JOURNAL OF BACTERIOLOGY, Aug. 1981, p. 340-3490021-9193/81/080340- 10$02.00/0

Vol. 147, No. 2

Assimilatory Sulfur Metabolism in Marine Microorganisms:Characteristics and Regulation of Sulfate Transport in

Pseudomonas halodurans and Alteromonas luteo-violaceustRUSSELL L. CUHEL,:t* CRAIG D. TAYLOR, AND HOLGER W. JANNASCH

Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543

Received 5 January 1981/Accepted 7 May 1981

Sulfate transport capacity was not regulated by cysteine, methionine, or glu-tathione in Pseudomonas halodurans, but growth on sulfate or thiosulfatesuppressed transport. Subsequent sulfur starvation of cultures grown on all sulfursources except glutathione stimulated uptake. Only methionine failed to regulatesulfate transport in Alteromonas luteo-violaceus, and sulfur starvation of allcultures enhanced transport capacity. During sulfur starvation of sulfate-growncultures of both bacteria, the increase in transport capacity was mirrored by adecrease in the low-molecular-weight organic sulfur pool. Little metabolism ofendogenous inorganic sulfate occurred. Cysteine was probably the major regula-tory compound in A. luteo-violaceus, but an intermediate in sulfate reduction,between sulfate and cysteine, controlled sulfate transport in P. halodurans.Kinetic characteristics of sulfate transport in the marine bacteria were similar tothose of previously reported nonmarine systems in spite of significant regulatorydifferences. Sulfate and thiosulfate uptake in P. halodurans responded identicallyto inhibitors, were coordinately regulated by growth on various sulfur compoundsand sulfur starvation, and were mutually competitive inhibitors of transport,suggesting that they were transported by the same mechanism. The affinity of P.halodurans for thiosulfate was much greater than for sulfate.

Sulfate transport systems of bacteria isolatedfrom marine environments have not been char-acterized. Since seawater contains over 25 mMsulfate and this saturating concentration hasexisted for geological time, sulfate transport maybe regulated in a different manner in marinebacteria than in terrestrial microorganisms,which experience low and variable environmen-tal sulfate concentrations. Study of the sulfateuptake systems of marine bacteria may thereforereveal features relevant to the understanding ofadaptation by microorganisms to the much moredilute terrestrial and freshwater environments.Sulfate is never a limiting nutrient in marineecosystems, so it is likely that sulfate transportand metabolism are tightly coupled to growth,and mechanisms of transport regulation may beinterpretable in terms of intracellular sulfatereduction and metabolism.

Possible regulatory mechanisms for sulfatetransport have been suggested through the useof organic sulfur compounds as sole sources of

t Contribution no. 4780 of the Woods Hole OceanographicInstitution.

: Present address: National Oceanographic and Atmos-pheric Administration, Atlantic Oceanographic and Meteoro-logical Laboratory, Miami, Fl, 33149.

sulfur for growth of microorganisms. Media con-taining good sulfur sources (e.g., sulfate, cys-teine, methionine, and glutathione) produce cul-tures with a low initial capacity for sulfate up-take (2, 7, 25, 28, 36), but transport rates can beincreased substantially by subsequent sulfurstarvation (2, 15, 31) or by growth on poor sulfursources such as djenkolic acid (7, 36). Theseobservations have been interpreted to indicatederepression of permease synthesis resultingfrom decreased intracellular pools of sulfate ora product of its metabolism which exerts tran-scriptional control on transport enzyme synthe-sis. The synthesis of the sulfate-binding proteinof Salmonella typhimurium during growth ondjenkolic acid has been directly demonstrated(22).Among the structural analogs of sulfate which

compete for transport in microorganisms, thio-sulfate is of particular interest. As a reducedsulfur compound, it may be responsible for large-scale chemical energy transport from saltmarshes and other anoxic water masses (13).Thiosulfate acts not only as an effective inhibitorof sulfate transport (2, 7, 21, 22, 29, 31, 34, 36); itis also an excellent source of sulfur for growth(11, 18, 23, 26). Demonstration of preferentialthiosulfate uptake by marine bacteria and algae

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SULFATE TRANSPORT IN MARINE BACTERIA 341

will aid in the determination of the fate of thiscompound near anoxic sediments and watermasses. Furthermore, in the open ocean, wherethiosulfate is absent, competition for sulfate up-take and metabolism by added [3S]thiosulfatemay provide a means of lowering the isotopedilution barrier which currently hinders themeasurement of sulfate uptake. It is thereforedesirable to extend the characterization of sul-fate transport systems to marine bacteria. Ifmarine sulfate transport systems are indeed sim-ilar to previously described transport proteins,generalizations based on these earlier findingscan be used with confidence in sulfur metabo-lism measurements directed towards determi-nation of bacterial growth in marine environ-ments.This paper presents evidence for the coinci-

dent regulation of sulfate and thiosulfate trans-port in a marine bacterium, Pseudomonas hal-odurans, which is consistent with genetic andnutritional studies of Salmonella typhimurium(18) and Chlorella pyrenoidosa (11). However,enhancement of transport capacity by growthwith cysteine, methionine, and glutathione wasin strict contrast to previously reported systems.A second marine isolate, Alteromonas luteo-uio-laceus, also demonstrated uptake stimulation bymethionine, but in other respects of regulationwas significantly different from the pseudo-monad. The observed regulatory features ofthese bacteria are considered in relation to theenvironmental conditions experienced in theirnatural habitat.

