacid reduction in the absence and the presence of two increasing V(V) concentrations. (C, D) Theoretical double reciprocal plots using thekinetic parameters obtained by fitting the data from the direct plots.J. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kineticsFEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3733OmcB inhibition by V(V) is characterized by adecrease in apparent Kmand Vmax. By plugging thevalues of the modeled apparent kinetic parameters intothe double-reciprocal Lineweaver–Burk equation andplotting the resulting linear functions, we obtained thegraph in Fig. 5D. The lines intersect at negative valuesof 1 ⁄ [S] and 1 ⁄ v, which is a characteristic signatureof noncompetitive inhibition. Thus, V(V) apparentlybinds both the free OmcB enzyme and the binaryOmcB–Fe(III)-nitrilotriacetic acid complex, and thebinding is kinetically favored upon Fe(III)-nitrilotriace-tic acid binding. We calculated Kicand Kiuvalues of65.9 lm and 11.5 lm, respectively, which again appearsto have physiologic significance. Hence, besides havingsignificantly different turnover rates, OmcA and OmcBmay also behave differently in terms of binding theirmetallic substrates.DiscussionIn the present study, we could not detect an-aerobic Fe(III)-nitrilotriacetic acid respiration foromcA–omcB–double mutant cells. Virtually no biomasswas generated in minimal medium containing lactateand Fe(III)-nitrilotriacetic acid as the electron donorand acceptor, respectively (Fig. 1), and baseline reduc-tion of Fe(III)-nitrilotriacetic acid was seen in the ferro-zine-based whole cell kinetic approach (data notshown). The collective action of both decaheme cyto-chromes c, OmcA and OmcB, appears to be crucial foranaerobic soluble Fe(III) respiration, and, becauseof their outer membrane localization, one or bothcytochromes probably function as terminal Fe(III)reductases. Both these outer membrane-localizedcytochromes are reduced through an as yet incompletelyidentified electron transport chain, which at an earlypoint receives electrons from the NADH pool, in ourstudy obtained by lactate supplementation. In a recentstudy, Marshall et al. [15] established almost equallyfast direct electron transfer from either dithionite-reduced MR-1 OmcA or OmcB to chelated Fe(III), pro-viding the first biochemical evidence that both decahemecytochromes c are in fact functional Fe(III) reductases.As an OmcAOmcB double mutant strain does notshow any Fe(III) reduction activity, our study not onlystrengthens, but also exceeds, this evidence, in thatOmcA and OmcB are found to be the sole Fe(III) reduc-tases present in MR-1. Furthermore, the outer mem-brane localization and partial extracellular exposure ofboth cytochromes c, combined with the fact that theresult of adding up the OmcA and OmcB Fe(III)-nitrilo-triacetic acid reduction curves conforms to the MR-1Rcurve, allow us to deduce that the electron transportchain does not bifurcate any further, but ends at thispoint before transferring electrons to the subject metalspecies, indicating that OmcA and OmcB are the ter-minal Fe(III) reductases in MR-1. Other MR-1 cyto-chromes c, previously shown to be ferric iron reductasesin vitro, such as MtrA [17] and Ifc3 in S. frigidimarina[18], appear to be not directly involved in the process ofanaerobic chelated Fe(III) respiration.Notably, the apparent maximal rate reported forFe(III)-nitrilotriacetic acid-dependent OmcB oxidationis approximately 50 times slower than the kcatforOmcB-dependent Fe(III)-nitrilotriacetic acid reduction(205 s)1), determined here using a whole cell kineticsapproach, which has the advantages of: (a) maintain-ing the complete electron transport chain used duringmetal respiration; and (b) keeping the terminal reduc-tases in their native cellular compartment. For OmcA,the in vitro Kobsvalues determined by Marshall et al.