APPLIED MICROBIOLOGY, June 1970, p. 966-972 Vol. 19, No. 6Copyright @ 1970 American Society for Microbiology Printed il U.S.A.

Bacteriology of Manganese NodulesIV. Induction of an MnO2-Reductase System in a Marine Bacillus'

R. B. TRIMBLE2 AND H. L. EHRLICH

Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181

Received for publication 19 February 1970

Bacillus 29, isolated from a ferromanganese nodule from the Atlantic Ocean,was shown to possess an MnO2-reductase system which is induced in the presenceof manganous ion. Maximal activity of the enzyme system was induced in about 5hr in the presence of 4.35 mM MnSO4 and was minimally dependent on the presenceof either glucose or peptone and oxygen. Induction of optimal activity required thesimultaneous presence of glucose and peptone. At least 30% of maximal activitywas induced in 5 hr in the presence of 0.4 ,M MnSO4. Actinomycin D (5 ,ug/ml) orchloramphenicol (35 ,ug/ml), when added to the induction medium, inhibitedapproximately 90% of MnO2-reductase synthesis and incorporation of uracil-2-'4Cor leucine-1-_4C. Cell-free extracts having MnO2-reductase activity were preparedby sonic disruption of cell suspensions of induced Bacillus 29. Such extracts usedglucose metabolism as a source of electrons. They had an average specific activityof 1.15 nmoles of Mn"I produced per mg of protein per hr at 25 C. They had atemperature optimum of 18 C for reductase activity and retained 50% of theiractivity at 4 C, the approximate temperature of the natural habitat of the organism.Extracts were stable for several days at 4 C but rapidly lost over 50% of theiractivity on freezing and thawing. Over 90% of the activity of the extract could bedestroyed by heating in a boiling-water bath for 5 min. At a concentration of 1mM, HgCl2 and atebrine dihydrochloride inhibited MnO2-reductase activity by atleast 50%, but sodium azide was ineffective. The MnO2-reductase activity of inducedcells and extracts from them was no greater in the absence of oxygen than in itspresence, confirming an earlier observation that MnO2 and 02 do not compete asterminal electron acceptors in the respiratory activity of this organism.

A number of heterotrophic and two autotrophicorganisms are known to transform large amountsof inorganic materials, such as sulfates, nitrates,and carbon dioxide, by using them as terminalhydrogen acceptors (12, 14, 17). A lesser knownphenomenon of microbial interaction with in-organic materials is the reduction and solubiliza-tion of oxides of iron (1, 2, 13, 16, 19, 20) andmanganese (4, 9, 11, 18, 19, 20). Although someof the earlier reports did not associate the solubili-zation of these metal oxides directly with enzy-matic activity, recent evidence has suggested thatit does occur (2, 5, 13, 18, 20; A. F. de Castro,Ph.D. Thesis, Rensselaer Polytechnic Institute,Troy, N.Y., 1969).

In previous work, it was shown that growingcultures of Bacillus 29, isolated from a ferro-manganese nodule from the Atlantic Ocean, are

1 Part of a dissertation submitted by the senior author to theDepartment of Biology of Rensselaer Polytechnic Institute inpartial fulfillment of the requirements for the Ph.D. degree.

2 Present address: Division of Laboratories and Research,New York State Department of Health, Albany, N.Y. 12201.

able to reduce MnO2 in the presence of glucoseafter a 1-day lag, and the lag can be eliminated byadding ferricyanide to the culture medium (5,18). It was also shown that resting cells of Bacillus29, previously grown in the absence of MnO2 , areunable to reduce MnO2 in 3 hr in the absence ofan electron carrier, either ferri- or ferrocyanide(5). They do possess the necessary enzyme systemto convey electrons from glucose oxidation toferricyanide and from ferrocyanide to MnO2 (5).From these observations, it was hypothesized thatgrowing cultures, initially unable to reduce MnO2without an added electron carrier, develop thisability during the 1-day lag as a result of an"adaptation" (5) by synthesizing one or morecomponents that in unadapted cells are replacedby the ferri-ferrocyanide couple. The enzymaticnature of MnO2-reduction by Bacillus 29 waspreviously suggested by the observation that 1mM HgCl2 rapidly stops MnO2 reduction bygrowing cultures and that boiled or HgC92-treated cells are unable to reduce MnO2 inresting-cell experiments (5, 18). The probable

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chemical mechanism of MnO2 reduction byBacillus 29 was previously discussed (18). Thepresent investigations show that Bacillus 29reduces MnO2 with the help of a partially in-ducible MnO2-reductase system.

