APPLIZD MICROBIOLOGY, Dec. 1975, p. 951-958Copyright © 1975 American Society for Microbiology

Vol. 30, No. 6Printed in U.SA.

Tetramethyl-p-Phenylenediamine Oxidase Reactionin Azotobacter vinelandii

PETER JURTSHUK, JR.,* 0. M. MARCUCCI, ANDDONALD N. McQUITTY

Department ofBiology, University ofHouston, Houston, Texas 77004

Received for publication 11 July 1975

It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD)oxidase reaction inAzotobacter vinelandii strain 0 using turbidimetrically stand-ardized resting cell suspensions. The Q(02) value obtained for whole cell oxida-tion of ascorbate-TMPD appeared to reflect the full measure of the high respira-tory oxidative capability usually exhibited by this genera of organisms. TheQ(02) value for the TMPD oxidase reaction ranged from 1,700 to 2,000, and thisvalue was equivalent to that obtained for the oxidation of the growth sub-strate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cellswas similar to that obtained for the "particulate"A. vinelandii electron transportparticle, that fraction which TMPD oxidase activity is exclusively associatedwith. Under the conditions used, there appeared to be no permeability problems;TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a ratecomparable to that of the growth substrate. This, however, was not true for theoxidation of another electron donor, 2,6-dichloroindophenol, whose whole cellQ(02) values, under comparable conditions, were twofold lower. The TMPDoxidase activity in A. vinelandii whole cells was found to be affected by thephysiological growth conditions, and resting cells obtained from cells grown onsucrose, either under nitrogen-fixing conditions or on nitrate as the combinednitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidaserates were also noted for chemically induced pleomorphic A. vinelandii cells,which suggests that modified growth conditions can (i) alter the nature of theintracellular terminal oxidase formed (or induced), or (ii) alter surface permea-bility, depending upon the growth conditions used. Preliminary studies on thequantitative TMPD oxidation reaction in mutant whole cells of both Azotobacterand a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxi-dase activity, showed this assay can be very useful for detecting respiratorydeficiencies in the metabolism of whole cells.

The para-phenylenediamines (particularlythe tetramethyl- and dimethyl-derivatives) areelectron donors that have been used in micro-biology primarily as qualitative (or partiallyquantitative) indicators of "oxidase" activity.Paul Ehrlich (3) was the first to use dimethyl-p-phenylenediamine and realize its significancein establishing the oxidizing capabilities ofvarious tissues. It was Gordon and McLeod(5), however, who first recognized the useful-ness of the p-phenylenediamines for measuringoxidizing activities in bacteria. Using a colorreaction, they were able to separate bacteriainto oxidase-positive or -negative groups. Thecolonies of oxidase-positive organisms, whenexposed top-phenylenediamines, develop a bluecolor. It was subsequently realized that alloxidase-positive bacteria possessed the "directoxidase" of Gordon and McLeod which referred

to an unidentified enzyme complex that carriedout p-phenylenediamine oxidations (7). Kovacs(19) standardized the oxidase test using thetetramethyl derivative N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD);later, Steel (22, 23), using Kovacs method,found the test taxonomically useful in classify-ing bacteria of the family Neisseriaceae andPseudomonadaceae. Since then, other modifi-cations have been utilized, such as using com-pounds like the oxalate derivative of p-amino-dimethylaniline (dimethyl-p-phenylenediamine)(4) or, by indirect methods, analyzing chemi-cally for the presence of intracellular heme orcytochromes with benzidine (2).

Paralleling the use of p-phenylenediamineoxidation in microbiological studies was its usefor quantitatively estimating cytochrome oxi-dase activity in tissues and subsequently in