[15] and the in vivo kcatvalues determined in our studyalso differ, although to a lesser extent (six-fold). This dis-crepancy can most likely be accounted for by the factthat the purified cytochromes used in the in vitroapproach lack some factor(s), such as one or more pro-tein partners or lipids that generate maximal activity.Reduced activity due to detergent-based solubilizationof the outer membrane cytochromes is an alternativeexplanation.Growth experiments as well as the whole cell Fe(III)reduction kinetics presented here agree with previousfindings that OmcB is more important than OmcA inanaerobic Fe(III) respiration [19]. Using a heme-quanti-fication approach, we have presented evidence showingthat this relative difference is not based on differentialprotein production profiles of either the omcA or omcBgene in the presence or absence of the other. Shi et al.[19] provided evidence for synergistic complex forma-tion between both decaheme cytochromes, which mayexplain the dominance of OmcB over OmcA in dissimi-latory Fe(III) reduction. Our whole cell-based kineticanalysis, however, refutes the possibility that OmcB isnecessary to reconstitute fully functional OmcA, as theFe(III)-reducing activities of omcA–and omcB–cells addup to the counterpart activities of MR-1R cells. A per-fect fit, however, only becomes possible after slightlyincreasing the affinity of OmcB for its chelated Fe(III)substrate (Fig. 3A). Complex formation may thus causesome synergism only at low micromolar and thereforephysiologically relevant substrate concentrations.The kinetics for OmcA- and OmcB-dependentFe(III)-nitrilotriacetic acid reduction (Table 1) dorationalize the different roles of these proteins in Fe(III)respiration. Both cytochromes have similar low micro-molar affinities for their Fe(III) substrate; however,Shewanella oneidensis MR-1 OmcA and OmcB kinetics J. Borloo et al.3734 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBScompletion of the electron transfer pathway takes$ 11.5 times longer for OmcA than for OmcB. Takinginto account the specificity constants, OmcA should out-number OmcB about six-fold if it is to substitute for thelatter in anaerobic Fe(III) respiration at physiologic fer-ric iron concentrations, a hypothesis that will be pursuedfurther in our laboratory. Note that the division of laborestablished here for OmcA and OmcB cytochromesshould not necessarily apply to homologs from differentbackgrounds; the OmcA homolog from S. frigidimarina,for example, has been found to be as fast (206 s)1)asthe S. oneidensis MR-1 OmcB reductase [20].It has previously been recognized that both cyto-chromes, OmcA and OmcB, appear to have some sub-strate specificity, as purified reduced batches lackactivity towards nitrite, nitrate and, in the case ofOmcA, uranyl acetate [15]. OmcB was shown to havesome activity towards U(VI); however, the turnovernumber (Kobs1¼ 0.039 s)1) is more than 100 timeslower than that for Fe(III)-nitrilotriacetic acid(Kobs1¼ 4.1 s)1) [15]. Our anaerobic growth experi-ments show that neither decaheme cytochrome c isnecessary for dissimilatory uranyl acetate reduction(Fig. 1). OmcA, as expected, but also OmcB does notbind U(VI) in the competition assay shown in Fig. 4.The 100-fold lower Kobs1for U(VI) reduction com-pared to Fe(III)-nitrilotriacetic acid reduction reportedby Marshall et al. [15] thus appears to result not fromdisturbed catalysis, but rather from hampered sub-strate binding. Of the other metals tested in this study[V(V) and Se(VI)], only vanadate was shown to be asubstrate for either OmcA or OmcB. Inhibition experi-ments suggest that Fe(III) and V(V) bind both cyto-chromes with similar efficiencies (Table 1). However,whereas omcA–omcB–double mutant cells did notgrow on chelated Fe(III), they do grow on V(V) toabout 50% of the MR-1R stationary-phase density(Fig. 1). In the case of V(V), the electron transportchain may thus bifurcate to one or several other, asyet unrecognized, terminal reductases. Redundancy interminal metal reductases has been clearly shown here,as MR-1 does not suffer from the omcA–omcB–dou-ble mutants in anaerobic growth on the terminal elec-tron acceptors Se(VI) and U(VI), and as none of thesemetals inhibits OmcA- and OmcB-dependent wholecell Fe(III)-nitrilotriacetic acid reduction. In summary,metal reduction appears to be a selective process inwhich the reduction potential and the topology andaccessibility of the presented metal play crucial roles interms of binding efficiencies and subsequent reductionby the appropriate enzyme. The identification andcharacterization of