A Pseudomonas stutzeri Outer Membrane Protein Inserts Copper

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1987, p /87/ $2./ Copyright 1987, Americn Society for Microbiology Vol. 169, No. 12 A Pseudomons stutzeri Outer Membrne Protein Inserts Copper into N2 Reductse KHOTSO MOKHELE,t YAJARAYMA J. TANG,t MARTA A. CLARK, AND JOHN L. INGRAHAM* Deprtment of Bcteriology, University of Cliforni, Dvis, Cliforni Received 17 July 1987/Accepted 14 September 1987 Among set of frmeshift mutgen (ICR-191; Polysciences, Inc.)-induced muttions tht confer inbility to grow nerobiclly with N2 s the sole electron cceptor, one clss ws found tht produced n inctive N2 reductse which lcked copper. All of these mutnt strins filed to produce 61,-Mr protein locted in the outer membrne. This protein, termed NosA, seems not to be responsible for bringing copper into the cell becuse the mutnt strins nd their prent were similrly sensitive to the copper content of the growth medium nd no intermedite copper concentrtion in the medium permitted the mutnt strins (nosa) to grow nerobiclly with N2 s the sole electron cceptor. We conclude tht NosA is necessry to insert copper into N2 reductse or to mintin it there. Biologicl denitrifiction, process medited exclusively by procryotes, is the route by which the dinitrogen content of the tmosphere is replenished t the expense of terrestril nd qutic nitrte. This pthwy leding from nitrte to N2 is composed of series of nerobic respirtory chins for which the terminl electron cceptors re sequentilly NO3-, NO2-, NO (nitric oxide), nd N2 (nitrous oxide) (12). The finl step of the pthwy, the reduction of N2O to N2, is ctlyzed by copper-contining terminl oxidoreductse (5). This enzyme ctlyzes the reduction in vitro of N2 to N2 by reduced benzyl viologen. Muttionl loss of the enzyme renders bcteril strins unble to reduce N2 nd to grow in medi in which this gs is present s the sole electron cceptor (15). Here we report tht the muttionl loss of second protein (NosA) confers the sme mutnt phenotype, becuse NosA-deficient strins synthesize inctive N2O reductse tht lcks copper. MATERIALS AND METHODS Bcteril strins nd medi. The strins of Pseudomons stutzeri used in this study were ll derived from strin JM3, smooth vrint of soil isolte, JM299 (4). Strin JM64, spontneous nlidixic cid-resistnt (nl-7) mutnt, ws the prent of ll of the Nos mutnts described here. The medi used included modified Luri-Bertni (LB) medium (4), LB medium supplemented with 1 ml of trce minerls per liter (LT medium), nd miniml succinte (MS) medium (B. A. Bryn, Ph.D. thesis, University of Cliforni, Dvis, 198). Trce-minerl stock contins (per liter of.1 M HCl) 2.5 g of EDTA, 5. g of FeSO4. 7H2O, 1.54 g of MnSO4- H2O,.1 g of CuS4 5H2,.24 g of Co(NO3)2 6H2O,.95 g of N2B4O7, 1.18 g of N2MoO4. 2H2O. LT medium used to score nerobic growth on N2 s the sole electron cceptor ws modified to be 5,uM with respect to copper. The medium used to grow cells in which the loction or presence of NosA ws to be determined ws modified LT medium mde from trce minerls tht lcked dded copper (low-cu LT medium); by tomic bsorption spectroscopy, this medium ws found to contin 1. p.m copper. * Corresponding uthor. t Present ddress: Deprtment of Biochemistry, University of Fort Hre, Alice 57, South Afric. t Present ddress: Microgenics Inc., Concord, CA Mutgenesis nd mpicillin counterselection. A culture of strin JM64 ws grown overnight in LT medium nd diluted 1:1 into MS medium tht contined 1 p.g of ICR-191 ( frmeshift mutgen obtined from Polysciences, Inc.) per ml. A set of 2-ml smples of this mixture ws incubted overnight in foil-covered test tubes on shker t 37 C. The culture ws then diluted 1:5 into 5 ml of LT medium plus 12 mm NNO3 in 125-ml Erlenmeyer flsk nd incubted overnight t 37 C shken t low rte (12 