951

952 JURTSHUK, MARCUCCI, AND McQUITTY

mitochondria (18, 25, 27). The oxidation-reduc-tion potential of TMPD (Eo' = +0.26 V at pH7.0) indicates that electrons enter the electrontransport chain at the level of cytochrome c (1,9), which then allows for TMPD oxidation bythe active cytochrome or terminal oxidase com-

plex. In enzyme assays, exogenously addedTMPD can be used to replace cytochrome c,

making it possible to quantitate the terminaloxidase activity particularly in those bacterialelectron transport systems where mammalian(horse heart) cytochrome c generally serves as

a poor electron donor (17, 21).Yamagutchi (28) first demonstrated that it

was possible to measure quantitatively theterminal (or cytochrome) oxidase activity inbacterial whole cells by using p-phenylenedi-amine and dimethyl-p-phenylenediamine. Amanometric assay system showed that both ofthese electron donors could be readily oxidizedby a wide variety of heterotrophic bacteria.This reaction, in whole cells, was also shownto be cyanide sensitive. In this report we

have continued with this type of study andexamined TMPD oxidation in Azotobacter vine-landii whole cells. We were able to showthat in organisms of this genera, which hadlong been suspected to possess one of themost active bacterial oxidases known, TMPDoxidation can be measured quantitatively withthe high rates usually observed for mostgrowth substrate oxidations. Unlike Neisseriaspp., which exhibit whole cell TMPD oxidaserates that are 10 to 20 times greater than thatobserved for growth substrate oxidations (15),in A. vinelandii the TMPD oxidase rate isequivalent to the rate of oxidation observed forthe growth substrate, particularly for restingcell suspensions grown on acetate, under nitro-gen fixing conditions, or on nitrate serving as

the nitrogen source. In both A. vinelandii (12,14, 17) and Neisseria catarrhalis (16), theTMPD oxidase activity concentrates exclusivelyin the subcellular electron transport particle.

In this report it will be shown that: (i) TMPDoxidase activity can be measured in A. vine-landii whole cells without permeability prob-lems; and (ii) TMPD oxidation by whole cellapparently does measure the cytochrome-de-pendent terminal oxidase reaction. These find-ings led us to conclude that the TMPD oxidaseassay, as first employed by Yamagutchi (28),will probably be very useful in examining thedegree to which TMPD oxidation occurs inwhole cells of other oxidase-positive bacteria,and that this assay will probably become a use-

ful technique for examining respiratory-defi-cient mutants in A. vinelandii as well as in

any other organisms that have previously beenshown to lack cytochrome oxidase activity.

MATERIALS AND METHODSPreparation of resting cells. All organisms

used in this study were grown at 30 C in low-formculture flasks placed on a reciprocal shaker (72cycles per min). Routinely, A. vinelandii strain 0cells were grown under nitrogen fixing conditionsusing modified Burk's medium with 1% (wt/vol)sodium acetate, or sucrose, as the sole carbonsource (17). On occasion, potassium nitrate (5 mm)or nutrient broth (Difco) was added as the exoge-nous nitrogen source. Pleomorphic A. vinelandiistrain 0 cells were induced by growth on 5% peptone(Difco) as described in detail by Vela and Rosenthal(26). Mucor bacilliformis was grown in a 1% pep-tone broth (Difco) supplemented with 0.5% yeastextract and 5% glucose, pH 5.9 to 6.0 (24).

All cells were harvested by centrifugation aftergrowth to the late logarithmic phase, suspended incold 0.02 M phosphate buffer, pH 7.5, and allowedto sit overnight at 4 C to reduce the endogenousintracellular reserve by starvation. The followingday the cells were washed with freshly prepared,cold phosphate buffer and standardized turbidi-metrically so that a 1/100 dilution of a bacterialsuspension (in 0.02 M phosphate buffer) gave anoptical density reading between 0.70 and 0.80 at420 nm.

Preparation of Azotobacter electron transportparticle (R3). The A. vinelandii electron transportfraction (13) was prepared as previously described(12-14, 17).

Chemicals and chemical methods. The sourcesof the electron donors used in this study are:TMPD; 2,6-dichlorophenolindophenol (DCIP); andL-(+)-ascorbic acid from Eastman Organic Chemi-cals, Rochester, N.Y. All other chemicals were ofreagent grade quality and were obtained from theusual commerical sources (12-17).A 0.1 M solution ofTMPD and a 0.04 M solution of

DCIP were prepared in deionized water immediatelybefore use and kept at 4 C in a low actinicglass test tube. A 0.1 M solution of ascorbicacid was always freshly prepared; the pH wasadjusted to 6.2 with KOH. Protein concentrationswere determined by using the biuret method ofGornall et al. (6).

