JOURNAL OF BACTERIOLOGY, Sept., 1965Copyright © 1965 American Society for Microbiology

Vol. 90, No. 3Printed in U.S.A.

Cyanide Resistance in AchromobacterI. Induced Formation of Cytochrome a2 and Its Role in

Cyanide-Resistant RespirationKEI ARIMA AND TETUO OKA'

Laboratory of Fermentation, Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan

Received for publication 4 June 1965

ABSTRACTARIMA, KEI (University of Tokyo, Tokyo, Japan), AND TETUO OKA. Cyanide resist-

ance in Achromobacter. I. Induced formation of cytochrome a2 and its role in cyanide-resistant respiration. J. Bacteriol. 90:734-743. 1965.-By following the cytochrome con-

centrations during the growth cycle and under various conditions (aerobic, aerobic plusKCN, reduced aeration, anaerobic plus NaNO3) in Achromobacter strain D, a close rela-tionship between the formation of cytochrome a2 (and a,) and the difficulty of oxygenutilization was demonstrated. Cytochrome o, which was the only oxidase found inaerobic log-phase cells, was present in bacterial cells grown under various conditions;the amount present had no relation to the degree of cyanide resistance. On the otherhand, cytochrome a2 (and a,) was inducible, and a close relation was observed betweenthe amount of cytochrome and resistance to cyanide. Spectrophotometric observationsindicated that, among the cytochromes present in resistant cells, cytochrome a2 couldbe oxidized most easily in the presence of cyanide and that cytochrome b1 could beoxidized without the oxidation of cytochrome a, . We concluded that cytochrome a2 is a

cyanide-resistant oxidase capable of catalyzing the oxidation of cytochromes in thepresence of cyanide. Cytochrome a2 is also resistant to azide, an inhibitor of cytochromeoxidase.

It was previously reported from this laboratorythat a strain of Achromobacter was found to giveadaptive growth and respiration in the presenceof 10-3 MKCN (Mizushima and Arima, 1960a, b,c). Because of the increased content of eachcytochrome, especially of cytochrome oxidases,it was postulated that cyanide-resistant respira-tion was caused by the increased amount of theoxidases (cytochromes a, and a2) which were in-hibited by cyanide. As the difference spectrum(reduced minus oxidized) obtained from cellsgrown in the presence of cyanide shows two dis-tinct peaks at 595 and 625 mu.i, it is necessary todetermine which oxidase plays the main role incyanide-resistant respiration. This report de-scribes studies on the terminal electron-transportsystems of this strain of Achromobacter undervarious growth conditions, and the role of cyto-chrome a2 in cyanide-resistant respiration.

MATERIALS AND METHODS

Organism and growth media. A doubly auxo-trophic mutant was induced by treatment ofAchromobacter strain 7 (Mizushima and Arima,1960a) with nitrous acid according to the method

l Present address: Kyowa Hakko Co., TokyoResearch Laboratory, Machida, Tokyo, Japan.

of Kaudewitz (1959). This mutant (Achromobacterstrain D), which requires isoleucine and adenine(or guanine) for growth, was used throughout thiswork.

Cells were cultivated at 30 C in four differentways: with vigorous aeration in a bouillon mediumcontaining 0.5% meat extract, 1.5% peptone, 0.5%NaCl, and 0.5% K2HPO4 (pH 7.0) (referred to asthe sensitive cells); with reduced aeration (one-tenth aeration); aeroblically in the bouillon me-dium containing W03 M KCN (the resistant cells);and anaerobically in the bouillon medium con-taining 2.0% NaNO3 (nitrate-grown cells). Vigor-ous aeration was achieved by passing through theculture a volume of air per minute equal to thevolume of the culture or by reciprocal shaking(120 oscillations per min) of 100 ml of the mediumin 500-ml Sakaguchi flasks. Growth was measuredturbidimetrically with a Kotaki nephelometer.