MATERIALS AND METHODS

Bacterial cultures. Bacillus 29, originally isolatedfrom an Atlantic ferromanganese nodule (H. L.Ehrlich, Bacteriol. Proc., p. 42-43, 1964), was usedexclusively in this work. The organism is a motile,gram-positive, sporeforming rod which produces acidfrom glucose but not lactose. It forms acetoin in glu-cose-peptone water and peptonizes litmus milk. Itdoes not hydrolyze starch, reduce nitrate, or produceindole. It is able to grow in nutrient broth containing10% NaCl. Stock cultures were grown and stored at25 i 1 C on Stock Culture Agar (Difco) prepared indistilled water.An "adapted" Bacillus 29 culture was prepared by

growing the parent strain in liquid induction medium(described below) and was maintained by subcultureon Stock Culture Agar (SCA) containing about 5mM MnSO4 (J. T. Baker Chemical Co., Phillipsburg,N.J.). This medium was prepared by adding 1 ml of aseparately sterilized 0.1 M MnSO4 solution to 20 ml ofSCA, after first cooling both to 43 to 46 C. The term"adapted" indicates that the culture was capable ofMnO2 reduction in the absence of the artificial electroncarrier, ferricyanide.

Both adapted and unadapted cultures of Bacillis29 were checked periodically for purity, as describedelsewhere (18).

Inocula. Cells of Bacillus 29 for experimentationwere grown on Roux bottle slants containing 100 ml ofseawater-nutrient agar (SWNA), prepared as pre-viously described (18). After 24 hr of incubation at25 i 1 C, the cells were harvested and washed threetimes by centrifugation at 1,500 X g in 10-fold dilutedseawater at room temperature, aseptically if necessary.

Cells of adapted Bacillus 29 for experimentationwere grown in the same manner as the parent strain,except that SWNA containing about 4.7 mm MnSO4was used. This medium was prepared by adding 5 mlof sterile 0.1 M MnSO4 solution to 100 ml of meltedSWNA in a Roux bottle. The cells were harvested likethose of the unadapted parent strain, except that cellswere washed repeatedly at 6,000 X g until 1 ml of thesupematant fluid was free from manganese by theassay described below (usually three to five washings).MnO2. Pure, reagent-grade manganese dioxide

(J. T. Baker Chemical Co.), mineralogically equivalentto pyrolusite, was prepared as previously described(18).

Anaerobiosis. Anaerobic incubation was carriedout in vacuum desiccators under nitrogen, as pre-viously described (18).

Liquid induction medium. For induction studies, abasal liquid medium consisting of natural seawatercontaining 0.88% glucose (Difco) 0.044% peptone(Difco), and an appropriate concentration of MnSO4was used. For radiolabel incorporation studies, one ofthe following was also added to the medium: 0.025 ,c

(34 pc/lgmole) of leucine-1-14C per ml of medium, oreither 0.21 or 0.08 ,uc (2.7 Mic/,.mole) of uracil-2-14Cper ml of medium. The leucine-1-14C was a generousgift from C. Hurwitz and the uracil-2-14C was fromD. E. Wilson, Jr. When studying the effect of inhibi-tors of ribonucleic acid (RNA) and protein synthesison MnO2-reductase induction, the basal mediumcontained 35 ,ug of chloramphenicol (CAP; Parke,Davis & Co., Detroit, Mich.) per ml or 5 ,g of actino-mycin D (AMD) per ml. The AMD was a generousgift from D. E. Wilson, Jr.

Induction of MnO2-reductase. To study MnO2-reductase induction, unadapted cells of Bacillus 29were suspended in liquid induction medium of ap-propriate composition, using 57.5 ml of medium foreach cell crop from a Roux slant to give a final titer ofabout 5 X 108 organisms per ml. This suspension wasincubated for a specific length of time at 25 ± 1 C.Then, if required, cells were harvested and washedrepeatedly by centrifugation at 6,000 X g in 10-fold-diluted seawater until 1 ml of the supernatant fluidwas free from manganese as indicated by the man-ganese assay. In experiments in which AMD treatmentfollowed CAP treatment, the cells were harvested andwashed at 37 C in 10-fold-diluted seawater supple-mented with 0.88% glucose, 0.044% peptone, and 1.5,ug ofAMD per ml until the supematant fluid was freefrom manganese.