Assay for TMPD oxidation. The conventionalmanometric technique, using Warburg vessels, wasused to measure TMPD oxidase activity with turbi-dimetrically standardized resting cell suspensions.All assays for terminal oxidase activity were initi-ated by the simultaneous addition (from separateside arms) of ascorbate and either TMPD or DCIP.The TMPD oxidase assay was used exactly in themanner previously described (12, 14) except that thereaction was carried out at pH 6.0 at 30 C. DCIPoxidase activity was measured in the identicalmanner employing 2.6 mM DCIP and 6.7 mMascorbate. The resting cell concentrations usedfor estimating TMPD oxidase activity ranged from0.09 to 0.12 mg dry weight and that for DCIP oxidase

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OXIDASE REACTION IN AZOTOBACTER 953

activity ranged from 0.18 to 0.21 mg dry weight.For both the TMPD and DCIP oxidase assays,

suitable controls were always incorporated into themanometric assay system to insure that ascorbateoxidation did not occur with resting cells prior tothe addition of TMPD (or DCIP) in the assay, andthat no chemical (non-enzymic) autooxidation reac-tion occurred (12) that would have obscured theterminal oxidase reaction with A. vinelandii wholecells.

The manometric assay technique was also em-ployed in measuring the resting cell oxidation ratesof the growth substrate, e.g., acetate or sucrose. Theconditions used are identical to those previouslydescribed for the Neisseria studies (15) which werecarried out at pH 7.5 employing a final substrateconcentration of 16.7 mM. This same techniqueand conditions were used for measuring the rates ofDL-lactate and succinate oxidation by the A. vine-landii R3 electron transport particle (13, 14).

RESULTS

The metabolic patterns for acetate oxida-tion as well as the two electron donors, TMPDand DCIP, by standardized whole cell sus-pensions of A. vinelandii are shown in Fig. 1.The cells used in this study were grown onacetate as the sole source of carbon undernitrogen-fixing conditions. The microliters of 02consumed was plotted as a function oftime. Thekinetics show that the rate of ascorbate-TMPDoxidation is slightly higher but almost identicalto that of acetate oxidation, both exhibitinghigh and comparable Q(02) values (1,764 versus1,692) that are a common metabolic feature inAzotobacter metabolism. This finding suggeststhat there are no permeability problems; re-duced TMPD readily enters the cell and isoxidized at a rate comparable to that of thegrowth substrate. There was no detectableascorbate-TMPD (or DCIP) oxidation inmA vine-landii cells inactivated by boiling and no de-tectable ascorbate oxidation in the absence ofeither TMPD or DCIP. The rate ofTMPD oxida-tion in the absence of ascorbate is decreased[Q(02) = 7571 to less than one-half the rate aswhen ascorbate is present in the assay. For theother electron donor system, which is comprisedof ascorbate-DCIP, the Azotobacter restingcells exhibited Q(02) values of about one-half[Q(02) = 806] of that noted for the ascorbate-TMPD or acetate oxidations. Therefore it ap-pears that the ascorbate-TMPD oxidation capa-bility, measured in resting cell suspensions ofA. vinelandii strain 0, truly reflects the fulloxidative-respiratory potential exhibited by thisorganism, and there appear to be no problemsconcerning electron donor permeability relativeto that noted for acetate metabolismThe results presented in Fig. 2 show the ef-

E

=U

la

a0

Resting Cells Oxidation

0 5 10 15 20Time (min)

FIG. 1. The metabolic patterns for oxygen con-sumption observed during the oxidation ofascorbate-TMPD, acetate, ascorbate-DCIP, and TMPD (in theabsence of ascorbate) by turbidimetrically stand-ardized resting cells ofA. vinelandii strain 0. Rest-ing cells were grown on acetate under N7fixingconditions. The oxygen consumed (microliters of 02uptake) was plotted as a function of time. The stand-ard manometric assay was carried out as describedin the text. The concentration of resting cells usedwas 0.097 mg dry weight, which was kept constantthroughout this study. The specific activities calcu-lated for the oxidation of acetate and the variouselectron donor systems are shown on this figure andexpressed using the conventional Q(0O) value(microliters of oxygen consumed per hour per milli-gram dry weight) at 30 C.

