Sulfur Metabolism in Beggiatoa alba

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1987, p /87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 169, No. 12 Sulfur Metbolism in Beggito lb THOMAS M. SCHMIDT,'t BOAZ ARIELI,2 YEHUDA COHEN,3 ETANA PADAN,2 AND WILLIAM R. STROHLl.4* Progrm of Environmentl Biology1 nd Deprtment of Microbiology,4 The Ohio Stte University, Columbus, Ohio 43210, nd Deprtment of Physiologicl Chemistry, The Hebrew University of Isrel, Jeruslem,2 nd Steinitz Mrine Biologicl Lbortory, Eilt,3 Isrel Received 18 My 1987/Accepted 12 September 1987 The metbolism of sulfide, sulfur, nd cette by Beggito lb ws investigted under oxic nd noxic conditions. B. lb oxidized cette to crbon dioxide with the stoichiometric reduction of oxygen to wter. In vivo cette oxidtion ws suppressed by sulfide nd by severl clssic respirtory inhibitors, including dibromothymoquinone, n inhibitor specific for ubiquinones. B. lb lso crried out n oxygen-dependent conversion of sulfide to sulfur, rection tht ws inhibited by severl electron trnsport inhibitors but not by dibromothymoquinone, indicting tht the electrons relesed from sulfide oxidtion were shuttled to oxygen without the involvement of ubiquinones. Intrcellulr sulfur stored by B. lb ws not oxidized to sulfte or converted to n externl soluble form under erobic conditions. On the other hnd, sulfur stored by filments of Thiothrix nive ws oxidized to extrcellulr soluble oxidtion products, including sulfte. Sulfur stored by filments of B. lb, however, ws reduced to sulfide under short-term noxic conditions. This nerobic reduction of sulfur ws linked to the endogenous oxidtion of stored crbon nd to hydrogen oxidtion. All freshwter Beggito strins thus fr tested hve the bility to grow heterotrophiclly on cette in the presence of oxygen (14, 25, 26). Members of the genus Beggito, however, lck ctlse (3, 26), so erobic growth under highly oxygented conditions is pprently limited. Moreover, lowered oxygen tension nd the presence of sulfide hve been shown to hve beneficil effects on the growth nd metbolism of Beggito spp. (13, 15, 17, 18, 26). Oxidtion of sulfide my eliminte the need for ctlse by detoxifying metboliclly formed hydrogen peroxide (3). Alterntively, oxidtion of sulfide my simply llevite the toxic effects of sulfide in the environment. Sulfide oxidtion my supplement energy for growth on cette by freshwter strins of Beggito (6, 27) nd is the sole energy source for the chemolithoutotrophic growth of t lest one mrine Beggito strin (15, 16). Whtever the effect of sulfide nd its oxidtion might be, mrked concentrtions of Beggito filments in nture virtully lwys coincide with the presence of hydrogen sulfide (26). Recently we showed tht Beggito lb B18LD nd other strins contin multiple electron trnsport system components (29). In this study, the functioning of the respirtory electron trnsport chin in the presence nd bsence of sulfide ws investigted with the use of electron trnsport inhibitors. It ws found tht under erobic conditions, sulfide slightly suppressed cette oxidtion nd ws converted to elementl sulfur, with oxygen serving s n electron cceptor. Under nerobic conditions, sulfur ws reduced either by endogenous substrtes or by dded hydrogen gs. MATERIALS AND METHODS Strins nd growth conditions. B. lb strins B18LD, B25RD, nd B15LD (12), Beggito sp. strins 75-2 (13, 14) nd SM-1 (S. Mier, unpublished), Vitreoscill beggitoides B23SS (28), Vitreoscill filiformis ATCC (28), * Corresponding uthor. t Present ddress: Deprtment of Biology, Indin University, Bloomington, IN Thiothrix nive JP3 (10), nd Chromtium vinosum (7) were used in this study. The medi were prepred in bsl slts solution (BSS) consisting of 4.7 mm NH4Cl, 1 mm CCl2, 73.5,uM KH2PO4, 40,uM MgSO4 * 7H20, nd 5 ml of microelement solution per liter (34). BH (heterotrophic) medium consisted of BSS plus 6.1 mm sodium cette (ph 7.3). BSO (sulfide oxidtion) medium contined BSS, 6.1 mm sodium cette, nd 2 mm neutrlized sodium sulfide, with ll components dded before utoclving (24). Cultures of Beggito nd Thiothrix strins were grown t 23 C in 2-liter flsks tht contined 1 liter of medium. The flsks were inoculted with pproximtely 100 ml of sttionry-phse culture nd shken t 100 rpm. The cultures were hrvested fter 24 h of incubtion by centrifugtion t 6,000 rpm nd wshed once in BSS. Concentrted cell suspensions were prepred by suspending the pellet to density of c. 0.3 to 0.4 mg of cell protein per ml in the pproprite buffer. Acette, sulfide, nd thiosulfte oxidtion. Acette-dependent oxygen consumption by B. lb B18LD ws mesured with the Wrburg respirometer s described by Umbreit et l. (32). Two milliliters of concentrted cell suspension from BH medium were dispensed into Wrburg flsks. The center well of the flsks contined pleted filter pper (5 by 25 mm) sturted with 0.2 ml of 20% potssium hydroxide. The side rm of ech flsk contined 222,ul of 60 mm sodium cette in BSS. Inhibitors were incubted with the cell suspension t the concentrtions designted in Tble 1 for 15 min before the ddition of sodium cette. Mnometric redings were tken every 15 min. The effect of inhibitors on the rte of [2-14C]cette oxidtion ws mesured in 25-ml siderm rection flsks (no ; Kontes, Vinelnd, N.J.) by dding 333,u1 of [2-14C]cette (1,uCi; 0.1,uCi/,umol) to the concentrted cell suspension s described previously (35). Inhibitors were dded to the rection flsks 15 min before the ddition of substrte. 14CO2 ws collected on KOH-sturted filter pper nd counted with Beckmn LS-6800 scintilltion counter. Dt were corrected for quenching by using n internl stndrd of [methyl-14c]toluene.