Test for MnO2-reductase activity. To test for MnO2-reductase activity in induced cells or cell-free extracts,3-hr activity assays were done by the short-term assayof Trimble and Ehrlich (18), but the 0.1 ml of 0.01 Mpotassium ferricyanide was replaced by an equalvolume of 10-fold-diluted seawater. Uninduced cellsor cell-free extracts were tested by the unmodifiedassay (18). When the activity of cell extracts wasmeasured, 1 ml of extract was used in place of cellsuspension. Cell suspensions tested by the assay wereadjusted turbidimetrically at 540 nm to contain afinal titer of about 8 X 108 organisms per ml. Allassays were incubated at room temperature (24 to25 C) or at 18 C, as specified in the experiments.

Radioactivity measurements. For determining radio-leucine incorporation, appropriately sized sampleswere collected on 0.45-Mum filters (Gelman Instru-ment Co., Ann Arbor, Mich.; Millipore Filter Corp.,Bedford, Mass.) by using an interchangeable syringeand a Swinney Filter Unit (Millipore Filter Corp.).Filters were washed first by twice passing 2-ml volumesof cold, 10% trichloroacetic acid and then distilledwater through them. For the determination of radio-uracil incorporation, cells were collected by centrif-ugation at 4,000 X g at room temperature and resus-pended for 15 min in a few milliliters of ice-cold, 10%trichloroacetic acid. A portion of this suspension wascollected on 0.45-,um filter membranes and washedtwice with 2-ml volumes of distilled water. After thefinal washing in distilled water, all filters were air-dried and then cemented by their periphery to copperplanchettes. In all radiolabel incorporation studies,cell titers of different suspensions were equalizedimmediately before sample collection. Radioactivitycounting was carried out in a model PC-1 Gas FlowProportional Counter (Nuclear Measurements Corp.,Indianapolis, Ind.) for a length of time which resulted

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20 24

FIG. 1. Levels of MnO2-reducing activity of restingcells of Bacillus 29 after different periods of incubationin liquid inductioni mediuim conitaininig 4.35 X ,03IMnSO4 . The per cenit activities were calculated on thebasis of a maximum of 24.0 nmoles of Mni+ per mlreleased in the 3-hr activity assay. Vertical barsinidicate spread of values about the meani for eachpoint.

in a 2-sigma counting error of less than 1%. All datawere corrected for background counts and nonspecificbinding of radioactive material to the filter membranes.

Cell-free extracts. Cell-free extracts of adapted andunadapted Bacillus 29 were prepared by sonic disrup-tion, by using a Biosonic BP-I11 generator equippedwith a BP-III probe assembly (Bronwill Scientific,Inc., Rochester, N.Y.). For disruption, the washedharvest from two to three Roux bottle slants ofBacillus 29 (adapted or unadapted) was suspended in5 to 7 ml of 10-fold-diluted seawater and sonicallytreated in an ice-water bath at an intensity of 55 to 60scale units for a total of 5 min in 1 -min intervals inter-spaced by 0.5-min rest periods to facilitate cooling.The crude sonic-treated material was centrifuged at10,000 X g for 20 min at 4 C. The supernatant fluid,which contained the enzyme activity, was separatedfrom the precipitate by decantation and designated"crude extract."Manganese determination. To measure the amount

of MnO2 reduced by cells or cell-free extracts in theMnO2-reductase assay, the method of Ehrlich (5) wasused, except that no 1.5-ml sample of supernatantfluid was removed before acidification with 0.05 mlof 10 N H2SO4 .

Protein determination. Protein content of crudeextracts was measured by the method of Lowry et al.(10). Developed color was read at 680 nm in a Cole-man Junior spectrophotometer (Coleman Instru-ments, Inc., Maywood, Ill.). The assay was stand-ardized by use of 5X recrystallized bovine serum

albumin (Nutritional Biochemicals Corp., Cleveland,Ohio).