MATERIALS AND METHODS

Organisms and culture conditions. P. halodur-ans was obtained from Galen E. Jones, University ofNew Hampshire, Durham. A. luteo-violaceus was iso-lated over the Puerto Rico Trench from a seawatertoilet on R/V Oceanus cruise no. 40 (May, 1978) byaerobic enrichment and streaking on seawater mediumcontaining sodium glutamate. It was identified on thebasis of its characteristic violet pigment, narrow nu-tritional capability, and DNA base composition (44mol% guanine plus cytosine) (9). The organisms weregrown in an artificial seawater medium similar to thesulfate-free modification of the Lyman and Flemingformulation (20). However, sulfate contaminationfrom reagent-grade NaCl was sufficient to permitgrowth of A. luteo-violaceus without added sulfate(background concentration, 6.0MM). The problem wasinexpensively overcome through neutralization ofNaOH, which is routinely available at one-tenth thesulfate contamination of NaCl. The resulting relativelylow-contamination (ca. 1.5 MM sulfate) artificial sea-water, designated RLC-water, contained (in grams perliter): NaOH, 16.3; HCl, 14.8; MgCl2.6H20, 10.6; CaCl2.2H20, 1.5; KCl, 0.66; NaHCO3, 0.19; KBr, 0.10; H;3BO3,0.03; and SrCl2.6H20, 0.04. Additional sterile supple-ments to the autoclaved basal salts medium were, with

their respective final concentrations: sodium gluta-mate (10 mM), NH4Cl (500 MM), KH2PO4 (50 MM),and I.M.R. trace elements solution (8) (1 ml/liter). Allcultures were grown at pH 7.8 to 8.0 at 20 ± 2'C on arotary shaker at 250 rpm.

Uptake studies were performed with cultures grownin complete medium containing 1 mM sodium sulfate.Cells were harvested by centrifugation (5,000 x g, 10min) in the late exponential phase of growth (1 x 108to 3 x 108/ml), washed once with basal salts medium,and suspended to the same density in complete me-dium minus sulfur. Cultures were starved for 2 h beforethe assay of transport activity as described below. Forderepression experiments, 1,500 ml of complete me-dium minus sulfur was inoculated at low density (ca.104 cells per ml) and shaken for 10 min to evenlydistribute the cells. Samples of 100 ml were asepticallytransferred to sterile flasks and supplemented withfilter-sterilized solutions of Na2SO4, Na2S203, L-CYS-teine-hydrochloride (neutralized just before use), DL-methionine, or reduced glutathione at a final sulfurconcentration of 500 MM (i.e., 250 MM Na2S203). Late-exponential-phase cultures were treated as describedabove.

Sulfate uptake assay. Transport activity was as-sayed as follows: 9.8 ml of the cell suspension wasadded to tubes containing 0.1 ml of the inhibitor orappropriate solvent and 0.1 ml of 35S-labeled sulfateor thiosulfate. Carrier-free sulfate (final activity, 1 to2 MLCi/ml) was supplemented with sterile solutions ofNa2SO4 to achieve the desired final concentration (11.5to 201.5 MM, including background sulfate). Thiosul-fate (sulfane S tagged) was used at concentrations of2.5 to 25 MM (specific activity, 10 to 30 dpm/pmol).The mixture was blended thoroughly on a Vortexmixer, and 1-ml subsamples were withdrawn at 60-sintervals for 5 min into tubes containing 0.1 ml of 1 MNa2SO4 or Na2S203 as appropriate to dilute the label(final concentration, over 100 mM). Subsamples of 1ml were filtered through Reeve Angel 984H Ultrafineor Whatman GF/F glass fiber filters and rinsed with0.5 M NaCl. Termination of the reaction by isotopedilution did not result in loss of label transported intothe cells, nor did further detectable uptake of labeloccur before filtration as determined in separate ex-periments (data not shown).

Determination of LMW compounds. Cultureswere grown as described for transport studies exceptthat [35S]sulfate was included in the medium at 11 to19 dpm/pmol. After more than 10 generations ofgrowth, cultures were harvested and suspended insulfur-free medium as described above. Samples (5 ml)were filtered as described above, and the filters wereground in cold 10% trichloroacetic acid, refrigeratedfor 30 min, and centrifuged (5,000 x g, 20 min). Thepellets were washed once with cold 10% trichloroaceticacid and centrifuged, and the supernatant fractionswere combined. Inorganic sulfate was separated fromlow-molecular-weight (LMW) organic sulfur com-pounds by barium precipitation in a mixture contain-ing, in 2.05 ml: 1.75 ml of sample, 0.05 ml of 5 mMNa2SO4, and 0.25 ml of 1 M BaCl2 (pH 2). After 2 h at40C, the precipitate was removed by centrifugation(5,000 x g, 10 min), and 1 ml of the supernatant fluidwas counted, giving the LMW organic sulfur fraction.