2 VOL. 169, 1987 Sulfide-dependent oxygen consumption by B. lb B18LD ws mesured in- the Wrburg pprtus by the method described for cette oxidtion, except tht the side rm of ech flsk contined 333,ul of freshly prepred nd neutrlized 10 mm sodium sulfide in plce of sodium cette. Duplicte mesuremhents of the chemicl oxidtion of sulfide were mde with respirometer flsks contining 2 ml of BSS without cells. The oxidtion of rdiolbeled sulfide to lbeled "intrcellulr" sulfur (24, 27, 35) ws mesured concurrently with identicl cell suspensions. Smples (10 ml) of the concentrted cell suspension were dispensed into 50-ml Erlenmeyer flsks, nd the vessels were shken t 120 strokes per min in Dubnoff metbolic shker t 23 C. When noxic conditions were required, oxygen ws removed from the flsks by flushing with nitrogen for 10 min, nd then the flsks wero crefully cpped with rubber stopper (35). Sodium [35S]sulfide (0.425,uCi/,umol) ws dded to ech flsk to finl concentrtion of 1 mm. The utooxidtion of sulfide nd the sorption of sulfide to the cells were mesured in control flsks with utoclved cells. The effect of respirtory inhibitors or 6 mm cette on sulfide oxidtion by B. lb B18LD ws mesured by dding the ffectors to the cell suspension 15 min before the ddition of sulfide. Smples (200,ul) were removed t timed intervls nd filtered through Whtmn glss fiber filters. The filters with cells contining 35S inclusions were wshed with BSS t ph 3 to remove ny externlly bound lbel (24, 27) nd then dried t 60 C for 2 h. The dried filters were counted in the scintilltion cocktil described previously (25). Quench corrections were mde with [14C]toluene s described previously. To confirm tht the cell-bound product of [35S]sulfide oxidtion ws 35S, replicte smples were filtered through Gelmn GA-3 glss fiber filters (Gelmn Sciences, Inc., Ann Arbor, Mich.) nd wshed with 2 ml of either BSS (ph 3), ethnol, benzene, cetone, or 5% queous trichlorocetic cid. Oxidtion of cellulr sulfur. In n ttempt to deplete the cellulr reserves of sulfur, B. lb B18LD nd T. nive JP3 were grown to lte log phse in BSO medium, hrvested, nd then suspended in sterile BSS to the sme volume nd incubted t 23 C on rotry shker t 150 rpm. After 12 h, the cells were exmined microscopiclly for the presence of sulfur inclusions nd prepred for respirometry s described previously. Cells tht were hrvested from BSO medium nd not strved for sulfur were lso prepred for respirometry. To mesure oxidtion products obtined from intrcellulr 35S stores, B. lb B18LD nd T. nive were grown in BSO mediuiii contining 1 mm [35S]sulfide (0.4 p.ci/4imol). Filments hrvested from the rdioctive medium were wshed twice in BSS nd suspended to their originl density in one of the following: norml BSS, BSS with chloride slts substituted for ll sulfte slts, BSS with chloride slts plus 1 mm freshly prepred nd neutrlized sodium sulfide, BH medium, or BSO medium (ll solutions were djusted to ph 7.2). The cultures were incubted erobiclly in 500-ml flsks shken t 200 rpm on rotry shker for periods of 12 h to 4 dys, depending on the experiment. Typiclly, four 200-,ul smples were tken from ech flsk t ech time point. Two of the smples were filtered through Gelmn glss fiber filters nd wshed with 2 ml of BSS t ph 3.0. The rdioctivity on the filters, mesuring the intrcellulr sulfur, ws counted s described previously. To mesure the mount of 35S-lbeled soluble compounds relesed from the filments, the other two smples were centrifuged for 2 min in microfuge, from which 100-,ul smples of the superntnt were dded to toluene-bsed scintilltion cocktil contining 5 g of PPO (2,5-diphenyloxzole) nd 50 mg of B. ALBA SULFUR METABOLISM 5467 POPOP [1,4-bis(5-phenyloxzolyl)benzene] per liter in 33% Triton X-100 in toluene (21) nd counted s described previously. An lterntive method for obtining B. lb B18LD lbeled sulfur inclusions ws lso used. B. lb ws grown in 500 ml of BH niedium for 16 h, fter which 40,uCi of N235S (1,uCi/mmol) ws dded. The filments were incubted with the lbeled sulfide for 6 h, pelleted septiclly by centrifugtion, wshed twice with BSS, suspended in 50 ml of BH medium, nd incubted erobiclly on rotry shker (250 rpm) for 72 h (25 C). Smples were tken s described bove to determine the mount of lbel remining in the filments nd the mount relesed into the medium. Microelectrode studies. A 5% inoculum of B. lb B18LD ws introduced into BSO medium, which upon incubtion yielded visible tufts of the orgnism in erly exponentil growth. Individul tufts were removed from the medium, rinsed gently in BSS, nd embedded in 2% gr cube mesuring pproximtely 3 mm