Preparation of cell-free extracts. Bacterial cellswere harvested at the growth phase indicated,washed twice with 0.8%G NaCl, and suspended inan appropriate volume of 0.1 M phosphate buffer,pH 7.4 [usually 50 mg (dry weight) of cells per ml];the cells were then exposed to sonic oscillation(Toyo Riko, 10 kc) for 10 min. The resultant turbidpreparation was centrifuged at 10,000 X g for 10min to remove cell debris and unbroken cells. Thesupernatant fluid was collected and used as crudecell-free preparation. For further fractionation,

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CYANIDE RESISTANCE IN ACHROMOBACTER. I

z

z

v;4

WAVE LENGTH (my )

FIG 1.)D ifference spectra of Achromobacter strain D grown under various conditions. (A) Cyanide-sensitit cells. (B) Cyanide-resistant cells. (C) Nitrate-grown cells. Solid lines, reduced minus oxidizeddifference spectra; dashed lines, reduced plus CO minus reduced difference spectra.

the supernatant fluid was again centrifuged at105,000 X g for 1 hr in a Spinco ultracentrifuge(model L), and the reddish precipitate was col-lected (particulate fraction).

Oxygen uptake. The activity of the succinoxidasesystem was measured by conventional manometrictechniques and by use of a rotatary platinumelectrode (Yanagimoto Manufacturing Co,Kyoto, Japan). Inhibition by cyanide was meas-ured manometrically with KOH equilibrated withcyanide in the center well, according to the method

of Krebs (1935). The activity of the reduced nico-tinamide adenine dinucleotide (NADH2) oxidasesystem was measured spectrophotometrically asdescribed by Mizushima and Arima (1960a).

Succinic dehydrogenase activity. Succinic de-hydrogenase was measured manometrically at30 C with phenazine methosulfate as an acceptoror spectrophotometrically with 2,6-dichloro-phenol-indophenol by the method of Guiditta andSinger (1959).Measurement of nitrate and nitrite reductase

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activities of cell-free preparations. Nitrate and The nomenclature of the cytochromes was that

nitrite reductase activities were measured by the used previously (Mizushima and Arima, 1960a).

increase or decrease of nitrite in the reaction mix- The definition of cytochrome o was that of Castorture. The reaction was stopped by the addition of and Chance (1959). Cvtochrome o wa maured

10% cold perchloric acid. After separating the from the at 572m7to the trough at 554 muprecipitate by centrifugation, nitrite was meas- or from4theoea o the trou m

ured by adding to the supernatant fraction 0.5 ml in _ yof 1% sulfanilamide and 0.5 ml of 0.02% N-(1-- measured from the peak at 416 m,u to the trough

naphthyl)-ethylenediamine. Absorbancy at 540 at 610 my in the reduced-oxidized spectra, but this

m,u was measured in a Hitachi EPU-2 spectro- estimation inevitably allows some error. Cyto-photometer. chrome a2 was measured from its maximum at 625

Nitric oxide formation by resting cells. Evolution mA to the trough at 650 m,u in the reduced-oxidizedof nitric oxide from nitrite was measured mano- ectra.metrically with nitrogen as the gas phase, accord-, Carbon monoxide spectra were obtained by

ing to the method of Najjar and Allen (1954). 'tubbling the gas into a cell-free preparation re-

Spectrophotometric measurements. The difference uced with Na2S204 and measuring the spectra

spectra of cell-free preparations were recorded- 'against a reduced preparation. The bubbling was

with a Cary recording spectrophotometer (model grepeated until saturation occurred. Neutralized