RESULTS

Induction of MnO2-reductase activity. Twenty-four-hour-old cells of Bacillus 29, unadapted forMnO2-reduction, were incubated aerobically at25 C for various lengths of time in liquid inductionmedium containing 4.4 mm MnSO4 and thenharvested, washed, and tested for their ability toreduce MnO2. The results (Fig. 1) show thatsome MnO2-reducing activity was formed after0.5 hr of contact with MnSO4 , reaching a peak ofactivity after about 4.5 hr of incubation. Controlexperiments in which the bacteria were incubatedfor 24 hr in modified induction medium showedthat, for MnSO4 to induce MnO2-reductaseactivity in Bacillus 29, both oxygen and glucose or

peptone were required (Table 1). When peptonewas the only organic substrate in the inductionmedium, the level of MnO2-reductase formed wastwice that found when glucose was the onlyorganic substrate, suggesting a need for proteinsynthesis for induction to take place. OptimalMnO2-reductase induction occurred in the com-bined presence of glucose and peptone. Cells in-cubated in the absence of glucose and peptonebut in the presence of MnSO4, although unin-duced, were still able to reduce MnO2 in thepresence of the electron carrier ferricyanide,indicating that their incomplete MnO2-reductasesystem was still active. In similar experiments,iron, furnished either as limonite (Fe2O3 nH20),a component of some manganese nodules, or asFeSO4, did not replace MnSO4 as an inducer of

TABLE 1. Level of MnO2-reduictase activity illBacillus 29 after 24 hr of incuibationi in

the presence of MnISO4 undcterdifferenit experimental

conditions

Condition Avg activity

Complete mediuma 100 (4)b

Complete medium air 3 (2)

Complete medium glucose ....... 67 (2)

Complete medium peptone 36 (2)

Complete medium - glucosepeptone ............ 0 (2)

Complete medium - glucosepeptonec 122 (2)

aComplete medium consisted of 4.4 mm MnSO,0.88%0 glucose, and 0.044% peptone in seawaterin air.

I Values in parentheses indicate number ofexperiments reported.

c Cells tested in the presence of Fe(CN)63.

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MnO2-reductase activity in the organism. Culturesof Bacillus 29 adapted in the liquid inductionmedium could be maintained in the induced stateby cultivation on SCA or SWNA containing 4.7mm MnSO4 (see above).

Effect of MnSO4 concentration on MnO2-reductase induction. When unadapted bacteriawere incubated for 5 hr at 25 C in liquid inductionmedium at various concentrations of MnSO4,the levels of MnO2-reductase activity shown inFig. 2 were obtained. The highest level of activitywas observed at an MnSO4 concentration of 4.35mM or 240 Mig of Mn2+ per ml. A gradual increasein activity occurred between 0.435 ,M and 4.35mM. Thereafter, a further 10-fold increase inMnSO4 concentration caused a strong suppres-sion of induction. It is noteworthy that 30% ofmaximal activity still formed after 5 hr of contactwith 0.435 uM Mn2+ or 0.024 Mg of Mn2+ per ml.This is within the range of maximum concentra-tion of 0.01 Mig of manganese per ml of openocean water and 6.6 Mug of interstitial water perml of an ocean sediment (15, 21).

Requirement for protein and RNA synthesis inMnO2-reductase induction. To determine whetherthe adaptation to MnO2-reduction was truly aninduction of missing enzyme protein (or proteins)

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-5 -4 -310 10 10

CONCENTRATION

-2 -I10 10(x4.35M)

FIG. 2. Induction of MnO2-reducing activity inresting cells of Bacillus 29 by different concentrationsof MnSO4 in liquid induction medium. Incubation ateach MnSO4 concentration was for 5 hr. The per centactivities were calculated on the basis ofa maximum of24.0 nmoles ofMn2+ per ml released in the 3-hr activityassay. Vertical bars indicate spread of values about themean for each point.

in the electron transport chain to MnO2, experi-ments were conducted in which either CAP orAMD was incorporated into the inductionmedium with an appropriate, radiolabeled com-pound. Prior control experiments showed thatCAP at a concentration of 35 Mg per ml of liquidinduction medium inhibited about 95% of proteinsynthesis in Bacillus 29 during 5 hr of incubationas measured by leucine-1_14C incorporation. Atthis concentration, CAP did not significantlyaffect RNA synthesis as measured by the incor-poration of uracil-2-14C into acid-insolublematerial.