per side. The gr cube contining the tuft of B. lb filments ws suspended in the middle of 500-ml glss bowl filled with BSS by using cpillry tubes tht were nchored to the bottom of bowl. Air, nitrogen, or hydrogen ws bubbled through the BSS solution t mbient temperture (c. 23 C). Microelectrodes, mde ccording to the methods described by Revsbech et l. (23), were positioned with micromnipultor to mesure sulfide nd oxygen grdients round nd through the tuft of B. lb filments embedded in gr while the vessel ws bubbled with ir. The electrodes were then relocted t the surfce of the tuft of filments in the gr cube, nd either nitrogen or hydrogen ws bubbled through the rection vessel. Mesurement of sulfide production. Reduction of sulfur to sulfide by mid-exponentil-phse cells of B. lb B18LD ws mesured in flsks which were continuously flushed with high-purity nitrogen. Sulfide ws trpped in two seril tubes, ech contining 10 ml of 2% zinc cette solution, which ws ssyed for sulfide by the method of Kline (9) t intervls. Prior to incubtion, cells grown in either BH or BSO medium were wshed nd suspended in BSS to their originl density. This procedure effectively eliminted the ccumultion of sulfide in cell suspensions. Hydrogen evolution nd uptke ssys. To mesure hydrogen evolution in Beggito nd Vitreoscill spp., concentrted cell suspensions of o.1 to 0.3 mg of cell protein per ml were prepred in BSS lso contining 25 mm HEPES (N-2-hydroxyethylpiperzine-N'--2-ethnesulfonic cid) buffer (ph 7.2). Two.milliliters of ech suspension ws dispensed into 9-ml serum bottles (Wheton Scientific, Millville, N.J.) which were seled with rubber stoppers nd luminum cps. Forty microliters of 100 mm methyl viologen in 10 mm phosphte buffer (ph 7.2) ws dded to ech flsk. The bottles were flushed with nitrogen for 15 min, nd the hydrogen evolution ssy ws initited by the ddition of 0.1 ml of 100 mm sodium dithionite (in distilled wter) to finl concentrtion of 10 mm. The bottles were shken t 120 strokes per min t 23 C in reciprocl metbolic shker. Hedspce gs smples of 100,ud were withdrwn from the bottles every 30 min nd nlyzed by injection into Vrin Aerogrph 3700 gs chromtogrph equipped with 3-m stinless steel column pcked with moleculr sieve SA (30-40 mesh). The following tempertures were used: injector, 100'C; column, 30 C; therml conductivity detector, 150 C; nd filment, 300 C. Nitrogen, t flow rte of 30 ml/min, served s the crrier gs. The output from the gs chromtogrph ws recorded on model 252A strip chrt recorder (Liner Instruments Corp., Cost

3 5468 SCHMIDT ET AL. Mes, Clif.). Pek heights were mesured nd compred with stndrd curve prepred from the heights mesured for hydrogen stndrds in nitrogen. Hydrogen consumption ws mesured by dispensing 1.5 ml of concentrted filment suspension into 9-ml serum bottles. After the bottles were flushed with nitrogen for 15 min, 4% of the hedspce ws replced with 1 tm ( kp) of hydrogen. The bottles were incubted s bove. The hedspce ws smpled t 60-min intervls nd nlyzed by gs chromtogrphy for remining hydrogen s described bove. Protein determintions. The concentrtion of totl cell protein ws estimted by the method of Lowry et l. (11) fter extrction of the sulfur with 95% ethnol for 1 h nd digestion of the cells by heting the smples t 90 C in 1 N NOH for 10 min, followed by reneutrliztion of the solutions with 1 N HCI. Bovine serum lbumin ws used s protein stndrd. Chemicls. Rdiochemicls were obtined from Amershm Corp. (Arlington Heights, Ill.). Sodium [35S]sulfide, prepred similrly to the procedure described by Vrgs nd Strohl (35), ws dissolved in deoxygented wter contining unlbeled sulfide so tht the finl concentrtion of sulfide ws 20 mm t ph 7.2. Unlbeled crystls of sodium sulfide were wshed in distilled wter nd dried before being weighed. The sulfide solution ws stored under nitrogen tmosphere t 4 C. The nitrogen tmosphere in the lbeled sulfide stock solutions ws replenished fter ech use. RESULTS Acette oxidtion. Acette-dependent oxygen consumption by Beggito lb B18LD ws liner for 90 min t rte of 3.60,l of 02 per min per mg of protein. This consumption ws equivlent to rte of 160 nmol of 02 per min per mg of protein. The initil rte of 14CO2 evolution from [2-14C]cette ws 65 nmol of 14CO2 per min per mg of protein. The cette oxidtion ws effectively inhibited by dibromothymoquinone, 8-hydroxyquinoline, 1,10-phennthroline, 4-N-hydroxyquinoline-n-oxide, KCN, NN3, nd 2,4- dinitrophenol s determined by mesuring the rtes of both cette-dependent oxygen consumption nd the relese of 14Co2 from [2-14C]cette (Tble 1). The rte of cette oxidtion, mesured