14), with the use of 0.1 to 0.2 optical-density slideraKCN (10-2 M) was added to the indicated concen-

wire. A cuvette with a 1-cm light path was used.The width of the slit was maintained under the rtions.limit of 0.3 mm. Physiological reduction and Protein. Protein was measured by the Folinm.dt_ p., yso garoduced bytnadding method, as modified by Hagihara (1956).OXidation of cytochromes were produced by adding Biceia egnsAH'a) hnzn

to the cuvette a few milligrams of sodium suc- Biochemical reagents. NADH2(Na2), phenazine

cinate and bubbling with air or by adding a few methosulfate, and 2-N nonyl-hydroxyquino

milligrams of KNO3 or NaNO2 . Oxidation by line-N-oxide (HQNO) were purchased from Sigma

nitrate or nitrite was measured in a Thunberg- Chemical Co., St. Louis, Mo.type cuvette with nitrogen as the gas phase. Thecytochromes were also reduced chemically by RESULTS

adding to the cuvette a few milligrams of Na2S204 Cytochrome systems of cells grown under variousand were oxidized by K3Fe(CN)6 . conditions. The bacterium was cultivated in four

Spectra were measured by use of the difference- cdi cecrium wascutriv inafourspectra methods described by Chance (1954). different ways. Spectrophotometric analysis

Absorption spectra were measured against a showed that the main respiratory pgments of

similar cell-free preparation containing the oxi- cells grown with vigorous aeration were cyto-dized cytochromes. chrome bi and cytochrome o (Fig. 1A and Table

TABLE 1. Cytochrome contents of Achromobacter grown under various conditions

Change in optical densitya of cytochromeGrowth conditions ation Maxim1 . al - - KCN concnc

time turbidity bi a as 0b(560-540) (595-610) (625-650) (418-432)

hrM

AerobicLog phase (sensitive

cells)..... 0.5 300 0.0144 A d 4 0.100 10-5Stationary phase ... 1,200 0.0164 0.0010 0.0037 0.100

Reduced aeration (one-tenth aeration)..... .. 2-3 180 0.0255 0.0015 0.0080 0.065 8-10 X 104

Aerobic plus 10-3 MKCN (resistant cells). 1 600 0.0260 0.0020 0.0100 0.060 1-2 X 10-3

Anaerobic plus 2%NO3- (nitrate-growncells).... 2-3 200 0.0365 0.0005 0.0044 0.0690 5 X 104

a The change in optical density, at the wavelengths (millimicrons) indicated in parentheses, wasmeasured from the difference spectrum (Na2S204 reduced minus oxidized) per 10 mg of protein per mlper cm of light path.

b Calculated from the CO difference spectrum.¢ Concentration of cyanide required for 50% inhibition of respiration. Experimental conditions were

the same as in Table 4.d May or may not be present.

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CYANIDE RESISTANCE IN ACHROMOBACTER. I

GROWTH CURVE

_ 1-11 (IV)- ~~~~~~(1ff)

(I)

I1 2 3 4hr

O.D 0).01

GROWTH CURVE

) k

C).D 0.01

550 000 650

WAVX 1: 1 X\( TI 111/1FIG. 3. Changes of the spectra (reduced minus

oxidized) after the addition of cyanide (10-3 m) toaerobic culture. 0, I, II: sampling points. Proteinconcentrations: 0, 15.0 mg/ml; 1, 13.9 mg/ml; II,

17.9 mg/ml.

550 600 650

WAVE LENGTH (m,u)

FIG. 2. Changes of the spectra (reduced minusoxidized) with the change of the growth phase inaerobic culture. I, II, III, IV: sampling points.Pr6ioin concentrations: I, 18.7 mg/ml; II, 18.7mg/ml; III, 16.7 mg/ml; IV, 16.1 mg/ml.

1). The same bacterium grown aerobically in thepresence of 10-s M KCN formed an electron-transport system consisting of cytochrome bl,cytochrome al, cytochrome a2, and cytochromeo (Fig. 1B). These resistant cells produced abouttwice as much cytochrome b1 and more than 10times as much cytochromes a, and a2, but thecontent of cytochrome o was lower than in aerobiccells.When the bacterium was grown with limited

aeration (one-tenth of vigorous aeration), thecytochrome pattern developed was the same asthose of the resistant cells, as reported previously(Mizushima, Oka, and Arima, 1960).

