sorbitol promotes zymomonas in environments evidence … · function of sorbitol in z. mobilis 7689...

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JOURNAL OF BACTERIOLOGY, Dec. 1994, p. 7688-7693 Vol. 176, No. 24 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology Sorbitol Promotes Growth of Zymomonas mobilis in Environments with High Concentrations of Sugar: Evidence for a Physiological Function of Glucose-Fructose Oxidoreductase in Osmoprotection HEIDI LOOS, REINHARD KRAMER, HERMANN SAHM, AND GEORG A. SPRENGER* Institut far Biotechnologie 1, Forschungszentnum JAlich GmbH, D-52425 Julich, Germany Received 25 July 1994/Accepted 14 October 1994 The gram-negative ethanologenic bacterium Zymomonas mobilis is able to grow in media containing high concentrations of glucose or other sugars. A novel compatible solute for bacteria, sorbitol, which enhances growth of Z. mobilis at glucose concentrations exceeding 0.83 M (15%), is described. Added sorbitol was accumulated intracellularly up to 1 M to counteract high external glucose concentrations (up to 1.66 M or 30o). Accumulation of sorbitol was triggered by a glucose upshift (e.g., from 0.33 to 1.27 M or 6 to 23%) and was prevented by the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone; 100 tIM). The sorbitol transport system followed Michaelis-Menten kinetics, with an apparent Km of 34 mM and a Vm.. of 11.2 nmol - min-' mg-' (dry mass). Sorbitol was produced by the cells themselves and was accumulated when growing on sucrose (1 M or 36%) by the action of the periplasmic enzyme glucose-fructose oxidoreductase, which converts glucose and fructose to gluconolactone and sorbitol. Thus, Z. mobilis can form and accumulate the compatible solute sorbitol from a natural carbon source, sucrose, in order to overcome osmotic stress in high-sugar media. No other major compatible solute (betaine, proline, glutamate, or trehalose) was detected. The gram-negative, strictly fermentative and ethanologenic bacterium Zymomonas mobilis tolerates high concentrations of both ethanol (up to 13% [24]) and sugar (16, 28) in the growth medium. An early report stated that Z. mobilis, (then called Termobacterium mobile), which had been isolated from pulque (fermented agave sap), could grow and ferment media con- taining up to 25% (1.39 M) glucose (16). Swings and De Ley tested various concentrations of glucose and found that all strains grew with 20% (1.11 M) glucose (within 34 h), 88% of the strains grew with 30% (1.66 M) glucose (within 2 to 5 days), and with 40% (2.22 M) glucose, 54% of the strains developed after lag phases of 4 to 20 days (28). In contrast, Z. mobilis is known to be rather sensitive to salt, as no strains grew at 2% NaCl (0.34 M) or 2% KCl in liquid media (22, 28). Therefore, sugar tolerance need not be linked to osmotolerance or salt tolerance in general. Possible mechanisms of adaptation to high glucose concentrations of Z. mobilis have been proposed earlier (10, 27). These authors stated that rapid equilibration of external and internal glucose, mediated by a glucose facili- tator system, should allow adaptation to high external glucose concentrations. However, in growing cells of Z. mobilis only low internal glucose concentrations were found (13), so that another compound should be needed for balance. During growth on sucrose or on mixtures of glucose plus fructose, Z. mobilis forms sorbitol as a major by-product to ethanol and CO2. Thus, up to 11% of the carbon source(s) was converted into sorbitol and afterwards detected in the media, the sorbitol yield being proportional to the increased sugar concentrations (2, 6, 11, 30-32). It was suggested (2, 31) that sorbitol formation resulted from an inhibition of fructokinase * Corresponding author. Mailing address: Institut fur Biotechnolo- gie 1, Forschungszentrum Julich GmbH, P.O. Box 1913, D-52425 Jilich, Germany. Phone: 49-2461-616205. Fax: 49-2461-612710. by glucose. This would lead to fructose accumulation and favor sorbitol formation (17) by glucose-fructose oxidoreductase (GFOR; EC 1.1.99.-), an enzyme which constitutes up to 1% of the soluble protein from Z. mobilis (34) and is located as mature protein in the periplasm of the bacterium (1, 15, 19, 20). As the physiological function of sorbitol formation by GFOR remained unknown (34), we assayed whether sorbitol had a protective function for Z. mobilis in the presence of high sugar concentrations. From the known habitats (fruit saps, honey, etc.) of Z. mobilis (26, 28), it may be inferred that the microorganism is frequently challenged by high-sugar media or by periods of desiccation (drought). Here, we present data that Z. mobilis forms sorbitol and accumulates it as a compatible solute to counteract detrimental osmotic effects. (A part of these results has been presented as a poster contribution at the 7th International Congress of Bacteriology and Applied Microbiology Division, International Union of Microbiological Societies, at Prague, Czech Republic, 3 to 8 July 1994). MATERIALS AND METHODS Bacterial strains and culture conditions. Strain Z. mobilis ZM6 (ATCC 29191) (28) was used. The complex medium consisted of (per liter) 5 g of yeast extract, 1 g of KH2PO4, 0.5 g of Mg2SO4, 1 g of (NH4)2SO4, distilled water, and various amounts of glucose, fructose, or sucrose at pH 5.0 (5). To achieve sugar concentrations higher than 50 g/liter, sugar stock solutions were autoclaved separately and mixed with the other ingredients afterwards. Media were inoculated with 1 to 20% of a preculture. Cells were grown anaerobically at 30'C in Bellco-Glass tubes (10 ml) or in 300-ml glass vessels filled with 200 ml of medium. 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Page 1: Sorbitol Promotes Zymomonas in Environments Evidence … · FUNCTION OF SORBITOL IN Z. MOBILIS 7689 filter. Growth was recorded turbidometrically in a Shimadzu UV-160 spectrophotometer