isotopiclly, ws decresed c. 18 to 20% by the presence of 2 mm sulfide. Sulfide oxidtion. The initil rte of [35S]sulfide ssimiltion by B. lb in BSS minus iron slts ws 35 to 65 nmol/min per mg of protein in the presence of oxygen; in the bsence of oxygen there ws no significnt mesurble sulfide uptke bove bckground (sorption of [35S]sulfide by utoclved cells; Tble 1). Sulfide uptke by T. nive ws lso oxygen dependent, nd the rtes were similr to those displyed by B. lb (27). Approximtely 90% of the lbeled internl sulfur ccumulted by B. lb B18LD nd T. nive in the presence of oxygen ws soluble in cetone, benzene, or ethnol (dt not shown). Tretment of 35S-contining filments collected on filters with 5% queous ice-cold trichlorcetic cid lwys resulted in recovery of c. 10% more counts thn mesured with filments wshed with BSS (dt not shown). The reltive effects of severl electron trnsport inhibitors on oxygen-dependent sulfide ssimiltion by B. lb B18LD re shown in Tble 1. The inhibitors thenoyltrifluorocetone, 8-hydroxyquinoline, KCN, nd NN3 suppressed sulfide oxidtion to sulfur by more thn 60%. Dibromothymo- J. BACTERIOL. TABLE 1. Effect of electron trnsport inhibitors on cette nd sulfide oxidtion in Beggito lb B18LD % of control vlues Affector or Concn Acette- CO2 Sulfur conditionb dependent 02 evolution ccumultion consumption from cette from sulfide None Ethnol 1% TTFA 0.5 mm C mm 3 DBMIB 20,M ,M p,M HQ 1.0 mm mm 34 PHEN 2.0 mm 10 6 HOQNO 50,uM KCN 1.0 mm NN3 1.0 mm Anerobiosis Acette 6.0 mm 50 Mlted 1.0 mm mM <1 Sulfidee 2.0 mm - 82 Three different methods were used to obtin these dt, s detiled in the Mterils nd Methods section. Control vlues: cette-dependent oxygen consumption, 162 nmol/min per mg of protein; [2-'4Cjcette oxidtion to 14CO2, 65 nmol/min per mg of protein; N235S oxidtion to 35S0, 53 nmol/min per mg of protein. All vlues re verges of t lest duplicte experiments nd represent the net vlues fter utoclved-cell control vlues were subtrcted from the originl gross dt. b Abbrevitions: TTFA, thenoyltrifluorocetone (flvoprotein inhibitor); DBMIB, dibromothymoquinone (ubiquinone inhibitor); 8-HQ, 8-hydroxyquinoline (inhibitor of b-type cytochromes); PHEN, o-phennthroline (inhibitor of b-type cytochromes); HOQNO, 4-N-hydroxyquinoline-n-oxide (inhibitor of cytochrome b-*cytochrome c couple). c-, Not done. d The dt for inhibition of sulfide oxidtion by mlte re courtesy of V. A. Vinci. e The sulfide ws neutrlized to ph 7.2 just prior to the experiment. quinone t concentrtions five times the mount necessry to inhibit cette oxidtion essentilly hd no effect on sulfide oxidtion (Tble 1). Sodium cette t finl concentrtion of 6 mm reproducibly inhibited sulfide oxidtion by c. 50%, nd 1 to 2 mm mlte strongly inhibited sulfide oxidtion (Tble 1). To determine the stoichiometry between sulfide oxidtion nd oxygen reduction, the rtes of sulfide-dependent oxygen consumption nd [35S]sulfide oxidtion were determined concurrently in duplicte flsks for ech mesurement. The rte of sulfur ccumultion, with cells grown in either BH or BSO medium, verged 38 nmol/min per mg of protein in this experiment. The concomitnt oxygen consumption ws 0.96,1/min per flsk. After ccounting for the oxygen consumption due to chemicl sulfide oxidtion (0.35,ul/min per flsk) nd endogenous oxygen consumption (0.30 p,l/min per flsk), the rte of sulfide-dependent biologicl oxygen consumption ws 0.31,ul of 02 per min per flsk. Ech flsk contined 0.8 mg of cell protein. The rte of sulfide-dependent oxygen consumption ws clculted from these dt to be 14 nmol/min per mg of protein. Sulfur oxidtion. Microscopic observtion of filments of B. lb B18LD nd T. nive JP3 grown in BSO medium reveled the presence of numerous refrctile sulfur inclusions. After such filments hd been hrvested, wshed, nd incubted in BSS for 12 h, the phse-bright inclusions were depleted from filments of T. nive but not from filments of B. lb B18LD. Moreover, numerous sulfur inclusions in