Cells grown in deep standing culture withnitrate contained three times as much cytochromebi and a few times as much cytochromes a, anda2 as the sensitive cells (Fig. 1C). The large con-tent of the cytochrome b, is thought to be relatedto the high level of activity of the nitrate reduc-

TABLE 2. Changes of the cytochrome contents*during adaptation to cyanide

Change in optical densityof cytochrome o at

Sampling time

418-432 mA, 572-554 mA

0 0.128 0.0056I 0.0915 0.0062II 0.0785 0.0055

After transfer from cyanide-containing tocyanide-free medium

Generations of cells Cytochrome Cytochrome Cytochromeharvested ~~0 a2 bharvested (418-432 mA) (625-650) mM) (560-540 mg)

0 0.0343 0.0094 0.0260Y2 0.0336 0.0089 0.0255(Y2)2 0.0300 0.0046 0.0196(½)3 0.0400 0.0035 0.0165(Y2)4 0.0348 0.0001 0.0140

* Change in optical density is given per 10 mgof bacterial protein per ml per cm of light path.Sampling times are explained in Fig. 3 and genera-tions of cells harvested are explained in Fig. 4.

tase which accepts electron from cytochrome b, .No c-type cytochrome was detected in the par-ticulate fraction or in the soluble fraction of cellscultivated under any of the conditions tested.

800600

400

200

H-

Hz

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ARIMA AND OKA

(;ROWTH CURAVE

-t-KCN -KCN

O(R) (1 2) (1 4) (1i 8)t: 300 -

= 200 - II

L 0 1 2 3hr

600 650

WAVE LENGTI H (mp)

FIG. 4. Changes of the spectra (reduced minusoxidized) after the transfer of the resistant cells tocyanide-free medium. Curves represent cells whichdoubled once (12), and three times (123) in the cya-nide-free medium.

Changes in the cytochrome contents with changesof the growth conditions. The changes in cyto-chrome content during the growth of a vigorouslyaerated culture are shown in Fig. 2. The aerobicculture in the logarithmic phase showed a promi-nent cytochrome b, (560 m,u) maximum in the aregion and a prominent Soret maximum at 430m,u. On entering the stationary phase, theD mi-tionofa was induced, andunally the spectrum became nearly the same asthat of the resistant cells. No significant changein cytochrome o content_wsQbseryed during thechaunge from one growth phaatqanother.The changes in the spectrum after the addition

of 10-3 M KCN to log-phase aerobic cells areshown in Fig. 3. The content of cytochromes a,and a2 was increased with the recovery of growth,which occurred about 2 hr after the addition ofcyanide. The cytochrome o content either didnot change or decreased a little (Table 2).When cells grown in the presence of 10-3 M

KCN were transferred into cyanide-free mediumand allowed to grow aerobically, the change in"degree of resistance to cyanide" was rather com-plex (Mizushima and Arima, 1960a; Oka andArima, 1965), and the change in the spectrumwas also complex (Fig. 4). Nonetheless, as thegrowth went on, the peaks of cytochromes a, anda2 faded away and the content of cytochrome b5

TABLE 3. Various respiratory activities of the cyanide-sensitive and cyanide-resistant cells*

Intact cells Sonic extract Succinic dehydrogenaseelectron acceptorType of cells

Glucose-oxidi- Succinate-oxidi- Succino-oxidase NADH2 oxidase DCPIt PMStzing activity zing activity activity activity

Sensitive... 100 210 38.4 0.173 10.0 37.0Resistant.15 150 140 23.0 0.280 5.6 10.0

* Results are expressed as Qo2 [microliters of O2 per milligram (dry weight) of cells per hour], exceptfor NADH2 oxidase activity which is expressed as change in optical density at 340 m,u per milligram ofnitrogen per minute.

t 2,6-Dichlorophenol-indophenol.Phenazine methosulfate.