fects of varying the electron donor concentra-tions for both the ascorbate-TMPD and ascor-bate-DCIP oxidation systems, again employingA. vinelandii resting cells as well as theelectron transport particle or R% fraction (14, 17).The resting cells used in this study also weregrown on acetate (under nitrogen-fixing condi-tions), and the sonic R, electron transport frac-tion was isolated from the same batch of cells.In this study the oxidation rate, expressed asthe Q(02) value, is plotted as a function ofthe molar concentrations of the electron donorpresent in the assay. The ascorbate concentra-tion was kept constant at the 6.7 mM concentra-tion level.The kinetics of the ascorbate-TMPD oxida-

tion is similar (almost parallel) between thestandardized resting cell suspension (dark cir-cles) and the R3 electron transport fraction(open circles), with the notable exception that

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954 JURTSHUK, MARCUCCI, AND McQUITTY

the R3 fraction exhibited a higher specific ac-

tivity value for TMPD oxidation. This would beexpected since the R3 fraction represents a

partially purified membrane preparation whichcontains the majority of the electron transportenzymes (12-14, 17). The similarity in theTMPD oxidation patterns that are exhibited bythese two curves again suggests that there is nopermeability problem regarding reduced TMPDentering the Azotobacter cell. The concentra-tion ofTMPD used for the standard manometricassay was 3.3 mM, which gives maximal specific

I 'I2400_ Avivnulondii 0

'o Acetote-NF grown cellsen MTPD +AeArbot,

2000

It ~~~~~~~~~~MPD+Ascorbot.