alternative terminal metal reductas-es will be the subject of future research.Experimental proceduresBacterial strainsS. oneidensis MR-1 was originally isolated from OneidaLake sediments (Oneida Lake, NY, USA) [21], and wasobtained from the LMG culture collection (LMG 19005;Ghent, Belgium). S. oneidensis MR-1R is a spontaneous rif-ampicin-resistant mutant of strain MR-1 that was isolatedin-house. Escherichia coli strain TAM1pir+and E. coli S17-1kpir cells were used for cloning purposes and conjugationexperiments, respectively.Growth conditionsMR-1R, omcA–, omcB–and omcA–omcB–S. oneidensis cul-tures were routinely grown overnight at 28 °C in LB brothand subsequently inoculated in M1 defined medium [22] sup-plemented with l-serine (1 lg ÆmL)1), l-arginine (1 lgÆmL)1),l-glutamate (1 lgÆmL)1), lactate (15 mm), and fumarate(20 mm). For growth experiments, fumarate was replacedby either Fe(III)-citrate (2 mm), Fe(III)-nitrilotriacetic acid(0.5 mm), Na2SeO4(1 mm), or UO2(CH3COO)2.2H2O(0.5 mm) (all products: Sigma-Aldrich, Bornem, Belgium).Growth on V(V) was studied using VM medium [23]. Anae-robicity was achieved using a Coy anaerobic chamber (CoyLaboratories, Grass Lake, MI) containing 90% N2,8%CO2,and 2% H2. The presence of H2in the anaerobic chamberdid not affect metal reduction (data not shown). Growthcurves were recorded by measuring the attenuance (D655)ofthe cultures at regular time intervals for 3 days. The averagerise in density after 3 days ± SEM for triplicate readings aresummarized in Fig. 1A, whereas the growth curves for MR-1R grown on the different metals are shown in Fig. 1B.Construction of the omcA–and omcB–singlemutants and of the omcA–omcB–double mutantstrains of MR-1Single omcA–and omcB–mutants and a double omcA–omcB–mutant strain of MR-1 were generated by insertional inacti-vation using the pKNOCK-based system [24]. The primersused in this study are summarized in Table 2. Briefly, internalPCR-amplified fragments of the omcA and omcB genes were5¢-phosphorylated and cloned into EcoRV-digested andcalf intestinal phosphatase-treated pKNOCK-Km andpKNOCK-Cm, respectively, using T4 DNA Ligase (allenzymes: New England Biolabs, Ipswich, MA), yieldingpKNOCK-Km-omcA and pKNOCK-Cm-omcB. These con-structs were transformed into E. coli S17-1kpir cells. Equalamounts of overnight-grown transformed E. coli S17-1kpircells and rifampicin-resistant S. oneidensis cells were mixedand spotted on LB ⁄ Rif plates (10 lgÆmL)1). After a 6 h incu-bation period (necessary for the conjugation to take place),the cells were resuspended in 500 lL of LB broth [25] andJ. Borloo et al. Shewanella oneidensis MR-1 OmcA and OmcB kineticsFEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBS 3735plated on LB ⁄ Rif plates containing either kanamycin(25 lgÆmL)1) or chloramphenicol (25 lgÆmL)1) (Duchefa,Haarlem, The Netherlands). After overnight incubationat 28 °C, colonies were analyzed via PCR using the oligo-nucleotides OMCA-F ⁄ OMCA-R and OMCB-F ⁄ OMCB-R(Table 2), designed to amplify the entire omcA gene andomcB gene, respectively. Homology-based insertional integ-ration of the pKNOCK constructs enlarged the omcA(2207 bp) and omcB (2015 bp) gene amplicons by 2700 and2500 bp, respectively (data not shown). The omcA–omcB–double mutant was constructed by applying a similar proce-dure to that described above, using the omcA–mutant as therecipient strain in conjugation. As omcA and omcB are partof the gene cluster mtrDEF–omcA–mtrCAB (omcB is alsoknown as mtrC), and the genes mtrCAB form a singleoperon, we expected polar effects to occur when disruptingomcB. RT-PCR experiments proved the absence of suchpolar effects (Fig. 2A) and confirmed that we had obtainedthe omcA–and the omcB–mutants.Complementation of the MR-1 omcA–and omcB–mutant strainsOligonucleotides OMCA-PBAD-F ⁄ OMCA-PBAD-R andOMCB-PBAD-F ⁄ OMCB-PBAD-R (Table 2) were used toamplify the omcA and omcB genes from MR-1 genomicDNA, respectively. These genes were subsequently clonedinto vector pBAD202 ⁄ D-TOPO (Invitrogen, Carlsbad,CA), and the constructs were transformed into the appro-priate omcA–or omcB–mutants of MR-1 by electropora-tion, generating the in trans complemented strains. AspBAD202 ⁄ D-TOPO carries a kanamycin