Additional control experiments revealed thatthe protein synthesis inhibition caused by CAPcould be removed by washing the cells in fresh,inhibitor-free medium consisting of 10-fold-diluted seawater containing 0.88% glucose and0.44% peptone and then incubating them in washmedium containing leucine-1-14C. The washedcells incorporated the radiolabel rapidly andcontinuously into cellular material. The rate ofreversal of drug inhibition measured by thistechnique was temperature-dependent, being atleast twofold greater at 37 C than at room tem-perature (20 C), which is in agreement withsimilar studies conducted with Escherichia coli(8).

Since uracil may act as a precursor in thesynthesis of the thymine component of deoxy-ribonucleic acid (DNA), a control experimenttesting for radiolabel incorporation intoDNA wasperformed. Two batches of Bacillus 29 (about 5X 109 organisms each), grown in the presenceof uracil-2-'4C, were collected by centrifugation.One batch was digested by suspension in 1 ml of0.3 N NaOH at room temperature, whereas theother was suspended in 1 ml of distilled water andmaintained in an ice bath. After 2.5 hr of incuba-tion, 5 ml of ice-cold, 10%O trichloroacetic acid wasadded to each batch, and an equal sample wasremoved from each and collected on 0.45-Mmfilter membranes. The membranes were washedwith 10% trichloroacetic acid and then distilledwater and prepared for counting. The results in-dicated that less than 6% of the incorporatedlabel was found in DNA.

After ascertaining that CAP functioned specifi-cally as an inhibitor of protein synthesis inBacillus 29 under the experimental conditionsemployed, the effect of this drug and that ofAMDwas tested on the formation of MnO2-reductaseactivity. Table 2 summarizes five similar experi-ments in which duplicate batches of unadaptedcells of Bacillus 29 were incubated for 5 hr at 25 Cin flasks with liquid induction medium containing4.35 mm MnSO4. One flask of each pair con-tained either 35 Mg of CAP per ml or 5 ,g of

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TABLE 2. Effect ofchloramphenicol and actinomycinD on the incorporationz of radioactive label andthe formation of MnO2-reductase activity in un-adapted Bacillus 29 during 5 hr of incubationunder inducing conditionsa

Avg per centinhibition

Inhibitorb Radiolabel R

aciiyreductaseincorpor- activitydation actiiy

Chloram- Leucine- 92 (2)e 89 (3)phenicol 1-14C

Actinomycin D Uracil- 91 (2) 95 (2)2_14C

a See footnote a, Table 1.b Chloramphenicol, 35 iAg/ml; actinomycin D,

5 Ag/ml.c Technique of determination of incorporation

described in Materials and Methods.d As determined by the 3-hr activity assay.e Numbers in parentheses represent number of

experiments reported.

AMD per ml as indicated, whereas the otherflask was free from inhibitor. At the end of theincubation period, the differently treated batchesof cells were harvested separately, washed at roomtemperature in 10-fold-diluted seawater, andtested for MnO2-reductase activity. The results(Table 2) are consistent with the notion that bothRNA synthesis and protein synthesis are requiredfor the formation of MnO2-reductase activity inunadapted Bacillus 29.

It was reasoned that if Bacillus 29 becameadapted for MnO2 reduction by a classicalinduction mechanism as implied by the results ofthe foregoing experiments, a demonstration of theappearance of MnO2-reductase activity in un-adapted cells as a result of sequential synthesis ofmessenger RNA followed by that of protein mightbe possible. The hypothesis was tested by firstincubating unadapted cells in the inductionmedium in the presence of 35 ,ug of CAP per mlfor 5 hr at 25 C. Then AMD was added to a finalconcentration of 5 ,ug per ml, and the cellswere quickly harvested and washed repeatedly at37 C with MnSO4-free medium supplementedwith AMD until the final wash fluid was freefrom manganese. These cells were now tested fortheir ability to reduce MnO2 . Results of repeatedexperiments indicated that the cells were, indeed,able to reduce MnO2 without ferricyanide afterthese sequential treatments. The level of MnO2-reductase activity in such cells was approximatelyhalf of the maximal level found when cells wereinduced in the absence of the drugs.