1200

~~~~0OCIP +

E 800

000<O DCIP

400 QAscorboe.

10 o-nMolar Concentration of Electron Donor

FIG. 2. Effect of varying concentrations of elec-tron donor on enzymatic oxidation of TMPD andDCIP by standardized suspensions of A. vinelandiiwhole cells (closed circles) and R3 electron transportfraction (open circles). The manometric assay was

used as described. The concentration of ascorbatewas kept constant at the 6.7 mM level. The dryweight concentrations of the Azotobacter R3 fractionused for measuring TMPD and DCIP oxidationswere 0.75 and 1.5 mg/ml, respectively. The dryweight concentrations for the Azotobacter restingcells was 0.12 mg/ml for TMPD oxidation and 0.21mg/ml for DCIP oxidation.

activities for both resting cell oxidation as wellas the oxidation carried out by the electrontransport particle. The kinetics ofTMPD oxida-tion by the Azotobacter R3 fraction has alreadybeen described in detail (12).

The kinetics of ascorbate-DCIP oxidationbetween resting cells and the R, fraction are

markedly different than that noted for TMPDoxidation. As shown by the two lower curves inFig. 2, the DCIP oxidase activities are also sub-stantially lower in comparison to TMPD oxi-dation for both resting cells (dark circles) andthe E electron transport fraction (open circles).At an electron donor concentration of 3 mM, theTMPD oxidation rate for resting cells was twotimes greater than that noted for DCIP oxida-tion, whereas TMPD oxidation by the Azoto-bacter R3 electron transport particle was approx-

imately four times greater than that observedfor DCIP oxidation. For the standard mano-

metric assay ascorbate-DCIP oxidase assay thefinal concentration of DCIP used was 2.6 mM.

Table 1 shows a composite summary compar-

ingA. vinelandii resting cell oxidations to thatof the R% electron transport fraction, using boththese electron donors as well as various sub-strates, two of which (D-lactate and succinate)are known to be oxidized directly by the non-

pyridine nucleotide-dependent electron trans-port system in A. vinelandii (13, 14). TheAzotobacter resting cells and the R3 fractionwere prepared from the same batch of cells foreach of the three experiments. All were grownon acetate under nitrogen-fixing conditions.The oxidation rates for the electron donors,TMPD and DCIP, are shown in a comparativerelationship to the oxidation rates of some ofthe substrates known to be directly involved inA. vinelandii electron transport reactions. Theresults also show the extent to which variabil-ity can occur during repetitive Q(02) estima-tions for both the resting cells and the R3 elec-tron transport fraction.

TABLE 1. Comparative substrate and electron donor oxidation rates for A. vinelandii resting cells and R3electron transport fraction

Q(02) values"

Substrate Resting cells R.3 fraction

1 2 3 1 2 3

None (endogenous) 10 9 12 0 0 0Acetate 1,453 2,075 1,764 0 0 0Sucrose 12 7 11 0 0 0D,L-Lactate 567 632 499 81 80 137Succinate 686 729 530 454 383 334Ascorbate-TMPD 1,851 1,841 1,840 2,582 2,747 2,559AscorbateDCIP 878 669

a Expressed in microliters of 02 consumed per hour per milligram dry weight at 30 C.

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OXIDASE REACTION IN AZOTOBACTER 955

Acetate was oxidized readily by the restingcells, but not by the R3 fraction which lacksnecessary soluble cofactors and enzymes. As ex-pected, sucrose was not oxidized by the restingcells or the R3 fraction. The oxidation of thiscarbohydrate by whole cells is inducible. D,L-Lactate and succinate, two substrates that canbe oxidized directly by the electron transportsystem ofA. vinelandii, were oxidized at higherrates by the resting cells than the H3 fraction,suggesting that the integrated intracellularsystem of whole cells oxidized these substratesgenerating other metabolizable products thatcould be further oxidized by other intracellularenzymes. Of interest, however, is the fact thatthe ascorbate-TMPD oxidation rate by theAzotobacter resting cell preparation was com-parable to that of acetate but was approxi-mately three- to fourfold higher than the rateobserved for either D,L-lactate or succinate oxi-dation. As expected, the oxidation of ascorbate-TMPD by the R3 fraction was higher than theactivity noted for the ascorbate-DCIP oxidationby the standardized resting cell preparation(also see Fig. 2). Again, the DCIP oxidationrate was twofold lower than that noted for theascorbate-TMPD oxidation rate for the A.vinelandii resting cells and fourfold lower inthe R3 fraction, but still this DCIP oxidationrate was higher than that noted for D,L-lactateor succinate oxidation by either of the twooxidizing systems (cells and membrane parti-cles). Because of the lower activity rates, anddifference in the oxidation kinetics (see Fig.2), the DCIP oxidase activity was not examinedfurther as a potential electron donor for meas-uring terminal oxidase activity. This electrondonor has been used extensively for studyingterminal or cytochrome oxidase reactions by theAzotobacter electron transport system (10, 11);in another instance menadione was also usedfor this purpose (20).Table 2 shows comparative Q(02) value data