resistance region,the ability to complement the omcA–mutant was shownusing a pKNOCK-Cm-based omcA–mutant, instead ofthe pKNOCK-Km-based mutant that was applied in allother experiments. Full complementation of either the omcAor omcB insertional mutation by the wild-type genes,controlled by an arabinose promoter [26], was achieved asvisualized by heme staining of SDS ⁄ PAGE gels (Fig. 2B),as well as at the level of activity (see further).Visualization of c-type cytochromes using hemestainingHigh and low molecular mass c-type cytochromes wereresolved by SDS ⁄ PAGE according to Laemmli [27] andSchaegger & von Jagow [28] (tricine gels), respectively. Ineither case, 4 · 107whole cells of anaerobically grown over-night cultures were applied to the gels, which were thenheme stained according to Thomas et al. [29]. The outermembrane cytochromes c OmcA and OmcB, the periplas-mic FR, and the cytoplasmic tetraheme cytochrome cCymA were unambiguously identified via MS from heme-stained Tris ⁄ glycine gels and tricine gels, respectively.Spectral quantification of the outer membranedecaheme cytochromes c OmcA and OmcBThe heme content of whole cells was determined using thedifference absorption coefficient of 21 400 m)1Æcm)1[16] at554 nm for the pyridine ferrohemochrome minus pyridineferrihemochrome spectrum. In that study, the differenceabsorption coefficient was determined at pH 8.0, whereasall our experiments were carried out at pH 7.5. We observedno differences between spectra measured at pH 8.0 and 7.5(data not shown). Sodium dithionite was used as the redu-cing chemical. Overnight anaerobically grown cells (with20 mm fumarate as the electron acceptor) were washed withand suspended in an equal volume of air-saturated NaCl ⁄ Pi(pH 7.5), and incubated at room temperature for 1 h toensure oxidation of the outer membrane cytochromes.Absorption spectra of 1 mL fractions were recorded at554 nm using a double-beam spectrophotometer (Uvikon,Kontron, Herts, UK) in the absence and the presence of afew crystals of sodium dithionite (Sigma-Aldrich). Thedecaheme cytochrome c concentration was calculated asexplained in Results, taking into account 10 heme groupsper molecule of either OmcA or OmcB and our experiment-ally derived correlation between D655and cell concentration(a 1 mL MR-1 culture with a D655of 1.0 contains1.44 · 109cells). The values presented are means of tripli-cate experiments ± SEM. To quantify FR, lysed MR-1RomcA–, omcB–and omcA–omcB–cells were assayed for thisspecific enzyme activity according to Maklashina et al. [30].Whole cell kinetics of ferric iron reductionThe Fe(III) reductase activity of whole cells was measuredusing the ferrozine-based method [31]. The chromophoreformed by ferrous iron and ferrozine was measured at562 nm [32]. Whole cells for the Fe(III) reductase assays wereTable 2. Synthetic oligonucleotides used in this study.Oligonucleotide name Sequence (5¢-to3¢)OMCA-KO-F CACACTGCAACCTCTGGTOMCA-KO-R ACTGTCAATAGTGAAGGTOMCB-KO-F CCCCATGTCGCCTTTAGTOMCB-KO-R TCGCTAGAACACATTGACOMCA-F ATGATGAAACGGTTCAATOMCA-R TTAGTTACCGTGTGCTTCOMCB-F CTGCTGCTCGCAGCAAGTOMCB-R GTGTGATCTGCAACTGTTOMCA-PBAD-F CACCGAGGAATAATAAATGATGAAACGGTTCAATTTCOMCA-PBAD-R TTAGTTACCGTGTGCTTCOMCB-PBAD-F CACCGAGGAATAATAAATGATGAACGCACAAAAATCAOMCB-PBAD-R TTACATTTTCACTTTAGTShewanella oneidensis MR-1 OmcA and OmcB kinetics J. Borloo et al.3736 FEBS Journal 274 (2007) 3728–3738 ª 2007 The Authors Journal compilation ª 2007 FEBSprepared as follows. Anaerobically grown cells (with fuma-rate as the terminal electron acceptor) were collected bycentrifugation at 10 000 g (Beckman Coulter Avanti J-301centrifuge, JA-30.50 rotor), washed twice with NaCl ⁄ Pisup-plemented with 1 mm lactate (unless otherwise mentioned),and placed on ice. These preparations retained full activityfor at least 4 h. Comparison of the reduced-minus-oxidizedspectra of anaerobically grown MR-1R cells washed withNaCl ⁄ Pi(pH 7.5) on the one hand, or water on the other,revealed no differences in heme content, indicating that thesalt treatment did not lead to unwanted release of outermembrane cytochromes. Assays were conducted in microtiterplates at 25 °C in a final volume of 200 lL of NaCl ⁄ Pi(pH 7.4), and were monitored using a Bio-Rad