TABLE 4. Effects of various inhibitors on the oxidation of succinate by intact cells*

Per cent inhibition of respirationInhibitor Conditions pH

Sensitive cells Resistant cells

KCN .............. 5 X 16-5M 7.4 100 0NaN3 .............. 2.5 X 10-4 M 5.2 88 46BALt 2 X 10-3 M 7.4 60 40AntimycinA S 50,-g/ml 7.4 0 0HQNO 50,ug/ml 7.4 0 20

* Succinate oxidatioin by intact cells was measured with a Warburg respirometer. Each Warburgvessel contained about 4 X 109 cells, 1.6 ml of 0.1 M phosphate buffer (pH 7.4), and 0.2 ml of an inhibitoror the buffer in the main compartment; 0.2 ml of 0.2 M succinate was in the side arm; and 0.2 ml of 20%KOH was in the center well. The total volume was 2.2 ml.

t 2,3-Dimercapto-propanol.

IO.D 0.01

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CYANIDE RESISTANCE IN ACHROMOBACTER. I

A.CONTROLJ ~5i1bYb, (560mu ) CQ{jL

550 600 650

C. 5 X 10 3I KCN

550 600 650

WAVE LENGTH (mp)FIG. 5. Oxidation and reduction of cytochromes

in the absence and in the presence of cyanide. Thereference cell contained the oxidized preparation andthe sample cell contained the reduced preparation.The reduction and the reoxidation of cytochromesin teeoxt7dzed preparation was followed spectro-photometrica7[y. AJ,control no cyanide); B, 1O-3 MKCN; C, 5 X 10-8 M KCN. Figures show the lapseof time in minutes. Dotted lins, lizht aeration,dashed ,l vgrous aner .era ion was carrjd

iele atmlase, and then v-igorously.

decreased to half, whereas the content of cyto-chrome o remained at nearly the same level(Table 2).Nature of the electron transport system of sensitive

and resistant cells. As described above, cyanide-resistant cells contain twice as much cytochromeb1, more than 10 times as much cytochromes a,and a2, and a little less cytochrome o than doaerobic cells in the log phase of growth. It waspreviously reported that there was no differencein total flavine content (Mizushima and Arima,1960a).

In Table 3, various respiratory activities ofintact cells and cell-free preparations are shown.Though the resistant cells contained manv cyto-chromes, the activity of the succinoxidase systemwas two-thirds of that of aerobic cells. This maybe the reflection of the low succinate dehydrogen-ase activity of the resistant cells.

Effects of various respiratory inhibitors areshown in Table 4. The inhibition caused by eachreagent was measured under its most effectivecondition, and the concentration shown gave themaximal difference between per cent inhibitionof the sensitive and the resistant cells. It is ofinterest that the resistant cells were also resistantto azide, but not to carbon monoxide. They werealso resistant to 2, 3-dimercapto-propanol (BAL).Similar results were obtained in the cell-freepreparations, except that BAL stimulated ratherthan inhibited the oxidation of succinate. Thedata cited above suggest that the electron-trans-port system of the sensitive cells is very differentfrom that of the resistant cells.

Identification of the oxidase active in resistantrespiration. As shown in the next paper (Oka andArima, 1965), the concentration of cyanide whichcauses 50% inhibition of the respiration of resist-ant cells was about 200 times as much as thatproducing the same effect in the sensitive cells.To identify the oxidase which could catalyze theoxidation of cytochromes in the presence of 10-3M KCN, the oxidation-reduction state of cyto-chromes in cell-free preparations from the resist-ant cells was studied.

Figure 5 shows the changes in the oxidation-reduction state of cytochromes after the additionof succinate to the air-oxidized preparation. Whenthe reduction occurred in the absence of cyanide,cytochromes bi, a1, and a2 were rapidly reduced(Fig. 5A). The peaks of cytochromes a, and a2vanished more rapidly than that of cytochromebl. When the preparation was exposed to mildaeration, cytochromes b1 , a1 , and a2 were oxidizedto the original level.When the reduction proceeded in the presence

of 10-3 M KCN, the peaks of cytochromes bi anda, disappeared more rapidly than that of cyto-

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ARIMA AND OKA

These data show that cytochrome a2 is oxidizedmost easily in the presence of cyanide and thatthere is no more resistant bypath of electron flowfrom cytochrome b1 to oxygen than that throughcytochrome a2. Therefore, we concluded thatcytochrome a2 is an oxidase relatively insensitiveto cyanide and that it can mediate the oxidationof the cytochrome system in Achromobacter strainD in a high concentration of cyanide. Other pos-sibilities that may explain the mechanism ofcyanide-resistant oxygen uptake, for example,autoxidation of cytochrome bi or participationof cytochromes o and a1, are excluded.