Effect of oxygen on MnO2-reductase activity.in whole, induced cells of Bacillus 29. The cells forthis experiment were grown on Roux bottleslants of SWNA containing 4.7 mm MnSO4.Activity tests for MnO2-reductase carried out inair or in a nitrogen atmosphere showed no differ-ence in activity (Table 3).MnO2-reductase activity in cell-free extracts

from induced Bacillus 29. A number of crude ex-tracts of induced cultures were tested for MnO2-reductase by the 3-hr activity assay. Enzymeactivity, expressed as nanomoles of manganesesolubilized per hour at 25 C in the 3-hr assay,was determined relative to protein concentrationin the extract. Although the total activity ofdifferent preparations varied, the specific activityconsistently fell in the range of 0.9 to 1.3 (average1.15) nmoles of Mn" per mg of protein per hr.The MnO2-reductase activity of a given crudeextract was directly proportional to enzymeconcentration over the range tested. It was thesame in air and under nitrogen (Table 3). Incontrol experiments, crude extracts from un-adapted bacteria failed to reduce MnO2 in 3 hrunless ferricyanide was present in the reactionmedium.The temperature optimum of MnO2-reductase

activity in the cell-free preparation was deter-mined by assaying the activity of 1-ml portionsof a given extract at different incubation tempera-tures. Figure 3 summarizes the results of severalexperiments. The optimal temperature was foundto be 18 C.The dependence of MnO2-reductase on an

oxidizable substrate (glucose) was determined by

TABLE 3. MnO2-reduction by induced cells ofBacillus 29 and extracts from them in the

presence and absence of oxygen

Manganese insupernatant fluid

Experimental conditions (nmoles/ml)a

Aerobic Anaerobic

Mediumb - cells + MnO2...... 3.2 0Medium + cells - MnO2...... 4.8 4.8Medium + cells + MnO2...... 27.5 27.5

Medium - extract +Mn......... 4.8 4.8Medium + extract - MnO2.... 6.4 6.4Medium + extract + MnO2.... 27.2 27.2

a After acidification of the reaction mixture atthe end of the 3-hr assay.

b The medium consisted of 1 ml of 25 mMNaHCO3 (pH 7) containing 1% glucose, 5 ml of10-fold diluted seawater, and 0.1 g of MnO2,where noted.

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TEMPERATURE (OC)FiG. 3. Relative MnO2-reductase activity ofcell-free

extracts ofinduced Bacillus 29 at various temperatures.Vertical bars indicate spread of values about the meanfor each point.

testing 1-ml portions of cell-free extract by the3-hr assay in the presence and absence of glucose.Initial experiments indicated that crude extractsof Bacillus 29, prepared immediately after harvest-ing, were only 35 to 45% dependent on glucosefor the reduction of MnO2 . This low dependencewas thought to be a result of utilization of reducedmetabolic intermediates as well as endogenousstores liberated into the extract during sonicdisruption. To test this hypothesis, cells werestored in 10-fold-diluted seawater at 25 C for 30min after harvesting. Crude extracts from cellstreated in this manner exhibited at least 60%glucose dependence in the reduction of MnO2 incomparison to substrate-free controls.The effect of temperature on the stability of

MnO2-reductase activity in cell-free preparationswas determined by heating the enzyme in aboiling-water bath and by storing it at 4 C andat -20 C. It was found that the extract lost 58%of its activity after 2 min and 91% after 5 min ofboiling. Further decrease in activity after thefirst 5 min of boiling was slow, probably due tothe presence of nucleic acid which may protectsome of the enzyme proteins from denaturation(6). The MnO2-reductase activity appeared stablefor at least 4 days at 4 C and still retained 54%of its activity after 7 days. It lost activity onfreezing, probably due to salt effects, as extractswere prepared in 10-fold-diluted seawater.

Inhibitor studies on MnO2-reductase activityin cell-free extracts showed that at a concentra-tion of 1 mM, HgC12 caused 75 to 85% inhibitionand atebrine dihydrochloride caused 50% inhibi-tion in 3-hr activity assays. Sodium azide had noeffect at this concentration.

DISCUSSION

These investigations show that manganous ion,or possibly some derivative thereof, acts as aninducer in the formation of a ferricyanide-independent MnO2-reductase system in Bacillus29. Accompanying sulfate ions can be ruled outas an inducer since seawater alone contains ahigher sulfate concentration than that containedin the added MnSO4. Yet, MnO2-reductaseactivity is not induced in cells growing in sea-water containing glucose and peptone but noMnSO4 .