4 VOL. 169, 1987 TABLE 2. Relese of rdiolbeled sulfur from erobiclly incubted filments of B. lb B18LD Rdioctivity Time of Expt Expt (cpm/ml) % mesurtoemeium(hd Relesed mesurement Mediumb Filments Medium (h) 1 52,000 2, BHA 2 45,200 2, BSS-Ac 3 44,200 4, BSS-Ac-N2S 4 1,620,000 76, BH 5 170,800 2, BH 6 141,580 1, BH-no S ,500 8, BSO-no S042- Time t which the mesurements were tken fter inititing incubtion of filments contining 35S inclusions in the medi described. b Abbrevitions for medi: BHA, BH medium (see text) plus 0.05% sprgine; BSS-Ac, BSS plus 0.001% sodium cette; BSS-Ac-N2S, BSS- Ac plus 0.03% neutrlized N2S; BH-no SO42- nd BSO-no So42-, BH nd BSO medi with Cl- slts replcing ll sulfte slts. B. ALBA SULFUR METABOLISM 5469 filments of B. lb B18LD were still visible 72 h fter the filments were removed from sulfide. Endogenous respirtion by B. lb contining sulfur inclusions (pregrown in BSO) ws c j.i of 02 per min per mg of cell protein. Endogenous respirtion of B. lb lcking sulfur inclusions (pregrown in BH medium) ws 0.48,ul of 02 per min per mg of cell protein. On the other hnd, sulfurcontining filments of T. nive consumed oxygen t rtes 20- to 25-fold greter thn sulfur-strved filments of T. nive (dt not shown). The oxidtion of intrcellulr sulfur by B. lb nd T. nive ws lso determined by mesuring the extrcellulr relese of 35S-lbeled compounds from rdiolbeled sulfur inclusions under erobic conditions. In seven seprte experiments conducted with B. lb B18LD, never more thn 5% of the 35S deposited in the filments ws relesed into the medium fter 22 h (or 11% relesed fter 60 to 72 h) of incubtion under erobic conditions (Tble 2). This bsence of significnt sulfur oxidtion ws observed regrdless of whether sulfte slts, phosphte slts, cette, or sulfide ws present or bsent in the medium. Filments of T. nive, on the other hnd, relesed pproximtely 40% of their 35S lbel into the medium within 21 h of incubtion (Fig. 1). The presence or bsence of 1 mm sulfide or sulfte in the BSS solution hd little effect on the solubiliztion of the lbel by T. nive filments (Fig. 1). Chemicl mesurements indicted tht sulfte ws mjor product of the oxygendependent sulfur oxidtion in T. nive, lthough stoichiometric vlues were not obtined (T. M. Schmidt, Ph.D. thesis, Ohio Stte University, Columbus, 1985). Microelectrode studies. While ir ws bubbled round tuft of B. lb B18LD filments embedded in n gr cube, the endogenous metbolism lowered the concentrtion of dissolved oxygen to n undetectble level t the surfce of the filment pellet (Fig. 2). At the point where the oxygen concentrtion nered zero, sulfide ws detected nd reched concentrtion of 10 p.m t distnce of 50 p.m inside the filment pellet. When nitrogen insted of ir ws bubbled through the microelectrode pprtus, constnt rte of sulfide production ws mesured t the surfce of the cell pellet (Fig. 3). When the nitrogen ws replced with hydrogen, the rte of sulfide production incresed (Fig. 3). Endogenous nerobic respirtion. In bubbling pprtus designed to provide short-term noxic conditions with continuous removl of sulfide, sulfide ws produced t rte of 6.7 nmol/min per mg of protein by sulfur-contining filments of B. lb B18LD (Fig. 4). B. lb filments tht lcked sulfur inclusions nd hd been grown in BH medium did not produce ny detectble sulfide over period of 4 h (Fig. 4). As positive control, sulfide ws produced t rte of 5.0 nmol/min per mg of protein by drk-incubted cells of C. vinosum (Fig. 4), purple phototrophic bcterium known to reduce sulfur to sulfide (33). Anerobic hydrogense ctivity. Becuse hydrogen ppered to stimulte the nerobic production of sulfide from sulfur-contining filments of B. lb B18LD (Fig. 3), tht orgnism ws tested for hydrogense ctivity. Sulfurcontining filments of B. lb B18LD, incubted nerobiclly, consumed hydrogen t constnt rte of 7.9 nmol/min per mg of protein. There ws no detectble hydrogen consumption by B. lb filments tht lcked sulfur inclusions or by B. lb filments tht were boiled for 2 min (not shown). Hydrogen production by B. lb B18LD ws detected only in the presence of both methyl viologen nd dithionite. The rte of methyl viologen- nd dithionite-dependent in vivo hydrogen production by B. lb B18LD ws 2.25 nmol/min per mg of cell protein nd ws insensitive to 50 p.m crbonylcynide-p-trifluoromethoxyphenylhydrzone. Filments of B. lb B18LD exposed to sulfide lone or to sulfide in combintion with either methyl viologen or dithionite produced no hydrogen. A survey of severl other strins of Beggito nd Vitreoscill ws crried out. The rtes of in vivo methyl viologen- nd dithionite-dependent hydrogen production found were (in nnomoles per minute per milligrm of cell protein): B. lb B25RD, 6.1; B. lb B15LD, 4.9; Beggito sp. clone 75-2, 6.7; Beggito sp. E C. G TIME IHOURSI FIG. 1. Relese under erobic conditions of 35S-lbeled compounds from filments of T. nive JP3 contining 35S inclusions. Decrese of I'S lbel in the filments (sulfur inclusions; solid symbols) nd increse in soluble, cid-stble 35S lbel (open symbols) during incubtion in the following medi: BSS contining sulfte slts (U, I); BSS with chloride slts replcing sulfte slts (A, A); or BSS contining sulfte nd 1 mm fresh neutrlized sodium sulfide (ph 7.5) (0, 0).