rpm) to crete seminerobic conditions for expression of the mutnt phenotype nd induction of denitrifiction enzymes. A 1-ml volume of this culture ws used s n inoculum for 5 ml of LT broth tht hd been sturted with N2 in 6-ml Wheton serum vil closed with rubber septum nd metl cp. Growth of the culture ws monitored by mesuring the A65 of 1-ml smples removed with syringe t 3-min intervls. When the density of the culture hd doubled twice, mpicillin ws dded to 2 mg/ml, nd incubtion ws continued overnight. Then 1 mg of DNse I (Sigm) ws dded, nd the cells were pelleted t 6, x g for 1 min. Induction nd counterselection were repeted twice. Survivors were plted on LT medium incubted t 37 C, nd the developing clones were scored for their bility to grow on pltes of LT medium plus 5 p.m copper incubted t 37 C in n tmosphere of N2- Seprtion of inner nd outer membrnes. Strin JM64 ws grown overnight t 37 C in 6 liters of low-cu LT broth supplemented with 4 mm KNO3. Cells were collected by centrifugtion t 6, x g for 1 min, wshed once in 5 mm Tris buffer (ph 8.) nd suspended in 25 ml of Tris buffer contining 5 mm MgCl2 nd 1 mg ech of DNse I nd RNse A. The ph of these nd ll other Tris buffers ws set t 4 C. The outer nd inner membrnes from these cells were seprted by modifiction of the Hncock nd Nikido (6) procedure. The suspension ws pssed through French press t 16, lb/in2, 2 mg of lysozyme ws dded, nd the mixture ws held t room temperture for 3 min. Then the cell debris ws removed by centrifugtion t 6, x g for 1 min, nd the superntnt fluid ws diluted with 15 to 2 ml of Tris buffer. A crude preprtion of membrnes ws obtined by centrifuging this mixture t 24, x g for 2 h. This preprtion ws suspended in 25 ml of 2% (wt/vol) sucrose in Tris buffer. Smples (2 ml) of this suspension were lyered on grdient consisting of 1 ml of 7% sucrose, 3 ml of 64% Downloded from on My 8, 218 by guest

2 5722 MOKHELE ET AL. sucrose, 3 ml of 58% sucrose, nd 3 ml of 52% sucrose (ll in Tris buffer) nd spun in n SW41 rotor for 18 h t 188, x g. The bnds of turbidity tht developed were collected from the top of the tube, using Psteur pipette. Ech turbidity bnd ws diluted in distilled wter nd centrifuged t 126, x g for 9 min. The resulting pellets were suspended in.5 to 1. ml of distilled wter nd frozen t -8 C. Enzyme ssys. Assys for succinte dehydrogense nd lctte dehydrogense were done by the method of Booth nd Curtis (2), with the exception tht 4% rther thn.4% lctic cid ws dded to the rection mixture. Assys of N2O reductse were performed on cell extrcts prepred s follows. An overnight culture of the strin to be ssyed ws diluted 1:5 into 4 ml of LT medium supplemented with 4 mm NNO3. Cultures were incubted in 6-ml Wheton serum vils without shking t 3 C, hrvested in erly sttionry phse by centrifugtion t 5, x g for 2 min, wshed once in 5 mm potssium phosphte (ph 7.2) contining 5 mm MgCl2, nd stored t -8 C for no more thn 2 dys before use. The centrifuge tube ws fitted with septum cp, nd ll subsequent mnipultions were done without llowing the preprtion to come in contct with ir. The thwed cell pste ws suspended in 5 ml of LT medium contining 3 mm succinte nd incubted t 3 C for 3 min to llow exhustion of endogenous N2. After centrifugtion t 5, x g for 5 min, the cell pste ws suspended to protein concentrtion of.5 to 1. mg/ml in 1 mm potssium phosphte (ph 7.6) contining 5 mm MgCl2 nd.1 mg of DNse I. The suspension ws rpidly frozen in dry ice-ethnol bth, thwed in lukewrm wter, nd centrifuged t 5, x g for 5 min to remove unbroken cells. The superntnt fluid ws trnsferred to nother centrifuge tube nd ssyed. The N2 reductse ctivities of these preprtions were ssyed by modifiction of the method of Kristjnsson nd Hollocher (7). The rection mixture consisted of 1 mm potssium phosphte (ph 7.6) contining.4 mm