Oxidation of cytochrome a2 by nitrite. When ni-trate was added to the cell-free preparation fromthe resistant cells, very slow oxidation of cyto-chrome a2 occurred anaerobically. On the otherhand, cytochrome a2 was rapidly oxidized by theaddition of nitrite, and this oxidation was not

20 40

MIINUTESFIG. 6. Anaerobic oxidation of cytochrome a2

with nitrite and nitrate. A 0.1-ml amount of 0.3 MNaNO2 or KNO3 was added to the crude cell-freepreparation (from the resistant cells) from the sidearm of a Thunberg-type cuvette with nitrogen as

the gas phase. Total volume, 3.0 ml; pH, 7.4; tem-perature, 20 C. Oxidation with nitrate in the absence(Z) and in the presence (a) of HQNO (10 ,ug/ml).Oxidation with nitrite in the absence (0) and inthe presence (0) of HQNO (10 ,ug/ml).

chrome a2. This indicates that there is a cross-

over point (Chance and Williams, 1956) betweencytochromes b1 and a2 or between cytochromesa, and a2. On mild aeration, cytochrome a2 was

oxidized to one-half of the original level, andcytochromes bi and a1 were oxidized to a lesserdegree. On successive vigorous aerations, cyto-chromes bi , a1, and a2 were oxidized nearly to theoriginal level (Fig. 5B).When the reduction took place in the presence

of 5 X 10-3 M KCN, the peaks of cytochromesa1 and b1 disappeared more quickly than that ofcytochrome a2, which was not completely re-'duced (Fig. 5C). Only cytochrome a2 was oxizedby mild aeration. With successive vigorous aera-

tions cytochrome b1 was oxidized, but cytochromea, remained reduced. The oxidation of cytochromea2 was nearly complete, whereas that of cyto-chrome b, was not complete, in the presence of5 X 10-3 M KCN. The fact that the oxidation ofcytochrome b1 occurred without the oxidation ofcytochrome a, indicates that there is at least one

pathway of electron flow from cytochrome b1 tooxygen that does not pass through cytochrome a, .

This pathway is resistant to cyanide and consistsof cytochromes b1 and a2 .

C)

z

10

5eo

10

HIQNO CONC.

20(7 ml)

FIG. 7. Effect of HQNO on the reduction ofnitrate by cell-free preparation from resistant cells.The reduction was conducted in a Thunberg tubewith nitrogen as the gas phase (30 C). Reactionmixture contained 40 MAmoles of sodium succinateand 60 ,umoles of KNO3 in 0.06 M phosphate buffer(pH 7.4).

N03-

CYTOCHRONIE b1 (560ma)|

OO.D 0.01

I

OXIDATION

1 MIIN

FIG. 8. Oxidation of cytochrome b1 with nitrate.A 2-mg amount of KNO3 was added anaerobicallyto the crude cell-free preparation from nitrate-grown cells. Cytochrome b1 was reduced with en-

dogenous substrate prior to the addition of nitrate.Conditions were the same as for Fig. 6.