5 5470 SCHMIDT ET AL. J. BACTERIOL. 14 *30 z X20 0 )IL DISTANCE lpmi FIG. 2. Microelectrode studies of oxygen (A) nd sulfide (0) grdients resulting from the endogenous metbolism of tuft of B. lb B18LD filments. Distnces re mesured in micrometers from the outer edge of the tuft, which is designted zero on the bsciss. Vlues to the left of zero indicte position inside the tuft of filments, nd vlues to the right represent positions in the gr just outside the tuft of filments. strin SM1, 2.2; V. filiformis ATCC 15551, 2.3; nd V. beggitoides B23SS, 7.2. DISCUSSION Acette oxidtion. B. lb (25) nd other freshwter Beggito strins (14, 29) utilize cette s sole crbon nd energy source in respirtory metbolism. The rte of [2-14C]cette oxidtion to 14CO2 (65 nmol/min per mg of protein) found in the present work grees closely with the Vmx vlue of 72 nmol/min per mg of protein determined by Strohl et l. (25). The methyl crbon is oxidized to CO2 t lower rte thn the crboxyl crbon, ccounting for only 34 to 40% of the cette oxidized by beggitos (25, 29). Becuse the components of typicl erobic, heterotrophic bcteril metbolism re present (29), the following stoichiometry for the complete oxidtion of cette is proposed: (160 nmol/min per mg of protein) '4CH312COOH C02 + 2H20 L[95 nmolmin per mg of protein] (1) (65 per mg protein) where the numbers given in prentheses re vlues obtined experimentlly nd the number given in brckets ws clculted from the expected stoichiometry. Severl known respirtory inhibitors (8) reduced the rte of cette oxidtion in cells of B. lb B18LD, s mesured by either the rte of cette-dependent oxygen consumption or the relese of 14C02 from [2-14C]cette. The suppression of cette oxidtion by sulfide is consistent with the results obtined by Strohl et l. (25) nd suggests tht perhps cette nd sulfide metbolism re competing for oxygen. 2: 12 I LL. _-4 lo1 F.. -J -i TIME (MIN) FIG. 3. Microelectrode experiments showing the effect of gssing tufts of B. lb B18LD filments, embedded in gr, with nitrogen or hydrogen. In one experiment, the filments were gssed with only nitrogen (0). In the second experiment (A), the filments were initilly gssed with nitrogen, nd t the first rrow the nitrogen ws replced with hydrogen gssing. After 5 min of gssing with hydrogen, the filments were gssed with nitrogen gin, nd t 10 min (second rrow; *) they were gssed once gin with hydrogen Sulfide oxidtion. The oxidtion of sulfide to sulfur is ctlyzed by filments of B. lb incubted in the presence of oxygen. We tested the possible involvement of electron trnsfer proteins in sulfide oxidtion by using electron trnsport inhibitors tht were shown to be effective inhibitors of cette oxidtion ih B. lb. Inhibitors of cytochromes or flvoproteins (1) suppressed sulfide oxidtion, but the ubiquinone nlog dibromothymoquinone (22, 30) ws ineffective, suggesting tht electrons from sulfide oxidtion do not interct t the level of ubiquinone in B. lb B18LD. If electrons enter n electron trnsport chin, the inhibitor dt suggest tht both flvoprotein nd cytochromes re in LU X: Lu -.6 Ii..4 -j LI, -J.2-0X TIME (MIN) FIG. 4. Production of sulfide by filments of B. lb B18LD contining (0) or lcking (A) sulfur inclusions incubted under nerobic conditions. Anerobic production of sulfide by drkincubted, sulfur-contining cells of C. vinosum (U) ws used s positive control.

6 VOL. 169, 1987 B. ALBA SULFUR METABOLISM 5471 volved. A pthwy in which electrons from sulfide oxidtion enter t cytochrome c vi flvocytochrome, s found in Chromtium spp. (5, 31), is possible for B. lb. This mechnism of sulfide oxidtion differs from the proposed pthwy for the cynobcterium Oscilltori limnetic, in which quinones re n essentil prt of the sulfide-oxidizing system (19). The stoichiometry for the chemicl oxidtion of sulfide to sulfur is 2:1 (H2S:02; eqution 2). The rte of [35S]sulfide ssimiltion to internl 35S0 (38 nmol/min per mg of protein) nd the rte of sulfide-dependent oxygen consumption (14 nmol/min per mg of protein), determined simultneously, indicte tht the molr stoichiometry for the biologicl oxygen-dependent sulfide oxidtion by B. lb ws 2.7:1 (H2S:02; eqution 3): 2H2S > 2S + 2H20 (2) 2.7H2S S + 2H H+ (3) Bsed on growth yields of mrine of Beggito, Nelson et l. (16) showed tht the stoichiometry of sulfide oxidtion to sulfur for energy purposes lone should be 2:1 (H2S:02; eqution 2), wheres the stoichiometry of oxygen-dependent sulfide oxidtion to sulfur for energetic nd reduction purposes would be 2.42:1 (H2S:02). Tht rtio would fll to pproximtely 0.5:1 if the sulfide were oxidized completely to sulfte (16). Our dt indicte molr rtio of 2.7:1, which, if compred directly with the utotrophic mrine strin of Beggito (16), should indicte tht B. lb obtins both energy nd reductive potentil from the oxidtion of sulfide. It is unlikely, however, tht B. lb B18LD requires