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342 CUHEL, TAYLOR, AND JANNASCH

The pellet was rinsed twice with 1 M BaCl2 andcounted as a water suspension, giving the inorganicsulfate fraction.

Filtration procedure. Due to the high activitiesof radiosulfate required fo some experiments, prob-lems with label adsorption to the filters can be consid-erable (16). We constructed a punch funnel (Fig. 1)which serves to excise only the cell-retaining portionof the filter. Use of the punch funnel reduced adsorp-tion blanks by over 10-fold, which increased the pre-cision of the assays significantly at low uptake rates.Other methods. Direct cell counts were made us-

ing acridine orange epifluorescence microscopy (5).Cell viability was determined by plating dilutions ofthe culture on complete medium containing 15 g ofagar (Difco) per liter. Protein was determined on 10%trichloroacetic acid-precipitable material dissolved in0.1 N NaOH by the method of Bradford (3). Radioiso-topes were counted in Aquasol in a Beckman LS-100Cliquid scintillation counter, using the channels ratiomethod of quench correction.

Radiochemicals. Carrier-free Na235SO4 andNa235S203 (10 to 30 dpm/pmol) were obtained fromAmersham (Chicago, Ill.).Other chemicals. Dicyclohexylcarbodiimide and

carbonylcyanide m-chlorophenylhydrazone were ob-tained from U.S. Biochemicals (Cleveland, Ohio).para-Hydroxymercuribenzoate and 2,4-dinitrophenolwere from Sigma (St. Louis, Mo.). Na2SeO4 was thegift of I. K. Smith. The protein dye reagent was

L

FIG. 1. Punch funnel used for filtration of radiois-otopically labeled samples. The funnel (upper por-tion) is 0.75-in. (ca. 19-mm) polycarbonate tubing, andthe lower portion is 316 stainless steel. The scale barrepresents 0.5 in. (ca. 12.7 mm). A small square ofWhatman no. 1 or similar filter paper is placed over

a 25-mm fritted glass base to preserve the frit surfaceand to prevent clogging by fibers from the glassfilters. A 25-cm Reeve Angel 984H or Whatman GF/F filter is placed on top, followed by the punch funnel.To reduce loss of vacuum for samples larger than 10ml, a thin piece ofrubberpunched with a hole slightlylarger than 0.5 in. (ca. 12.7 mm) may be placed on topof the filter. This funnel may also be used with mem-brane filters.

purchased from Bio-Rad Laboratories (Richmond,Calif.). All other chemicals were reagent grade. Specialprecautions were taken to obtain components of RLC-water with the lowest possible sulfate contamination.

RESULTSCultures of P. halodurans grown with 1 mM

sulfate as the sole sulfur source exhibited lowinitial rates of sulfate and thiosulfate uptakewhich could be enhanced by sulfur starvation(Fig. 2). The rapid increase in transport capacityduring the first 2 h of sulfur starvation wasmirrored by a decrease in LMW organic sulfurcompounds (Fig. 3), but little metabolism ofendogenous inorganic sulfate occurred. After 2h of constant transport activity, a slow increasein uptake rate was accompanied by a linearincrease in cell numbers (Fig. 2) and a similarlyslow utilization of LMW organic sulfur com-pounds (Fig. 3). In a similar experiment usingcultures grown in medium containing only 250

140

E 120

i 100

0 80

0 600 40

20

0

3 t-c

E

c00

0 2 4 6 6 10 12Sulfur Starvation Time (Hours

FIG. 2. Viable cell counts and sulfate and thiosul-fate uptake rates during sulfur starvation of P. hal-odurans. Final assay concentrations and specific ac-tivities were 200 PM S042- (17 dpm/pmol) and 20 AiMS2032- (17 dpm/pmol). Uptake rate data are the av-erages of duplicate determinations; error bars areshown when larger than the symbol. Viable countsare the mean ± 1 standard error of two plates at eachof three dilutions.

a) 700L A

,600

E 500- \A4Q \ LVW Organic S

A-A

0 300 A Ay

200 A,

100 SO4L

0-H_0__________ 3 0 0

0 2 3 4 5 6 7 8 9 0Inc-Ubat on TIrne (Hours)

FIG. 3. Disappearance ofLMW sulfur compoundsfrom soluble pools of P. halodurans during sulfurstarvation. Experimental details are in the text.