methyl viologen, 38 mm triethnolmine, nd 4 FM proflvine. Endogenous oxidtion of methyl viologen ws mesured in solutions mde free of 2 by sprging with Ar for 3 min; enzyme-dependent reduction of N2 t the expense of methyl viologen ws mesured in solutions tht hd been sprged with the substrte for 6 min. A 4.3-ml volume of ech gssed solution ws trnsferred to 4.5-ml cpped serum cuvette in which methyl viologen ws reduced to n A64 of 2. to 2.2 by exposure to 15-W Sylvni fluorescent lmp for 3 to 4 min. Endogenous nd enzyme-ctlyzed rtes of oxidtion were mesured in seprte cuvettes by injecting 5 to 1,ul of cell extrct nd monitoring the subsequent decrese in A64 with Beckmn DU spectrophotometer provided with recorder. The Ew4 of methyl viologen ws found to be 11,5 cm-1 M-1. The rte of the rection ctlyzed by N2 reductse ws tken to be the difference in the rte of methyl viologen oxidtion in the Arnd N2-sprged solutions. One unit of N2 reductse is defined s the mount of enzyme tht ctlyzes the oxidtion of 2 p.mol of reduced methyl viologen per min t 25 C. Specific ctivity is expressed s units per milligrm of protein. Ech reported ctivity is the verge of three determintions. Chemicl determintions. Protein ws determined by the method of Lowry et l. (9), except for studies of N2 reductse, in which cse the method of Brdford (3) ws used, nd for studies of frctions from chromtogrphic columns, in which cse the A28 ws mesured. In both the Lowry nd the Brdford methods, bovine serum lbumin J. BACTERIOL. ws used s the stndrd. Determintion of 2-keto-3- deoxyoctonic cid ws done s described by Osborn (11). Copper ws determined with n tomic bsorption spectrophotometer (Perkin Elmer 5) t nm. Reltive bundnce of prticulr protein seprted by sodium dodecyl sulfte-polycrylmide gel electrophoresis (SDS-PAGE) nd stined with Coomssie blue ws determined by scnning gels with n LKB Ultroscn XL lser densitometer. Prtil purifiction of N2 reductse. Cells were cultured nd hrvested s described for the N2 reductse ssy except tht totl volume of 3.6 liters of culture contined in three 1,-ml Wheton bottles ws used for ech purifiction. The cells were wshed once with 5 mm potssium phosphte (ph 7.2) contining 5 mm MgCl2. In ll subsequent steps, the buffer used ws 1 mm Tris (ph 7.5). The cell pste ws suspended in 4 ml of buffer supplemented with 5 mm MgCl2 nd 1 mm phenylmethylsulfonyl fluoride. To ech grm of cells,.1 mg of DNse I nd.1 mg of RNse A were dded. The cells were broken by two pssges through French pressure cell t 16, lb/in2 nd centrifuged t 24, x g for 9 min. For N2 reductse from nosa strins, the superntnt fluid ws frctionted with solid (NH4)2SO4. The precipitte formed between 5 nd 8% sturtion ws dissolved in 1 ml of buffer, dilyzed, nd pplied to column (1.6 by 45 cm) of DEAE-cellulose (DE52; Whtmn, Inc.). The column ws eluted with 2 ml of to.4 M liner grdient of NCl. Frctions (2.5 ml) were collected, ssyed for protein, nd exmined by one-dimensionl SDS-PAGE. N2 reductse eluted just fter one column volume. The N2 reductsecontining frctions were pooled nd concentrted in dilysis tubing (12,4 Mw) surrounded by polyethylene glycol. For extrcts from the prent, the superntnt ws loded directly onto the DEAE-column; N2 reductse eluted in the void volume. Then the preprtion ws frctionted with (NH4)2S4- Gel seprtions. One-dimensionl SDS-PAGE (8) nd O'Frrell gel electrophoresis (1) were done s described before. RESULTS Isoltion of mutnt strins. After mutgenesis with ICR- 191, the mutnt phenotype ws llowed to express itself by diluting the culture 1:5 into LT-nitrte medium nd incubting it overnight t 37 C on rotry shker turning t 12 rpm. Under these conditions of incubtion, the culture becomes sufficiently nerobic to induce fully the denitrifiction enzymes. This culture served s n inoculum for three cycles