reduction potentil from the oxidtion of sulfide, becuse it contins NAD(P)H dehydrogense (29) nd oxidizes cette (25, 29). These processes should generte ll of the reduction potentil required by B. lb, s differentited from utotrophs, which do not generte reducing power from orgnic metbolism. Sulfur oxidtion by Beggito nd Thiothrix spp. The initil observtions on Beggito by Winogrdsky included descriptions of the depletion of sulfur inclusions in the filments, which ws ttributed to the oxidtion of intrcellulr sulfur to sulfte (36). This hypothesis ws recently confirmed for mrine strin of Beggito which ws cpble of utotrophic growth (16). On the other hnd, the microscopic, respirometric, nd isotopic dt presented here indicte tht the freshwter strin, B. lb B18LD, does not oxidize sulfur to sulfte. Experiments with B. lb B15LD hve yielded comprble results (W. R. Strohl, unpublished dt). It is possible, then, tht other freshwter strins of Beggito re incpble of sulfur oxidtion to sulfte, which would mke them metboliclly very different from the mrine strins. Our bility to detect sulfur oxidtion to sulfte ws checked by mesuring sulfur oxidtion by T. nive JP3. Under the sme experimentl conditions s used for B. lb, we showed tht sulfur in T. nive ws trnsient compound tht ws converted to soluble form under erobic conditions. The resolubiliztion of sulfur in T. nive ws coupled with oxygen reduction (27), nd sulfte hs been mesured s one of the oxidtion products. Thus, it ppers tht the freshwter Thiothrix strin my hve sulfur metbolism more similr to tht of mrine Beggito strins thn to tht of the freshwter strins. Sulfur reduction. The inbility of B. lb B18LD to oxidize sulfur leves potentil source of electrons unused. This stored sulfur, however, might be used s terminl electron cceptor under noxic conditions. Sulfur-contining filments of B. lb B18LD reduce sulfur to sulfide, nd this reduction is pprently coupled to the oxidtion of endogenous crbon reserves, possibly poly-,-hydroxybutyric cid (6, 25). Desulfuromons cetoxidns lso linked nerobic sulfur reduction with the oxidtion of orgnic compounds such s ethnol nd cette (2). Filments of B. lb B18LD tht lck sulfur inclusions do not produce sulfide under noxic conditions, eliminting the possibility tht the sulfide ws product of sulfte reduction or the degrdtion of sulfur-contining proteins. Anerobic sulfide production by Beggito spp. ws first observed in strin 75-2 by Nelson nd Cstenholz (13) nd ws thought to be the mens by which the cells survived periods without oxygen. It is possible tht nerobic reduction of sulfur my be common to ll beggitos nd certinly to the freshwter strins tht do not oxidize sulfur to sulfte. When B. lb B18LD filments were plced under shortterm noxic conditions, the presence of hydrogen stimulted sulfide production, suggesting tht B. lb contined hydrogense nd tht hydrogen oxidtion might be coupled with sulfur reduction. Anerobic hydrogen consumption ws detected only when B. lb B18LD filments contined microscopiclly visible sulfur inclusions. Further experiments showed tht the hydrogense in B. lb B18LD ws n uptke hydrogense. The oxidtion of hydrogen is coupled to the reduction of periplsmiclly locted sulfur (27) to sulfide. The coupling of hydrogen oxidtion to sulfur reduction hs been observed with Desulfuromons spp. (4) nd is lso evident in experiments conducted with Chlorobium spp. (20). It is not certin tht hydrogen is vilble in the nturl hbitt of Beggito spp., but since severl strins of Beggito contin nitrogense (18), the hydrogense my function in the recycling of hydrogen produced by side rection of the nitrogense. The metbolic flexibility offered by the coupling of sulfur reduction to nerobic oxidtion of endogenous crbon reserves or hydrogen my be essentil to n orgnism tht exists in chnging environment such s tht of Beggito spp. (13, 17). While the orgnism ppers to grow best in the presence of low concentrtions of oxygen, it is pprently cpble of surviving t lest short periods of noxi (17). In nture this nerobic respirtion of sulfur my be the mens by which filments produce mintennce energy nd the energy required to glide to the oxic-noxic interfce. ACKNOWLEDGMENTS We sincerely thnk Lrs Peter Nielsen nd Mogens Michel Miller for use of microelectrodes mde in Dr. Jorgensen's lbortory. We lso thnk Ricrdo Guerrero (Autonomous University of Brcelon, Spin) for the culture of C. vinosum nd for informtion on growing it, Dougls Nelson (University of Cliforni, Dvis) for the culture of Beggito sp. strin 75-2, Siegfried Mier (Ohio University, Athens) for the culture of Beggito sp. strin SM-1, nd John M. Lrkin (Louisin Stte University, Bton Rouge) for the culture of T. nive. This reserch ws supported by grnt PCM from the Ntionl Science Foundtion. LITERATURE CITED 1. Brtsch, R. G., T. E. Meyer, nd A. B. Robinson Complex c-type cytochromes with bound flvins, p In K. Okunuki, M. D. Kmen, nd I. Sekuzu (ed.), Structure nd function of cytochromes. University of Tokyo Press, Tokyo. 2. Biebl, H., nd N. Pfennig Growth of sulfte-reducing bcteri with sulfur s electron cceptor. Arch. Microbiol. 112: Burton, S. D., nd R. Y. Morit Effect of ctlse nd