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SULFATE TRANSPORT IN MARINE BACTERIA 343

1LM sulfate (a concentration nearing sulfate-lim-ited growth for this bacterium), no increase incell numbers occurred (data not shown). Thisindicated that the utilization of endogenous sul-fur-containing compounds, rather than the up-take of contaminating sulfate from the medium,was responsible for the cell division shown inFig. 2.The low initial rate of sulfate uptake and its

enhancement by sulfur starvation as describedabove were also found for A. luteo-violaceus(Fig. 4). This organism did not take up thiosul-fate at rates sufficient for assay (4a). As with P.halodurans, sulfur starvation resulted in a rapiddepletion of the LMW organic sulfur pool (Fig.5) which was virtually the mirror image of thetransport activity time course. However, a dif-ferent response to prolonged sulfur starvationwas manifest in the absence of a plateau andfurther increase of transport capacity. After 2 hof sulfur starvation the uptake rate began todecrease, accompanied by a loss of cell viabilitywhich decreased to 16% of the initial value after4 h. Viability estimates were complicated bygrowth of the cells into tangled filaments, as

evidenced by the increase in optical density inFig. 4, but the ultimate decrease in direct countsdue to autolysis of this organism (9, 10) sup-ported the lower plating efficiency. Transportactivity and apparent viability began to declineas the LMW organic sulfur pool reached theminimum size shown in Fig. 5.The correlation of transport activity with dis-

appearance ofLMW organic sulfur from solublepools suggested that one or more organic sulfurcompounds may repress synthesis of transportproteins. Therefore the transport capacity of

E 26 -

"I 22 -

0t(' 18 -cnIV

14 -

Q 0.9 -

0.8 -

t 0.7 -C

b 06

0.5 -

'DU0

3 -

2 -I, I I I0 2 4 6 8 10 12

Sulfur Starvation Time (Hours)FIG. 4. Sulfate uptake capacity during sulfur star-

vation of A. luteo-violaceus. Final assay concentra-tion of 35so42 was 26.5 ELM (45 dpm/pmol). Cellnumbers are single direct count determinations.

s 4000 -

, 3500-u

E 3000-

0 2500-

> 2000-

a 1500 -

0 1000-

-' 500-

0-

X LMW Organic S

SO4T

0 2 3 4 5 6

Sulfur Starvation Time (Hours)

FIG. 5. Disappearance ofLMW sulfur compoundsfrom soluble pools of A. luteo-violaceus. Experimen-tal details are in the text.

cultures grown on various sole sources of sulfurwas investigated. Surprisingly, organic sulfurcompounds were poor sources of sulfur for thegrowth of P. halodurans, yielding cultures withgrowth rates half or less than half of those grownon sulfate or thiosulfate (Table 1). The methio-nine-grown culture reached 107 cells per ml witha lag time of 18 h relative to those grown onsulfate. This lengthy delay cannot be attributedto the slower growth rate alone and suggeststhat synthesis of enzymes required for the con-version of methionine to cysteine must be in-duced for growth on methionine to occur. Thesecells also exhibited a markedly higher proteincontent. Cultures provided with glutathionegrew at the slowest rate; however, extrapolationof the direct count regression line to the time ofinoculation indicated a more rapid growth ratebefore the appearance of turbidity. It is notknown whether this was due to dimerization ofglutathione and concomitant difficulty of trans-port or to other effects.

In contrast to previously studied sulfate trans-port systems (7, 21, 36), growth on all organicsulfur sources stimulated both sulfate and thio-sulfate uptake effectively. Uptake rates beforesulfur starvation were highest in cultures grownon methionine and glutathione, with transportcapacity greater than that observed for sulfate-and thiosulfate-grown cells which had beenstarved for sulfur for 8 h. However, the charac-teristically low initial uptake rates for both sul-fate and thiosulfate by sulfate-grown cells (Fig.2, Table 1) were also observed for the culturegrown on thiosulfate.

Further enhancement of sulfate transport ca-

pacity occurred as a result of sulfur starvationfor all cultures except that growth on glutathi-one. Sulfate and thiosulfate-grown cells showeda primary increase of about sixfold during thefirst 2 h of sulfur starvation, followed by a smallincrease. The cysteine-grown culture exhibited

(f0

/ \ SOz Uptake

0.420,0'

0'*

Direct C Dunts

Direct Counts \.

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344 CUHEL, TAYLOR, AND JANNASCH

TABLE 1. Sulfate and thiosulfate transport capacity after growth on various sulfur sources and subsequentsulfur starv.ation of P. halodurans"

Sulfur source (k) (Starvation Cells per ml Protein per 10i cells tJptake rate"(h)(x101) (pg ~~~~~~~Sulfate Thiosulfate

Sulfate (0.87 h-1) 0 1.45 18.4 ± 0.3 6.1 ± 1.1 5.9 ± 0.02 1.49 21.2 ± 0.8 32.6 ± 0.5 16.7 ± 0.18 2.08 20.7 ± 0.3 49.9 ± 0.1 14.9 ± 2.1

Thiosulfate (0.89 h ') 0 1.40 17.8 ± 1.2 4.8 ± 0.2 5.8 ± 0.32 1.55 19.7 ± 0.5 32.0 ± 0.6 16.5 ± 0.18 2.35 17.9 ± 0.3 47.2 ± 3.3 18.9 ± 0.1

Cystine (0.45 h ') 0 2.01 16.3 ± 0.6 44.1 ± 4.8 23.4 ± 0.22 2.46 19.5 ± 0.1 91.5 ± 0.0 43.4 ± 1.28 4.97 13.9 ± 0.1 45.2 ± 1.2 21.0 ± 1.1