of mpicillin counterselection in LT-N2 medium, which resulted collectively in from 2.4 x 13- to 2.9 x 14-fold killing. Of the survivors of vrious counterselections, 1 to 63% filed to grow or grew poorly on pltes of LT medium incubted in n tmosphere of N2, but grew well on LT-nitrte pltes under the sme conditions of incubtion. A totl of 22 independent mutnts unble to grow with N2 s the sole electron cceptor, designted Nos, were selected for further exmintion. Exmintion of Nos mutnts. Ech of the 22 mutnt strins ws exmined (Tble 1) with respect to the rte t which it grew nerobiclly in LT-N2 medium. In ddition, extrcts of ech mutnt were exmined by using two-dimensionl O'Frrell gels. By compring gels of extrcts of prentl nd Nos mutnt strins grown erobiclly nd nerobiclly on LT-nitrte medium, we found three protein spots ssocited Downloded from on My 8, 218 by guest

3 VOL. 169, 1987 Cu IN N2O REDUCTASE OF P. STUTZERI 5723 A * _WP B 4 FIG. 1. O'Frrell gels showing the loction of proteins ssocited with the reduction of N2 to N2. An extrct contining 23,ug of protein from the prentl strin (JM64) grown nerobiclly on LT-N2 medium ws seprted in two dimensions by the method of O'Frrell nd stined with Coomssie blue (see the text). The loctions of NosA (A) nd N2 reductse (13) re indicted by the rrows. Some strins tht filed to synthesize NosA synthesized new protein tht ws not present in extrcts of the prentl strin; this protein ppers in the region indicted by the circle (E). with the Nos phenotype (Fig. 1). On the bsis of moleculr weight, isoelectric point, nd comigrtion with the purified protein, one of these protein spots ws identified s being N2 reductse. The other protein spot ws designted NosA. Some of the Nos mutnt strins filed to produce N2 reductse; others filed to produce NosA. Some produced neither protein; others produced both of them. The muttions in strins lcking NosA were designted nosa; those lcking N2 reductse were designted nosb. Of the 12 mutnt strins tht did not produce NosA, three produced new protein, designted protein E (Fig. 1). These strins s well s those tht produce both NosA nd N2 reductse were designted nos. Enzymtic ctivity of mutnt strins lcking NosA. Tht strins lcking N2 reductse re unble to'grow nerobiclly with N2 s the sole electron cceptor ws expected, but we were surprised to find tht second protein, NosA, is lso needed. Some mutnts lcking NosA re completely incpble of reducing N2 s well s being unble to utilize it s electron cceptor becuse when grown nerobiclly on nitrte they produce exclusively N2 (dt not shown). To determine wht role NosA might ply in the metbolism of N2, extrcts of mutnt strins lcking this protein were ssyed for their bility to reduce N2 t the expense of methyl viologen (Tble 2). All strins lcking NosA, regrdless of whether they produced n N2 reductse protein s judged by O'Frrell gels, were inctive in this ssy. N2 reductse from nosa strins lck copper. Since the methyl viologen ssy mesures N2 reductse ctivity, we presumed tht the N2 reductses produced by nosa strins were defective in some uniform wy. A likely possibility for this defect ws lck of copper in the protein. N2 reductse hs been shown to contin copper (5), nd mutnt strins tht produce copper-free N2 reductse hve been shown to lck ctlytic ctivity for N2 reduction by in vitro ssys (15). To determine whether the enzyme from vrious mutnt strins contined copper, N2 reductse from them ws prtilly purified by mmonium sulfte precipittion nd ion-exchnge column chromtogrphy, using Whtmn DE52 cellulose (see bove). Purifiction ws guided by one-dimensionl SDS-PAGE. It ws pprent during purifiction tht the properties of N2 reductse from wild-type nd nosa mutnt strins differed: this protein from the wild type cme off the DE52 column within the void volume of the column; the