obtained for both ascorbate-TMPD and growthsubstrate oxidations by standardized restingcell suspensions of A. vinelandii grown underdifferent nutritional conditions. The ascorbate-TMPD oxidation rate levels were high and com-parable for resting cells grown on acetateregardless ofwhether or not nitrate was used asa nitrogen source or if growth had occurred un-der nitrogen-fixing conditions. However, rest-ing cells prepared from cells grown on sucrose,either in the presence of nitrate or under nitro-gen-fixing conditions, showed relatively lowTMPD oxidation activities which were de-pressed when compared to the oxidase rate ob-tained for sucrose oxidation. This observationstrongly suggests that the high sucrose oxidase

rates observed for sucrose-grown resting cells(either with N03 or N2 as nitrogen sources)might be mediated by a different type of termi-nal oxidase, or that the sucrose-grown cells hada new cell surface impermeable to TMPD. Rest-ing cells of A. vinelandii grown on nutrientbroth, with sucrose present, again exhibitedrelatively high ascorbate-TMPD oxidase ratescomparable to those found for acetate-growncells. In these cells, the TMPD oxidase ratewas twofold higher than the sucrose oxidaserate, the latter also being relatively active[Q(02) = 5411, but approximately one-half thesucrose oxidase rates noted for the resting cellsgrown on sucrose with either N2 or N03[Q(02) = 1,159 or 1,341, respectively] servingas the nitrogen source. The study strongly sug-gests that a major change in TMPD oxidaserates can occur in resting cells grown underdifferent physiological conditions, e.g., using adifferent carbon source.

In Table 3, Q(02) values are presented forascorbate-TMPD oxidase activities for the par-ent Azotobacter strain 0 resting cells, and thisvalue is compared to those obtained for somerespiratory-deficient mutants. The latter repre-sent A. vinelandii strain 0 strains that wereexposed to nitrosoguanidine. Selection wasmade for slow growing and petite-like colonies(JIG series). Also shown are the TMPD oxidaseQ(02) values for A. vinelandii strain 0 pleo-morphic cells that were chemically induced bygrowth on 5% peptone (26) as well as a M.bacilliformis strain from which a respiratory-deficient mutant was isolated and shown to belacking the cytochrome oxidase enzyme com-plex (24).

The results in Table 3 show that strain 0respiratory-deficient resting cells, i.e., mutantstrains JIG-1 and JIG-2, exhibit lower (approxi-mate fivefold) TMPD oxidation rates when com-pared to the wild-type strain 0 cells. This signif-icant decrease in TMPD oxidase activity mightpossibly indicate a major alteration in the typeofterminal oxidase formed. Pleomorphic restingcells ofA. vinelandii also show a markedly de-creased TMPD oxidase rate. Pleomorphic cellsobtained after 48 h of growth show an eightfolddecrease in TMPD oxidase activity, whereaspleomorphic cells obtained after 72 h of growthshow a 28-fold decrease in TMPD (terminal) oxi-dase activity. An analysis of the electron trans-port components in A. vinelandii pleomorphiccells will be made at a future date.

Table 3 also shows the results in the quanti-tation of the TMPD oxidase reaction usingwhole cells suspensions of eukaryotic cells,specifically M. bacilliformis. The two Mucorstrains were analyzed for TMPD oxidase activity

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956 JURTSHUK, MARCUCCI, AND McQUITTY

TABLE 2. The effects of different growth components on the oxidation ofascorbate-TMPD and sucrose byresting cells ofA. vinelandii

Q(02) valuesaGrowth components

pH 6.0 pH 7.5

Carbon source Nitrogen source Endogenous5 TMPD Endogenousb Sucrose

Acetate N2 (air) 36 1,247 2 4Acetate NO3 20 1,126 8 9Sucrose N2 (air) 8 132 25 1,159cSucrose NO3 7 181 15 1,341cSucrose NBd 22 1,221 19 541

a Microliters of 02 per hour per milligram dry weight at 30 C.b Endogenous value represents the cellular respiration rate obtained in the absence of any added

substrates or electron donor at the pH indicated.c From the data of Zehner (Masters thesis, University of Houston, 1972).d NB, Nutrient broth (Difco).

TABLE 3. Ascorbate-TMPD oxidase activities for standardized resting cell suspensions of wild-type andrespiratory-deficient mutants ofAzotobacter vinelandii and Mucor bacilliformis

Growth components Q(02) valuesaOrganisms

Carbon source Nitrogen source Endogenousb TMPD

A. vinelandii strain 0Wild type Sucrose NBC 22 1,221Mutant JIG-ld Sucrose NB 2 243Mutant JIG-2d Sucrose NB 5 293Pleomorphic cellse (48 h) Peptone (5%) Peptone (5%) 2 167Pleomorphic cellse (72 h) Peptone (5%) Peptone (5%) 1 45

M. bacilliformisWild type Glucose Peptone (1%) 8 32Mutant AY1' Glucose Peptone (1%) 1 1

a Microliters of 02 per hour per milligram dry weight at 30 C.b Endogenous value represents the cellular respiration rate obtained at pH 6.0 in the absence ofany added

electron donor.c NB, Nutrient broth (Difco).d Respiratory-deficient mutants of strain 0.e Pleomorphism induced by nutritional control (see reference 26).' Respiratory-deficient mutant of M. bacilliformis which lacks cytochrome oxidase (see reference 24).

(using yeast-phase cells) and one of these strainsis a well-known respiratory-deficient mutant(AY1) which was previously characterized (24)and shown to lack a cytochrome oxidase en-zyme complex. As shown by the data, the wild-type Mucor strain had a low but measurableTMPD oxidase rate. The respiratory-deficientmutant strain AY1, from M. bacilliformis,exhibited a TMPD oxidation rate that wasbarely measurable and equivalent to the Q(02)value obtained for the endogenous respiration.Again, it appears that the TMPD oxidase as-say described here will probably serve as auseful tool in quantitating respiratory de-ficiencies employing standardized resting cellsuspensions.