Glutathione (0.32 h-1) 0 1.67 18.6 ± 0.2 61.7 ± 0.3 31.5 ± 0.52 2.07 20.2 ± 0.0 61.7 ± 0.4 41.3 ± 0.08 4.43 13.1 ± 0.1 24.9 ± 0.7 14.9 ± 0.2

Methionine (0.46 h-1) O 1.30 23.8 ± 0.4 63.8 ± 2.1 34.8 ± 0.22 2.00 21.1 ± 0.5 89.5 ± 0.6 37.3 ± 0.68 3.31 18.6 ± 0.2 62.2 ± 1.3 22.4 ± 1.0

a Cultures were grown, harvested, and assayed as described in the text. Assay concentrations were 100 1iMsulfate and 20 jtM thiosulfate. The uptake rate is expressed as picomoles of SO,2 or S2032 per 10' cells perminute. The standard error for duplicate measurements is shown.

The growth rate, k, was determined from direct counts taken at intervals during exponential growth.The reaction mixture contained, in 10 ml, 9.9 ml of cell suspension and 0.1 ml of either 3-SO,2 (final

concentration, 100 pM, 32 dpm/pmol) or 35 s202 (final concentration, 20 pM, 8 dpm/pmol). The uptake rate isexpressed as picomoles of SO42- or S20:32 per 10' cells per minute.

a doubling of transport capacity, but an increaseof less than 50% accompanied sulfur starvationof the methionine-grown cells; both returned tothe initial rate as a result of cell division anddilution of transport proteins among daughtercells. Glutathione-grown cells demonstrated noincrease during the first 2 h, but declined byover 50% during the next 6 h as a result of celldivision. It should be noted that under no cir-cumstances did the total transport capacity (permilliliter of culture) decrease; all reductions inactivity reported in Table 1 were due to in-creases in cell numbers. This was also true fortotal protein.

Thiosulfate uptake rates followed the samepattern as those for sulfate. All three organicsulfur sources yielded cultures with enhancedthiosulfate transport capacity. The trends forboth sulfate and thiosulfate uptake stimulationby growth on various sulfur sources and subse-quent sulfur starvation were the same in 12 ofthe 15 cases.

In contrast to P. halodurans, the three or-ganic sulfur compounds tested were equally goodsulfur sources for A. luteo- violaceus. The growthrate (i.e., reciprocal doubling time, average 0.27h-') varied less than 10% among cultures. Table2 shows that enhancement of sulfate uptake

capacity occurred only in the culture utilizingmethionine. Initial uptake rates of cells grownwith cysteine or glutathione were lower than inthe sulfate-grown culture. Based on initial celldensity, all cultures demonstrated increased up-take as a function of sulfur starvation.The significantly different patterns of regula-

tion of sulfate transport in the marine bacteriacompared to previously studied systems war-ranted the investigation of other characteristicscommon among them. We therefore tested forsubstrate specificity, using sulfate analogs (15,22, 23, 29, 34, 36), for adherence to Michaelis-Menten kinetics, and for the ability to accumu-late sulfate in excess of growth requirements(32).A variety of active transport inhibitors de-

creased sulfate uptake rates in both marine bac-teria (Table 3). Sulfate and thiosulfate uptakeby P. halodurans were affected identically.

Also consistent with previous studies was theeffect of group VI oxyanions on sulfate transport.Table 4 lists the kinetic constants for sulfateuptake by the marine bacteria and for thiosul-fate uptake by P. halodurans, and Table 5 givesthe K'1 values for competitive inhibition of up-take by the sulfate analogs. In both bacteria, theeffectiveness of inhibition was inversely propor-

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SULFATE TRANSPORT IN MARINE BACTERIA 345

TABLE 2. Sulfate transport capacity ofA. luteo-violaceus after growth on various sulfur sources andsubsequent sulfur starvation a

Sulfate transporth with sulfur source for growth:Sulfur starvation (h)

Sulfate Methionine Cystine Glutathione

0 4.8±0.4 20.8±4.2 0.9±0.1 01 23.3 ± 0.4 68.3 ± 1.9 16.2 ± 0.2 9.4 0.52 33.7 ± 0.4 109 ± 0.2c 29.6 ± 0 45.4 6.8

" Cultures were grown, harvested, and assayed as described in the text. The assay sulfate concentration was100 pM. Initial cell densities were 1.1 x 108 to 1.7 x 108/ml.

Based on initial cell density and expressed as picomoles of S042- per i0' cells per minute.First 4 min of uptake only.