Since Mg2+ and Mn2+ may substitute for oneanother in the activation of some enzymes (3),Mg2+ may be expected to substitute for Mn2+ asan inducer of MnO2-reductase. This possibilitycan be ruled out because ofthe high concentrationof Mg2+ in seawater, which is about 125,000 timesgreater by weight than that of manganese (21).Contact with it in an MnSO4-free medium doesnot induce the enzyme.

If manganous ion as such is the inducer, itwould represent an unusual example of inductionbecause manganous ion is a product of the in-duced enzyme reaction rather than the sub-strate. This is, however, not impossible since innature manganous manganese is usually asso-ciated with manganic oxides through adsorption,and we assume that manganous manganese fromthis source is the natural inducer. The actualinduction may involve a derepression as a resultof reaction of the repressor with Mn2+ and itsresultant inactivation.

It may be argued that the true inducer isactually an oxidation product of Mn2+, namelytri- or tetra-valent manganese formed by oxida-tion of Mn2+, since oxygen was required in theseexperiments. However, Mn3+ ions can be ruledout since they are very unstable in aqueoussolution (7), and Mn4+ ions can be ruled outsince they are not known to occur in aqueoussolution (7). One way in which oxidation ofMn" may be involved in the induction phenome-non is by conversion of an MnI"-repressor com-plex to an Mn"I- or Mnlv-repressor complex,which may be more stable than the MnII-repressor complex. On the other hand, the oxygenrequirement may be related to the energy needfor the synthesis of the one or more missingenzyme components. The results of the present

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study do not permit a choice among these alter-native explanations.The inducibility of MnO2-reductase explains

why growing cultures of unadapted Bacillus 29are able to release Mn" from MnO2 after a 1-daylag (18); namely, they are induced by smallamounts of manganous ion normaUy associatedwith MnO2 in the first day of growth. Manganesedioxide itself is too insoluble to act as inducer.The observations that the appearance of MnO2-

reductase in Bacillus 29 incubated in inductionmedium was prevented when RNA or proteinsynthesis was inhibited show that a classicalinduction mechanism, rather than a mutationand selection phenomenon or an allosteric rear-rangement of an existing cellular component, wasinvolved. The induction of maximal activity in alarge population of unadapted cells in a briefspan of 5 hr also rules out a mutation and selec-tion phenomenon. No significant change in cellnumbers took place during induction of Bacillus29 in the liquid induction medium, as measured byturbidity changes. Since the initial cell concentra-tion in the induction medium was usually around10' to 109 cells per ml, which is the maximum cellconcentration achieved by a growing culture ofBacillus 29 in this medium, we would not expectsignificant multiplication during the inductionperiod. The finding that extracts from inducedbut not uninduced bacteria contain ferricyanide-independent MnO2-reductase activity is consistentwith the induction hypothesis.The enzymatic nature of the reductase system

is indicated by the susceptibility of cell-free ex-tracts to heat inactivation and their inhibition byHgCl2 and atebrine dihydrochloride, and also bythe dependence of activity on extract concentra-tion.The ability of induced whole cells or extracts

prepared from them to carry out MnO2-reductionat the same rate whether oxygen was present ornot confirms the earlier finding that oxygen andMnO2 do not compete as terminal electron accep-tors in the respiratory activity of Bacillus 29 (18).This represents an interesting case with respect toother organisms which use inorganic terminalelectron acceptors. Whereas the sulfate reducersand methane formers are obligately anaerobic,Bacillus 29 is unaffected by the presence or ab-sence of oxygen in its ability to reduce MnO2 oncethe cells are induced. Dissimilatory nitrate re-ducers represent an intermediate case in that they

are competitively inhibited in their ability toreduce nitrate by the presence of oxygen.

ACKNOWLEDGMENTSThis investigation was supported by contract 591 (22), work

unit number NR 137-655, between the Office of Naval Research.Department of the Navy, and Rensselaer Polytechnic Institute.We thank H. M. Clark for permission to use the counting

equipment in his radiochemistry laboratory. The helpful advice o(S. H. George Allen, H. M. Clark, C. Hurwitz, and D. E. WilsorJr., is gratefully acknowledged.

LITERATURE CITED1. Bromfield, S. M. 1954. The reduction of iron oxide by bac-

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