7 5472 SCHMIDT ET AL. culturl conditions on growth of Beggito. J. Bcteriol. 88: Fuque, G., D. Herve, nd J. LeGll Structure-function reltionship in hemoproteins: the role of cytochrome C3 in the reduction of colloidl sulfur by sulfte-reducing bcteri. Arch. Microbiol. 121: Gry, G. O., nd D. B. Knff The role of cytochrome c-552-cytochrome c complex in the oxidtion of sulfide in Chromtium vinosum. Biochim. Biophys. Act 680: Gude, H., W. R. Strohl, nd J. M. Lrkin Mixotrophic nd heterotrophic growth of Beggito lb in continuous culture. Arch. Microbiol. 129: Guerrero, R., J. Ms, nd C. Pedr6s-Ali Buoynt density chnges due to intrcellulr content of sulfur in Chromtium wrmingii nd Chromtium vinosum. Arch. Microbiol. 137: Heinen, W Inhibitors of electron trnsport nd oxidtive phosphoryltion, p In J. R. Norris nd D. W. Ribbon (ed.), Methods in microbiology, vol. 6. Acdemic Press, Inc., New York. 9. Kline, J. D Spectrophotometric determintion of hydrogen sulfide in nturl wters. Limnol. Ocenogr. 14: Lrkin, J. M., nd D. L. Shinbrger Chrcteriztion of Thiothrix nive. Int. J. Syst. Bcteriol. 33: Lowry, 0. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: Mezzino, M. J., W. R. Strohl, nd J. M. Lrkin Chrcteriztion of Beggito lb. Arch. Microbiol. 137: Nelson, D. C., nd R. W. Cstenholz Use of reduced sulfur compounds by Beggito sp. J. Bcteriol. 147: Nelson, D. C., nd R. W. Cstenholz Orgnic nutrition of Beggito sp. J. Bcteriol. 147: Nelson, D. C., nd H. W. Jnnsch Chemoutotrophic growth of mrine Beggito in sulfide-grdient cultures. Arch. Microbiol. 136: Nelson, D. C., B. B. J0rgensen, nd N. P. Revsbech Growth pttern nd yield of chemoutotrophic Beggito sp. in oxygen-sulfide microgrdients. Appl. Environ. Microbiol. 52: Nelson, D. C., N. P. Revsbech, nd B. B. J0rgensen Microoxic-noxic niche of Beggito spp.: microelectrode survey of mrine nd freshwter strins; Appl. Environ. Microbiol. 52: Nelson, D. C., J. B. Wterbury, nd H. W. Jnnsch Nitrogen fixtion nd nitrte utiliztion by mrine nd freshwter Beggito. Arch. Microbiol. 133: Oren, A., nd E. Pdn Induction of nerobic, photoutotrophic growth in the cynobcterium Oscilltori limnetic. J. Bcteriol. 133: Pschinger, H., J. Pschinger, nd H. Gffron Photochemicl disproportiontion of sulfur into sulfide nd sulfte by J. BACTERIOL. Chlorobium limicol form thiosulftophilum. Arch. Microbiol. 96: Ptterson, M. S., nd R. C. Greene Mesurement of low energy bet-emitters in queous solutions by liquid scintilltion counting of emulsions. Anl. Chem. 37: Poole, R. K., nd B. A. Hddock Dibromothymoquinone: n inhibitor of erobic electron trnsport t the level of ubiquinone in Escherichi coli. FEBS Lett. 52: Revsbech, N. P., B. B. Jorgensen, T. H. Blckburn, nd Y. Cohen Microelectrode studies of the photosynthesis nd 02, H2S, nd ph profiles of microbil mt. Limnol. Ocenogr. 28: Schmidt, T. M., V. A. Vinci, nd W. R. Strohl Protein synthesis by Beggito lb B18LD in the presence nd bsence of sulfide. Arch. Microbiol. 144: Strohl, W. R., G. C. Cnnon, J. M. Shively, H. Gtide, L. A. Hook, C. M. Lne, nd J. M. Lrkin Heterotrophic crbon metbolism by Beggito lb. J. Bcteriol. 148: Strohl, W. R., nd J. M. Lrkin Enumertion, isoltion, nd chrcteriztion of Beggito from freshwter sediments. Appl. Environ. Microbiol. 36: Strohl, W. R., nd T. M. Schmidt Mixotrophy in Beggito nd Thiothrix, p In W. R. Strohl, nd 0. H. Tuovinen (ed.), Microbil chemoutotrophy. The Ohio Stte University Press, Columbus. 28. Strohl, W. R., T. M. Schmidt, N. H. Lwry, M. J. Mezzino, nd J. M. Lrkin Chrcteriztion of Vitreoscill beggitoides nd Vitreoscill filiformis sp. nov., nom. rev., nd comprison with Vitreoscill stercorri nd Beggito lb. Int. J. Syst. Bcteriol. 36: Strohl, W. R., T. M. Schmidt, V. A. Vinci, nd J. M. Lrkin Electron trnsport nd respirtion in Beggito nd Vitreoscill. Arch. Microbiol. 145: Sun, I. L., nd F. L. Crne Coordinted, coenzyme Q reversible, 2,5-dibromothymoquinone inhibition of electron trnsport nd ATPse in Escherichi coli. Biochem. Biophys. Res. Commun. 68: Truper, H. G., nd U. Fischer Anerobic oxidtion of sulphur compounds s electron donors for bcteril photosynthesis. Phil. Trns. R. Soc. Lond. B 298: Umbreit, W. W., R. H. Burris, nd J. F. Stuffer Mnometric techniques. Burgess Publishing Co., Minnepolis. 33. vn Gemerden, H On the ATP genertion by Chromtium in drkness. Arch. Mikrobiol. 64: Vrgs, A., nd W. R. Strohl Ammoni ssimiltion nd metbolism by Beggito lb. Arch. Microbiol. 142: Vrgs, A., nd W. R. Strohl Utiliztion of nitrte by Beggito lb. Arch. Microbiol. 142: Winogrdsky, S Uber Schwefelbkterien. Bot. Zeit. 45: , , , , , ,