TABLE 3. Effects of active transport inhibitors on sulfate uptake by P. halodurans and A. luteo-violaceusand on thiosulfate uptake by P. haloduransa

P. halodurans A. luteo-violaceus S042-Additionh (concn) S042- uptake S2032- uptake uptake

Rate' % Inhibition Rate' % Inhibition Rate' %. Inhibition

Distilled water 60.7 0 15.0 0 53.4 0NaN3 (100 AM) 53.4 12 12.0 20d 46.2 14dNaN3 (1 mM) 29.0 52 6.8 55 10.8 80Glutamate omitted 6.9 89 4.2 72 20.8 61

Ethanol (1%) 62.0 0 15.7 0 52.8 0DCCD (100 AM) 48.0 23e 13.5 14e 23.7 5562,4-DNP (100 jM) 52.2 16 13.1 17 42.6 192,4-DNP (1 mM) 0 100 1.0 94 0 100CCCP (5 jiM) 57.0 8 12.9 18 29.2 45CCCP (50 AM) 26.5 57 7.4 53 0 100

" Starved cell suspensions were prepared as described in the text. Thiosulfate uptake was assayed at 15 AM.Sulfate was assayed at 200 jiM (P. halodurans) or 100 AM (A. luteo-violaceus). The cell density was 4.1 x 108/ml for P. halodurans and 1.9 x 108/ml for A. luteo-violaceus.

h DCCD, Dicyclohexylcarbodiimide; 2,4-DNP, 2,4-dinitrophenol; CCCP, carbonylcyanide m-chlorophenylhy-drazone.

' Rates are expressed as picomoles of SO42- or S2032- per 10' cells per minute.d Inhibition relative to distilled water control for NaN3 added and glutamate omitted.'Inhibition relative to 1% ethanol control for 2,4-DNP, CCCP, and DCCD.

TABLE 4. Kinetic constants for sulfate andthiosulfate uptake by P. halodurans and for sulfate

uptake by A. luteo-violaceus"

V,,,ax (pmolStrain Compound Ka, (pM) per 10' cells

per min)

P. halodurans Thiosulfate 14.7 ± 1.5 (8) 30.5 ± 3.0 (7)Sulfate 214 ± 34 (9) 108 ± 22 (9)

A. luteo-violaceus Sulfate 186 ± 18 (5) 146

" Numbers in parentheses indicate the number of experi-ments if greater than one.

tional to the molecular weight of the analog,demonstrating size selectivity. Additionally, sul-fate and thiosulfate were mutually competitiveinhibitors in P. halodurans, with the Km forthiosulfate uptake equal to the K', for thiosulfateinhibition of sulfate uptake. Sulfate analogs alsocompetitively inhibited thiosulfate uptake withthe same specificity as for sulfate, but higher

TABLE 5. Inhibition constants for competition bysulfate analogsa

K', (pM)Competi- P. halodurans A. luteo-

vlolaceusThiosulfate Sulfate sulfate

CrO42- 52 3 1 73SeO42- 1,989 ± 174 (2) 569 238MoO42 9,109 ± 562 (2) 1,327 + 120 (3) 12,200S2032- 16.6 ± 1.8 (2) NDSO42- 397 ± 75 (3)

a Computed according to the procedure of Webb (35). Num-bers in parentheses indicate the number of experiments ifgreater than one. ND, Not determined.

concentrations of inhibitors were required. Thisis consistent with the more than 10-fold higheraffinity of the transport system for thiosulfaterelative to sulfate.A more subtle regulatory feature was discov-

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346 CUHEL, TAYLOR, AND JANNASCH

ered during determination of kinetic constantsfor uninhibited sulfate uptake. The competitiveinhibition studies described above were con-ducted in the range from 10 to 200 1iM addedsulfate. In this range, values for K,, and V,.ax,derived from 1/u versus 1/S, v versus v/S, andS/u versus S plots, agreed within 5%. To verifyMichaelis-Menten kinetics, it was desirable toextend the range of sulfate concentrations tohigher values.With P. halodurans it was found that the

sulfate uptake rate ceased to increase in propor-tion to added sulfate above 250 (uM S042. In-deed, at 1 mM S042 the observed rate was only12% higher than at 250 1iM and only 62% of therate expected from extrapolation of the 1/L ver-sus 1/S plot used to derive the kinetic constantsin Table 4. Uptake was linear with time at allconcentrations. As previously mentioned, 250MM S042- borders on sulfate-limited growth inthis organism. Although the V,,,ax calculated forsulfate uptake would permit uptake in excess ofgrowth requirements, the observed maximumrate was just sufflcient to meet cellular demandsbased on the sulfur quota derived from equilib-rium labeling studies (R. L. Cuhel, C. D. Taylor,and H. W. Jannasch, Arch. Microbiol., in press).A close coupling between growth require-

ments for sulfur and sulfate uptake is achievedthrough a different mechanism in A. luteo-z0io-laceus. Initial sulfate uptake rates increasedwith added sulfate throughout the range of con-centrations tested and were within experimentalerror of the values predicted from a simple hy-perbolic relationship. However, at higher con-centrations the initial uptake range was main-tained for only a short period (Fig. 6), until 500to 600 pmol of S042- was taken up per 108 cells.This phenomenon was observed whenever initialuptake rates (based on the first 1 to 2 min) weregreater than 60 pmol of So42- per 10' cells permin regardless of the sulfate concentration, as inthe methionine-grown cells in Table 2 after 1and 2 h of sulfur starvation.Nonlinear uptake of the type shown in Fig. 6

suggests feedback inhibition of uptake by endproducts of sulfate metabolism, since the reduc-tion in transport rate is too rapid to be attrib-utable to repression of permease synthesis andsubsequent turnover of existing proteins. Thiswas supported by inhibition of sulfate uptake byorganic sulfur compounds. When assayed at 100MM added sulfate, 1 mM methionine had littleor no detectable effect, whereas cysteine andglutathione strongly inhibited sulfate uptake(100 and 85%, respectively). A. luteo-violaceusis insensitive to the sulfhydryl reagent para-hydroxymercuribenzoate with respect to sulfate