protein from nosa mutnts ws retined on the column, being eluted by the beginning of to.4 M NCl grdient. The copper contenlt of the prtilly purified preprtions of N2 reductse ws determined by tomic bsorption spectroscopy, nd the proportion of N2 reductse in the preprtions ws determined from densitometer scns of one-dimensionl SDS-PAGE. In' ll cses, the copper content of N2 reductse from nosa mutnts ws less thn 3.5% of the copper content of N2 reductse from the wild type (Tble 3). Concluding tht the inctivity of N2 reductse from nosa strins ws consequence of the bsence of copper, we performed vriety of experiments to restore N2 ctivity to extrcts of nosa strins by dding copper ions to them. All these ttempts were unsuccessful. Cellulr loction of NosA. Suspecting tht NosA might ply role in bringing copper ions into the cell, we sought to determine the cellulr loc'tion of this protein. We found, using O'Frrell gels to nlyze the content of NosA in the Downloded from on My 8, 218 by guest

4 5724 MOKHELE ET AL. TABLE 1. Properties of ICR-191-induced Nos mutnts Strin Relevnt Proteins produced Growth rte on Strin genotype A B E N2Ob (h-) JM64 Prent JM752 nos JM753 nosal NDc JM755 nos JM757 nosaj ND JM759 nosa ND JM761 nosbj ND JM764 nosaj ND JM765 nosb ND JM766 nosa ND JM769 nosa ND JM771 nos ND JM772 nos ND JM773 nos ND JM774 nos ND JM779 nosb ND JM78 nosa ND JM782 nosa ND JM786 nosa ND JM788 nosa JM789 nos ND JM79 nosa ND JM792 nos ND Presence (+) or bsence (-) of spots on O'Frrell gels corresponding to NosA (A), N2 reductse (B), nd new protein (E). b Specific growth rte on LT-N2 medium t 37 C. cno growth detected over period of 12 h. soluble nd insoluble frctions of cells broken in French pressure cell, tht this protein ws ssocited exclusively with the pellet derived by centrifugtion t 24, x g for 2 h. To determine whether NosA ws ssocited with the inner or the outer membrne, the membrnes were seprted by using modifiction of the method of Hncock nd Nikido (6). Centrifugtion of the membrne preprtion in sucrose grdient reveled three distinct bnds of turbidity. The top bnd, which ws red, ly in the region between 1.5 nd 2.6 cm from the top of the tube; the middle bnd, which ws drk ornge, ly between 5.4 nd 6.2 cm; the bottom bnd, which ws light ornge, ly between 7.5 nd 8. cm. The top TABLE 2. Strin Activity of N2 reductse in extrcts of the prentl strin nd certin Nos mutnts N2 reductse sp ct Prent (JM64).13.6 nosb JM JM nosa JM JM JM JM JM JM <.2 <.2 <.3 <.3 <.2 <.2 <.2 <.2 TABLE 3. Copper content of N2 reductse from the prentl strin nd certin mutnt strins (nosa) tht filed to produce NosA Copper content N2 reductse Copper content of smple content Strin (ng/mg of of of smple"(b) reductsec N2 protein) (ng/mg) Prent (JM64) 1, ,83 nosa JM JM JM JM Copper in the totl smple subjected to SDS-PAGE. b The frction of the smple tht ws N2 reductse s determined by scnning with densitometer. c Clculted by dividing the copper content of the totl smple by the frction of the smple tht ws N2 reductse. bnd hd those properties usully ssocited with the inner membrne-high lctte dehydrogense nd succinte dehydrogense ctivities nd low concentrtion of 2-keto-3- deoxyoctonic cid. The lower bnd hd those properties usully ssocited with the outer membrne-low lctte dehydrogense nd succinte dehydrogense ctivities nd high concentrtion of 2-keto-3-deoxyoctonic cid (Tble 4). O'Frrell gels reveled tht lmost ll of the NosA ws in the lower bnd (outer membrne frction) (Fig. 2). Is NosA necessry to llow copper ions to enter the cell? The loction of NosA in the outer membrne cused us to suspect tht it might ply role in bringing copper into the cell. This did not seem to be the cse. In n experiment in which vrious levels of CuS4 (1, 2, 4, 6, nd 8,uM nd 1., 2., nd 4 mm) were present in LT-N2 