0.02

6

m~ 0.01

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CYANIDE RESISTANCE IN ACHROMOBACTER. I

PREP 0.1ml

-I) 2 p mole NO2--\\

ECM

N)1 MIN

A. B.

FIG. 9. Effect of nitrite and nitrate on the oxygenconsumption by the crude cell-free preparation fromnitrate-grown cells. (A) Nitrate; (B) nitrite. Reac-tion mixture contained 20 ,imoles of succinate (Na).Total volume, 2.5 ml; pH, 7.4; temperature, 20 C.

inhibited by HQNO (Fig. 6). As the oxidation ofcytochrome a2 by nitrate was completely inhib-ited by HQNO, it was assumed that the oxidationof cytochrome a2 on addition of nitrate was dueto nitrite, which was the product of nitrate re-

ductase. In fact, the cell-free preparation fromthe resistant cells contained the activity of thenitrate reductase system which was inhibited byHQNO (Fig. 7), but this preparation did not con-

tain the activity of the nitrite reductase system.In addition, cells grown on nitrate did not formNO from NO2-. Thus far, we have not obtainedany physiological explanation for the oxidationof cytochrome a2 by nitrite, but it is of interestthat cytochrome a2, which functions as a cyto-chrome oxidase, also has some relation to thereduction of nitrite. We will discuss this pointlater.

Nature of the electron-transport system of nitrate-grown cells. The difference spectrum of nitrate-grown cells is shown in Fig. 1C. These cells hadan electron-transport system consisting of cyto-chromes b1, a,, a2, and o, and showed more

cyanide-resistant respiration than the sensitivecells. On the anaerobic addition of nitrate, thecytochrome bi of nitrate-grown cells was rapidlyoxidized to the level attained by oxidation withair (Fig. 8). The activity of the nitrate reductasesystem of nitrate-grown cells was inhibited byHQNO and was more sensitive to cyanide thanwas the respiration of the resistant cells.

Figure 9 shows the inhibition by nitrate andnitrite of oxygen consumption in the cell-freepreparation from nitrate-grown cells. The in-hibition by the limited amount of nitrate was

soon ended, and the rate of oxygen consumptionrecovered to nearly the original level; the modeof inhibition by nitrite was quite different. The

low concentration of nitrite did not inhibitoxygen consumption immediately after addition,but it gradually became effective with the de-crease of the oxygen concentration and finallycaused complete inhibition. The higher the con-centration of nitrite was, the higher was thelevel of oxygen at which the consumption stopped.Moreover, the inhibition remained in effect.These facts suggest that nitrite acts as a competi-tive inhibitor of the cytochrome oxidase foroxygen, and that nitrite is not utilized as anelectron acceptor in this bacterium.

DISCUSSIONThe above data suggest that the formation and

function of cytochrome a2 is closely related tothe environment in which the utilization ofoxygen is limited. The induced formation ofcytochrome a2 occurs on addition of potassiumcyanide to the aerobic culture, during the transi-tion from log to stationary phase, under limitedaeration, or in the anaerobic culture with nitrate;the resultant cells, containing cytochrome a2in various concentrations, are more or less re-sistant to cyanide. In this connection, the workof Castor and Chance (1959) is very interestingto us. They reported that in Escherichia colithe oxidase activity of cytochrome a2 earedonly in the stationary p-ase and that, for therespiration iinhibited (T,-cy;tochroime a2actdVifThipedominated over cytochrome o ac-ti~T~-There are two well-known mechanisms thatcontrol the formation of cytochromes in micro-organisms; one is "oxygen adaptation" (forma-tion of cytochromes in response to oxygen as inyeast; Ephrussi and Slonimski, 1950), and theother is "oxygen suppression" as in Pseudomonas(Lenhoff, Nicholas, and Kaplan, 1956) and indenitrifying bacteria (Verhoeven and Takeda,1956). Our data suggest that oxygen tensiondetermines not only the amount of cytochromesformed but also the nature of the terminal elec-tron-transport system. This information mayhelp to explain the fact that the main differencebetween the bacterial and the mammalian cyto-chrome systems seems to be the presence ofseveral oxidases in some bacteria in contrast toa single oxidase in mammalian tissue (Smith,1961). In Fig. 10, the electron-transport systemsof Achromobacter strain D grown under variousconditions and the electron flow in them aresummarized.Cytochrome a2 is a very interesting cytochrome

in many respects. First of all, its function as acytochrome oxidase was doubted (Smith, 1954),because there was no correlation between cyto-chrome a2 content and the respiratorv activity