900 -1002 zo

800-

700

0 - / o 5015" 600

0 500

0400 /1015

300D

b 200 265 o

100 -o-0 - 6 5

2 4 6 8 10Incubation Time(Minutes)

FIG. 6. Sulfate uptake by A. luteo-violaceus at sul-fate concentrations of 6.5 to 1,002 p.M. The cell sus-pension (3.2 x 108/ml) uas starved for sulfur andassayed as described in the text.

uptake (4a), so it is not likely that these com-pounds inhibited transport through binding ofcysteine sulfhydryl groups to the active site ofthe transport protein. It is more likely that theinhibition was a rapid response to an increase inorganic sulfur pool size. Additionally, in the pres-ence of glutathione the sulfate uptake rate de-creased continuously during the 5-min transportassay, suggesting metabolic conversion to cys-teine through the action of a peptidase. Thesedata are consistent with the sulfate uptake rates,tested as a function of the sulfur source forgrowth, that are presented in Table 2.

DISCUSSIONThe failure of growth on end products of sul-

fate metabolism to produce a significant regula-tory effect on sulfate transport capacity in P.halodurans is extremely unusual if not uniqueamong microorganisms. Some LMW sulfur com-pound must either repress permease synthesisor exert feedback control on existing enzymes,since cultures grown on sulfate or thiosulfateexhibited low initial uptake capacity. The con-current enhancement of uptake during sulfurstarvation and disappearance of LMW organicsulfur compounds strongly suggest that a com-ponent of this fraction is responsible for trans-port regulation. Sulfate itself is an unlikely can-didate because of the relative constancy of itsconcentration during the development of trans-port capacity. Growth on cysteine resulted inhigh initial rates of sulfate uptake, suggesting acompound in the cysteine biosynthetic pathwayafter sulfate, but not cysteine itself. Since thesulfate reduction sequence occurs via a pathway

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of enzyme-bound intermediates (1), it is likelythat adenosine 5'-phosphosulfate or adenosine3'-phosphate 5'-phosphosulfate regulated sulfatetransport capacity in P. halodurans.An unambiguous distinction between repres-

sion of permease synthesis and feedback inhibi-tion of existing transport proteins cannot readilybe made. In vivo inhibition of sulfate reductionenzyme activities by growth on S-containingamino acids (14, 27) cannot be assumed to bedue to repression of specific protein synthesis,since the activity of such enzymes has beenshown to be regulated in vitro by immediate endproducts of metabolism (6, 27). For example,Anacystis possesses no transcriptional controlfor synthesis of homoserine-O-transsuccinylase,an enzyme of methionine biosynthesis (6). Feed-back inhibition by methionine in vitro explainedthe apparent repression observed in activity as-says in cell-free extracts after growth on methi-onine; passage of the extract through a columnto remove LMW material restored enzyme ac-tivity. Concentrations of intracellular metabo-lites can therefore be high enough to controlenzyme activity in vivo through feedback inhi-bition. In addition, both repression of cystathi-onase synthesis in Escherichia coli by growthwith methionine and feedback of catalytic activ-ity in vitro by homocysteine have been demon-strated simultaneously (27).

It is probable that feedback inhibition by cys-teine is important in sulfate transport regulationin A. luteo-violaceus. This was suggested by thenon-linearity of uptake when initial rates weregreater than growth demands for sulfur and bythe rapid inhibition of sulfate uptake in thepresence of cysteine and glutathione, which alsoprevented expression of sulfate transport activ-ity when used as the sole source of sulfur forgrowth. In both bacteria it is clear that theactivity of enzymes catalyzing reactions in theopposite direction from normal cysteine and me-thionine biosynthetic pathways was sufficientlyrate-limiting to prevent build-up of intracellularpools to levels adequate to exert metabolic con-trol on sulfate uptake.

In contrast to regulatory differences, the ki-netic characteristics of sulfate uptake in themarine bacteria, i.e., size-selective competitiveinhibition by sulfate analogs (29, 34), energydependence (12, 15, 25, 29, 36), and adherence toMichaelis-Menten kinetics, were very similar tothose reported for terrestrial microorganisms.The 5- to 10-fold lower affinity of the marinetransport systems for sulfate was not surprisingin view of the consistently saturating concentra-tion experienced by marine bacteria in situ, al-though it suggests that selective pressure has

been brought to bear on their nonmarine coun-terparts.