DISCUSSION

It has been shown that a TMPD oxidationassay can be used to quantitatively estimateterminal or cytochrome oxidase activity in A.vinelandii whole cells. The whole cell TMPDoxidase Q(02) value for A. vinelandii was equiv-alent to that of the growth substrate oxidationwhen an oxidizable substrate-like acetate wasemployed as the primary carbon source forgrowth. This was not the case, however, whenresting cells were grown on a fermentable sub-strate, i.e., sucrose, utilizing either N2 or NO3as the nitrogen source for growth. With thelatter type resting cell preparations, there maybe another terminal oxidase present, one that

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OXIDASE REACTION IN AZOTOBACTER 957

exhibits high turnover rates for sucrose (but notTMPD) oxidase activity or sucrose-grown cellsare impermeable to reduced TMPD (see Table2). This type ofTMPD oxidase analysis, whichemployed turbidimetrically standardized rest-ing cell suspensions, also was successfullyused in studies in terminal oxidase activity invarious Neisseria spp., which are universallyknown to possess a strongly positive oxidase re-action (15). With the Neisseria spp., it wasshown that the enzymic whole cell TMPD oxi-dase activities were 10 to 20 times more activethan the growth substrate oxidation rates. Thisdata strongly suggests that electrons (fromTMPD) are capable of entering the terminaloxidase site in Neisseria whole cells at ratesfaster than the electrons internally generatedby substrate oxidations. This again suggeststhat the TMPD oxidase assay, even in Neisseriasp., can be effectively used to measure terminaloxidase activity in whole cells with negligiblepermeability problems. This might be expectedsince the enzymes that would carry out TMPDoxidation would be the membrane-bound ter-minal oxidases that are localized on the cyto-plasmic membrane. The localization of theterminal oxidase complex on the cytoplasmicmembrane suggests that reduced TMPD neednot penetrate far into the interior of the cell tobe enzymically oxidized. This, however, doesnot appear to be the case for reduced DCIP,whose oxidation by A. vinelandii whole cells,under the conditions described and published(10, 11), appears to be restricted (see Fig. 2).The fact that growth or environmental condi-

tions may alter terminal oxidase activity is awell known fact and is extensively described intwo review articles (8, 16a). The effect ofoxygenconcentration (during growth) is known tomarkedly alter the biosynthesis (and activity)of terminal oxidase enzymes (8), and the effectof growth substrate, whether it is capable ofbeing oxidized directly by molecular oxygen oris fermentable, would also play a major role inthe induction of terminal oxidase activity (16a).This point has already been clearly shown ex-perimentally by the results presented in Table2 with sucrose-grown resting cells. Finally, thephase of growth at which the cells are har-vested has also been shown to be a considera-tion in the type of terminal oxidase formed(16a), and this difference in terminal oxidaseactivity might reflect on the age of cells, whosebasic oxidative physiology (in the extreme form)might be reflected by data presented for thepleomorphic Azotobacter cells (Table 3). Pleo-morphic Azotobacter cells oxidize TMPD verypoorly in relation to the high turnover rates

observed for acetate-grown whole cells har-vested at the late logarithmic phase.The usefulness of the TMPD oxidase assay,

with resting cells, extends even further. It ap-parently can be used to analyze and select forrespiratory-deficient mutants. By use of thisassay it was possible to show with M. bacilli-formis (yeast phase) whole cells that a knownmutant (AY1), deficient in the cytochrome oxi-dase complex (24), did not have the ability tooxidize TMPD, whereas the wild-type parentreadily carried out this oxidation (see Table 3).The usefulness of this test could also be ex-tended to A. vinelandii cells, where it appearedthat respiratory-deficient mutant resting cellshad much lower TMPD oxidase rates. Verylittle as yet is known on the basic physiology ofthese respiratory-deficient A. vinelandii mu-tants.The TMPD oxidase assay as described in this

communication can serve as a useful tool to sur-vey large number of organisms and establishthe degree to which they may be respiratory de-ficient (or sufficient) in terminal oxidase ac-tivity. This implies, however, that such orga-nisms would have to be oxidase positive andhave as one of its major electron transportpathways a terminal oxidase that can be as-sayed for using the TMPD oxidation reactiondescribed. This would exclude the oxidase-negative organisms, many of which are knownto be obligately aerobic.

ACKNOWLEDGMENTSWe wish to thank R. L. Storck, of Rice University, for

providing the Mucor bacilliformis cultures used in thisstudy.

This study was supported in full by Public HealthService grant GM 17607-04 from the National Institute ofGeneral Medical Sciences.

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of certain phenols and of dimethyl-p-phenylenedi-amine by bacterial suspensions. Biochem. J. 24:1737-1743.

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