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ARIMA AND OKA

of bacteria (Tissieres, 1951, 1952; Moss, 1952).However, Castor and Chance (1959) concludedthat cytochrome a2 was a true oxidase and thatit was capable of catalyzing a large part of therespiration in the bacteria in which it was found.We also reported previously that there was aparallelism between cytochrome a2 content andcyanide-resistant respiratory activity, and weproposed that cytochrome a2 was the cytochromeoxidase in this Achromobacter strain D (Mi-zushima et al., 1960). In the present paper, wehave shown that cytochrome a2 is an oxidaserelatively insensitive to cyanide and that itplays the main part in cyanide-resistant respira-tion in Achromobacter strain D.The purest cytochrome a2 thus far obtained

is that of P. aeruginosa (Horio et al., 1961;Yamanaka and Okunuki, 1963). This cytochromea2 in its crystalline form has two prostheticgroups (heme a2 and c-type heme) and has twoactivities, a cytochrome oxidase activity and anitrite reductase activity. As it is produced onlyin the presence of nitrate, physiologically it is anitrite reductase. In contrast, the cytochromea2 of Achromobacter strain D, which is also oxi-dized by nitrite, is formed and acts as a cyto-chrome oxidase which catalyzes the oxidationof cytochromes in the presence of cyanide orunder low oxygen tension. In addition, Castorand Chance (1959) reported that in E. coli B,Aerobacter aerogenes, and Proteus vulgaris theoxidase activity of cytochrome a2 was found onlyin the stationary phase; this appearance in thestationary phase might be related to changes inoxygen concentration during growth of thecultures. Thus, it is of interest that these cyto-chromes, which are commonly called cytochromea2 from the position of their a peaks, have some

VIGOROUS AERATION

POOR AERATIONSTATIONARY

10 AERATION

KCN ( 10i3 M)

ANAEROBIC(2%NO3)

- bl O~ ~ 02-b-0

-O"',

bl---~ a;, °2|-b

I

%% 2\ --,

NO3 N02

FIG. 10. Schematic representation of the terminalelectron-transport systems of Achromobacter strainD grown under various conditions. The thicker lines

> ypa;pathway; dashed lines, a possbiepathway.

CN

NADH

SUC

N.02

NO3FIG. 11. Terminal electron flow and the site of

inhibition of respiratory inhibitors in cyanide-resistant cells. Dotted lines indicate possible path-ways.

relation to anaerobic or nearly anaerobic energymetabolism.As seen in Fig. 1B this bacterium contains

another a-type cytochrome, cytochrome a1. Insome bacteria, cytochrome a1 can function as acytochrome oxidase (Castor and Chance, 1955),and in Haemophilus species cytochrome a, isreported to have some relation to nitrate reduc-tion (White and Smith, 1962); Williams (1961),on the other hand, suggested the possibility thatcytochrome a1 functions similarly to cytochromec. We found that one strain of Pseudomonaspseudomallei, which was able to grow in thepresence of 2 X 10-2 M arsenite (Arima andBeppu, 1964), contained cytochromes bl, a1,and a2 without cytochrome o. This bacteriumwas very suitable for studying the role of cyto-chrome a1 occurring with cytochrome a2. Wefound that cytochrome a,, like cytochrome a2,acted as an oxidase in this bacterium, though itwas more sensitive to cyanide than was cyto-chrome a2 (unpublished data). As the relation ofcytochrome a1 to nitrate reduction was not foundin this strain of Achromobacter, it may be con-cluded that the resistant cells contained threedifferent kinds of oxidase, cytochromes a1, a2,and o.We may summarize the electron flow and the

inhibition sites of some respiratory inhibitors inthe cyanide-resistant cells as shown in Fig. 11.

ACKNOWLEDGMENTSWe thank G. Tamura of this laboratory and

S. Mizushima of the Institute of Applied Micro-biology, University of Tokyo, for participatingin many stimulating discussions ,and for criticalreviews of the manuscript.

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