medium, the prent strin (JM64) filed to grow t concentrtions of copper in excess of 1 mm, presumbly owing to toxicity. The nosa mutnt JM753 filed to grow in ny of these medi. Strin JM753 cn grow in LT-nitrte medium if it contins less thn 1 mm copper but not if it contins more thn this mount. Thus, we conclude tht copper probbly penetrtes prentl nd nosa mutnt strins, but it does not permit the growth of nosa mutnts t the expense of N2. DISCUSSION Mutnt strins tht do not produce NosA synthesize ctlyticlly inctive form of N2 reductse tht lcks copper. Thus, NosA must be essentil either to the bringing of copper ions into the cell or to the insertion of them into N2 reductse. The findings tht strins possessing nd TABLE 4. Properties of the three bnds of membrnes seprted by centrifugtion in sucrose grdient Protein Sp ctb KDOc Frction content (nmol/mg (%) LDH SDH of protein) Top (inner membrne) Middle Not done Bottom (outer mnembrne) Percentge of protein pplied to the grdient tht ws found in the bnd indicted. " Specific ctivity of lctte dehydrogense (LDH) nd succinte dehydrogense (SDH) is expressed in micromoles of substrte oxidized per minute per milligrm of protein. C KDO, 2-Keto-3-deoxyoctonic cid. J. BACTERIOL. Downloded from on My 8, 218 by guest

5 VOL. 169, 1987 Cu IN N2 REDUCTASE OF P. STUTZERI A * "I. _. X 1 e e I.. U I *~~~ -lo.t _~. I _n _ m _w q X _%6 *4.. r _... _S * AP e 46 em wo -dp e81 B Downloded from on My 8, 218 by guest. FIG. 2. O'Frrell gels of proteins from the purified outer (A) nd inner (B) membrnes of P. stutzeri JM64 grown nerobiclly in LT-nitrte medium. The loction of NosA is indicted by the rrow. Equl mounts of membrne protein (3,ug) were pplied to the two gels. To identify the loction of NosA in these gels, totl cell extrcts of strin (JM753) tht produces no NosA were coelectrophoresed with the extrcts of the membrnes (not shown); the resulting pttern of spots identified NosA. We lso rn O'Frrell gels (not shown) of extrcts from the outer membrne of strin JM753; these gels lcked the spot identified s NosA. lcking NosA exhibit the sme sensitivity to exogenous cupric ions nd tht rnge of lesser concentrtions of cupric ions fils to llow nosa strins to grow with N2 s the sole electron cceptor seem to eliminte the possibility tht NosA is necessry to bring sufficient copper into the cell to synthesize ctive N2 reductse. Rther, we conclude tht NosA plys the role of inserting copper into N2 reductse or mintining it there during ctive metbolism. There is no evidence tht copper in N2 reductse is ssocited with cofctor (5). Mutnt strins of P. stutzeri tht produce copper-free N2 reductse hve been previously described (15) nd ttributed to being defective in chromophore synthesis nd processing or copper uptke. Our results estblish tht NosA is essentil for insertion of copper into the protein or mintining it there. This conclusion is supported by our inbility to

6 5726 MOKHELE ET AL. restore in vitro ctivity to the copper-free reductse by dding copper ions under vriety of conditions. Similrly, Coyle et l. (5) could reintroduce copper into N2 reductse from which it hd been removed by cynide only by dding the copper-free enzyme to high concentrtions of copper (1 M) for prolonged periods (8 h), conditions tht were clerly not physiologicl. Snyder nd Hollocher (13) hve reported tht the in vitro ctivity of N2 reductse is chrcterized by wht they clled turnover inctivtion; i.e., the enzyme loses ctivity s it ctlyzes the reduction of N2 but not if it is held under similr environmentl conditions in the bsence of the substrte. It seems cler tht turnover inctivtion is n in vitro rtifct. The loction of NosA in the outer membrne seems to be curious one for protein tht inserts copper into n enzyme with metbolic function. We speculte tht N2 reductse might be locted