Thiosulfate was an extremely effective com-petitive inhibitor of sulfate uptake in P. halo-durans, with a greater than 10-fold higher affin-ity for the reduced sulfur compound. The simi-larity of the Km for thiosulfate uptake (14.7 AM)to the K', for its inhibition of sulfate uptake (16.6,LM), the similar reponse of sulfate and thiosul-fate uptake to inhibitors, and the coordinateregulation of sulfate and thiosulfate uptake bygrowth on various sulfur sources and sulfur star-vation leave little doubt that the two compoundsare transported by the same system. These dataare consistent with genetic and nutritional stud-ies of sulfate metabolism in Chlorella (11) andSalmonella (18).The much higher affinity of P. halodurans

for thiosulfate concurs with the involvement ofsulfhydryl groups in the transport mechanism,as demonstrated through inhibition bypara-hy-droxymercuribenzoate (4a, 21, 33). A sulfhydrylgroup or disulfide bond would provide a site forcovalent binding of thiosulfate through the sul-fane moiety; such binding is not possible withthe unreactive sulfate molecule and would in-crease the effective concentration of thiosulfateat the transport site. Combined with the identi-cal response of sulfate and thiosulfate transportto the conditions examined in this paper, as wellas the virtually unidirectional transport ob-served in this and other microorganisms (21, 32,36), the active transport model of Kaback andBarnes (17) is appealing. The absence of effec-tive thiosulfate transport by A. luteo-violaceusis the subject of another communication (4a).The major regulatory difference between P.

halodurans and A. luteo-violaceus was the re-duction of sulfate transport capacity by growthusing cysteine or glutathione in A. luteo-viola-ceus and its apparent rapid feedback inhibitionof transport by these compounds. This organismproduces an extracellular protease (9), especiallyduring stationary phases, as evidenced by therapid decline in transport rates and viabilityshortly after the cessation of protein synthesisduring sulfur starvation. The potency of theprotease was confirmed by addition of chloram-phenicol to an exponentially growing culture;total protein decreased by 50% in 1 h, less thanone-half a generation time. Frequent isolation ofA. luteo-violaceus from surfaces such as Sar-gassum weed and fish skin suggests that it de-rives much of its nutrition from proteolysis,which would provide cysteine and methionine atrelatively high concentrations compared to sea-water.

It should be noted that A. luteo-violaceus was

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348 CUHEL, TAYLOR, AND JANNASCH

chosen for study on the basis of its unusualnutritional characteristics and distinctive pig-mentation and should not be taken as repre-sentative of marine bacteria. Unlike most marinebacteria, it can grow on very few organic carboncompounds, and it was the only one of 12 isolateswhich could not grow on thiosulfate as the solesource of sulfur.

It was stimulating to find that the kineticcharacteristics of P. halodurans sulfate trans-port were virtually identical to those previouslyreported for the more extensively studied non-marine microorganisms, in spite of the com-pletely different regulatory mechanism. Thepredictability of sulfate uptake systems indicatesthat it may still be possible to use competitiveinhibition of sulfate uptake by [3'S]thiosulfateas a means of lowering the isotope dilution bar-rier for studies of marine bacterial sulfur metab-olism and protein synthesis.The lack of sulfate transport system regula-

tion by end products of sulfate metabolism in P.halodurans is both interesting and gratifyingwhen we consider the use of sulfate metabolismas a measurement of marine bacterial growth.Sulfate transport is energy-requirng, and itsreduction is even more so. If P. halodurans hadbeen exposed to utilizable concentrations of S-containing amino acids in its recent evolutionaryhistory, it would be expected to take advantageof this by reducing the activity or synthesis ofthe enzymes of precursor metabolism. The con-centrations of total dissolved amino acids inopen-ocean seawater are very low (19), but theyincrease in near-shore and estuarine envorn-ments (4), which are the habitat of P. halodur-ans (A. Rosenberg, Ph.D. thesis, University ofNew Hampshire, Durham, 1977). Nonetheless,S-containing amino acids, when detectable atall, rarely rise above 5 x 10-8 M (4, 19), scarcelya significant amount. This is largely due to thelow to undetectable amounts of these com-pounds in microbial cells (30) and the conse-quent small input resulting from death and au-tolysis of marine organisms. Furthermore, thelag time and slow growth rate of P. haloduranswhen using end products of sulfate metabolismas sole sources of sulfur indicates a poorly de-veloped capability to reverse the pathways ofsulfate reduction and S-amino acid biosynthesis.These observations strongly support the con-

tention that inorganic sulfur, probably sulfate, isthe only source of sulfur for marine microorga-nisms in aerobic waters. Constant, saturatingsulfate concentrations and regulation of trans-port rates to meet but not exceed cellular growthrequirements render it likely that sulfate trans-port and metabolism will provide an accurate

measure of marine bacterial growth. Supportingstudies on the intermediary metabolism of sul-fate and its relation to de novo protein synthesisin P. halodurans and A. luteo-uiolaceus havebeen submitted elsewhere.

ACKNOWLEDGMENTS

We are grateful to M. Mandel for the base compositionanalysis of A. luteo-violaceus DNA. Special thanks are ex-tended to Galen Jones for the culture of Pseudomonas halo-durans.

This work was supported by National Science FoundationGrants OCE77-12172, OCE79-19178, and OCE79-19264. Fur-ther support for R.I.C. was provided by the Education De-partment of the Woods Hole Oceanographic Institution.

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