model 680microplate reader (Bio-Rad, Hercules, CA). A standard reac-tion mixture contained 1 mm 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine monosodium salt (ferrozine;Sigma-Aldrich), 1 mm lactate (unless otherwise mentioned),a 1 : 100 dilution of the washed cell preparation, and Fe(III)-nitrilotriacetic acid at concentrations ranging from 0.5 lm to1.5 mm. Phosphate did not interfere with the reduction assay(data not shown), which is in accordance with the resultsreported by Ruebush [33]. For inhibition studies, the stand-ard reaction mixture containing 100 lm Fe(III)-nitrilotriace-tic acid (unless otherwise mentioned) was supplemented witheither V(V) (as V2O5), Se(VI) (as Na2SeO4) or U(VI) [asUO2(CH3COO)2.2H2O], ranging in concentration from0.5 lm to 1 m m. Inhibition curves were fitted using a leastsquares algorithm (graphpad prism Version 4.00; GraphPadSoftware, Inc., San Diego, CA) to the equation:mr¼ 100 ÀðImax½Me=ðIC50þ½MeÞÞwhere vris the relative activity, Imaxis the maximalresponse amplitude, [Me] is the supplemented initial con-centration of inhibiting metallic substrate, and IC50is thehalf-maximal concentration of inhibiting metallic substrate.To analyze kinetic data, we used Monod-based kineticmodels [34] that actually simplify to a Michaelis–Menten for-mulation under the applied conditions. The kinetic rate isdetermined solely by the electron acceptor, as the electrondonor used (lactate, 1 mm) is supplied in excess. The effect ofbacterial growth on Fe(III)-nitrilotriacetic acid reduction canbe neglected, as the initial cell concentration used was high,and growth-supporting nutrients were excluded. We alsoassumed that cell decay can be neglected, because the activityproceeded linearly during our 1 h analyses. Therefore, theMonod model takes a form similar to the Michaelis–Mentenexpression v ¼ VmS ⁄ (Ks+ S), where Vmequals the maximalactivity for the initial bacterial concentration, S is the initialFe(III)-nitrilotriacetic acid concentration, and Ksis the half-velocity constant. As we have determined the OmcA andOmcB concentrations present in omcB–and omcA–cells,respectively, and because omcA–omcB–double mutant cellscompletely lack Fe(III) reductase activity, we can, using thesingle mutants, convert Vmvalues to kcatvalues, and safelyassume Ksto be Km, the familiar Michaelis–Menten constantfor enzyme-catalyzed reactions. Activity data were fitted tothe regular Michaelis–Menten equation using graphpadprism Version 4.00. For MR-1R- and OmcA-dependentkinetics, the Michaelis–Menten equation was adjusted forsubstrate inhibition.AcknowledgementsThis work was supported by a personal grant toJ. Borloo from the Institute for the Promotion ofInnovation by Science and Technology in Flanders(IWT-Vlaanderen). J. Van Beeumen and B. Devreeseare indebted to the Fund for Scientific Research (FWO-Vlaanderen) for granting research project G.0190.04, aswell as to the Bijzonder Onderzoeksfonds of Ghent Uni-versity for Concerted Research Action GOA 120154.References1 Krause B & Nealson KH (1997) Physiology and enzy-mology involved in denitrification by Shewanella putre-faciens. Appl Environ Microbiol 63, 2613–2618.2 Moser DP & Nealson KH (1996) Growth of the faculta-tive anaerobe Shewanella putrefaciens by elemental sul-fur reduction. Appl Environ Microbiol 62, 2100–2105.3 Myers CR, Carstens BP, Antholine WE & Myers JM(2000) Chromium(VI) reductase activity is associatedwith the cytoplasmic membrane of anaerobically grownShewanella putrefaciens MR-1. 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GTGTGATCTGCAACTGTT OMCA- PBAD-F CACCGAGGAATAATAAATGATGAAACGGTTCAATTTC OMCA- PBAD-R TTAGTTACCGTGTGCTTC OMCB- PBAD-F CACCGAGGAATAATAAATGATGAACGCACAAAAATCA OMCB- PBAD-R TTACATTTTCACTTTAGT Shewanella. CCCCATGTCGCCTTTAGT OMCB- KO-R TCGCTAGAACACATTGAC OMCA- F ATGATGAAACGGTTCAAT OMCA- R TTAGTTACCGTGTGCTTC OMCB- F CTGCTGCTCGCAGCAAGT OMCB- R GTGTGATCTGCAACTGTT OMCA- PBAD-F
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Xem thêm: Báo cáo khoa học: A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB potx, Báo cáo khoa học: A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB potx, Báo cáo khoa học: A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB potx