in the periplsm nd, therefore, be in close proximity to NosA. In such loction, NosA might tke copper ions directly from the medium nd insert them into N2 reductse. Our preliminry results support periplsmic loction of N2 reductse. On theoreticl grounds, Boogerd et l. proposed (1) tht the H`-consuming side of N2 reductse is locted on the periplsmic side of the inner membrne of Prcoccus denitrificns. Zumft et l. (15) reported three phenotypes of TnSgenerted mutnts tht were unble to grow nerobiclly with N2 s the sole electron cceptor. One type filed to synthesize the N2 reductse protein; second type produced inctive protein tht lcked copper; nd third type produced low levels of reductse. Using frmeshift mutgenesis, we found the first two clsses nd ttributed the second to n inbility to produce NosA. We filed to find the third clss described by Zumft et l. but found two others. One of these did not produce NosA; rther, it produced new proteih tht we clled protein E. The other new clss produced both NosA nd N2 reductse s judged by O'Frrell gels. Since these mutnts were generted by frmeshift mutgenesis, the production of these two proteins with wild-type mobility on gels suggests tht the inbility of the mutnts to grow on N2 must be consequence of defect in gene other thn nosa or nosb. ACKNOWLEDGMENTS We re grteful to Curt Crlson for introducing us to the running of O'Frrell gels, to Tony Frnke for operting the tomic bsorption spectrophotometer, nd to Trcy Bennett for operting the lser densitometer. J. BACTERIOL. This work ws supported by grnt DE-FG3-85ER13356 from the Deprtment of Energy nd grnt 84-CRCR from the Deprtment of Agriculture. LITERATURE CITED 1. Boogerd, F. C., H. W. Vn Verseveld, nd A. H. Stouthmer Respirtion-driven proton trnsloction with nitrite nd nitrous oxide in Prcoccus denitrificns. Biochim. Biophys. Act 638: Booth, B., nd N. A. C. Curtis Seprtion of the cytoplsmic nd outer membrne of Pseudomons eruginos PAO.1. Biochem. Biophys. Res. Commun. 74: Brdford, M. M A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye binding. Anl. Biochem. 72: Crlson, C. A., L. S. Pierson, J. J. Rosen, nd J. L. Ingrhm Pseudomons stutzeri nd relted species undergo nturl trnsformtion. J. Bcterjol. 153: Coyle, C. L., W. G. Zumft, P. M. H. Kroneck, H. Korner, nd W. Jkob Nitrous oxide reductse from denitrifying Pseudomons perfectomrin. Purifiction nd properties of novel multicopper enzyme. Eur. J. Biochem. 153: Hncock, R. E. W., nd H. Nikido Outer membrnes of grm-negtive bcteri. XIX. Isoltion from Pseudomons eruginos PAO1 nd use in reconstitution nd definition of the permebility brrier. J. Bcteriol. 136: Kristjnsson, J. K., nd T. C. Holiocher First prcticl ssy for soluble nitrous oxide reductse of denitrifying bcteri nd prtil kinetic chrcteriztion. J. Biol. Chem. 255: Lemmli, U. K Clevge of structurl proteins during the ssembly of the hed of bcteriophge T4. Nture (London) 227: ".Lowry,. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: O'Frrell, P. H High resolution two dimensionl electrophoresis of proteins. J. Biol. Chem. 25: Osborn, M Studies on the grm-negtive cell wll. I. Evidence for the role of 2-keto-3-deoxyoctonte in the lipopolyscchride of Slmonell typhimurium. Proc. Ntl. Acd. Sci. USA 5: Pyne, W. J Denitrifiction. John Wiley nd Sons, New York. 13. Snyder, S. W., nd T. C. Hollocher Purifiction nd some chrcteristics of nitrous oxide reductse from Prcoccus denitrificns. J. Biol. Chem. 262: Stewrt, G. J., C. A. Crlson, nd J. L. Ingrhm Evidence for n ctive role of donor cells in nturl trnsformtion of Pseudomons stutzeri. J. Bcteriol. 156: Zumft, W. G., K. D4hler, nd H. Korner Isoltion nd chrcteriztion of trnsposon Tn5-induced mutnts of Pseudomons perfectomrin defective in nitrous oxide respirtion. J. Bcteriol. 163: Downloded from on My 8, 218 by guest