JOURNAL OF BACTERIOLOGY, Dec. 1994, p. 7688-7693 Vol. 176, No. 240021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

Sorbitol Promotes Growth of Zymomonas mobilis inEnvironments with High Concentrations of Sugar:

Evidence for a Physiological Function ofGlucose-Fructose Oxidoreductase

in OsmoprotectionHEIDI LOOS, REINHARD KRAMER, HERMANN SAHM, AND GEORG A. SPRENGER*

Institut far Biotechnologie 1, Forschungszentnum JAlich GmbH, D-52425 Julich, Germany

Received 25 July 1994/Accepted 14 October 1994

The gram-negative ethanologenic bacterium Zymomonas mobilis is able to grow in media containing highconcentrations of glucose or other sugars. A novel compatible solute for bacteria, sorbitol, which enhancesgrowth of Z. mobilis at glucose concentrations exceeding 0.83 M (15%), is described. Added sorbitol wasaccumulated intracellularly up to 1 M to counteract high external glucose concentrations (up to 1.66 M or30o). Accumulation of sorbitol was triggered by a glucose upshift (e.g., from 0.33 to 1.27 M or 6 to 23%) andwas prevented by the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone; 100 tIM). The sorbitoltransport system followed Michaelis-Menten kinetics, with an apparent Km of 34 mM and a Vm.. of 11.2nmol - min-' mg-' (dry mass). Sorbitol was produced by the cells themselves and was accumulated whengrowing on sucrose (1 M or 36%) by the action of the periplasmic enzyme glucose-fructose oxidoreductase,which converts glucose and fructose to gluconolactone and sorbitol. Thus, Z. mobilis can form and accumulatethe compatible solute sorbitol from a natural carbon source, sucrose, in order to overcome osmotic stress inhigh-sugar media. No other major compatible solute (betaine, proline, glutamate, or trehalose) was detected.

The gram-negative, strictly fermentative and ethanologenicbacterium Zymomonas mobilis tolerates high concentrations ofboth ethanol (up to 13% [24]) and sugar (16, 28) in the growthmedium. An early report stated that Z. mobilis, (then calledTermobacterium mobile), which had been isolated from pulque(fermented agave sap), could grow and ferment media con-

taining up to 25% (1.39 M) glucose (16). Swings and De Leytested various concentrations of glucose and found that allstrains grew with 20% (1.11 M) glucose (within 34 h), 88% ofthe strains grew with 30% (1.66 M) glucose (within 2 to 5 days),and with 40% (2.22 M) glucose, 54% of the strains developedafter lag phases of 4 to 20 days (28). In contrast, Z. mobilis isknown to be rather sensitive to salt, as no strains grew at 2%NaCl (0.34 M) or 2% KCl in liquid media (22, 28). Therefore,sugar tolerance need not be linked to osmotolerance or salttolerance in general. Possible mechanisms of adaptation tohigh glucose concentrations of Z. mobilis have been proposedearlier (10, 27). These authors stated that rapid equilibrationof external and internal glucose, mediated by a glucose facili-tator system, should allow adaptation to high external glucoseconcentrations. However, in growing cells of Z. mobilis onlylow internal glucose concentrations were found (13), so thatanother compound should be needed for balance.During growth on sucrose or on mixtures of glucose plus

fructose, Z. mobilis forms sorbitol as a major by-product toethanol and CO2. Thus, up to 11% of the carbon source(s) wasconverted into sorbitol and afterwards detected in the media,the sorbitol yield being proportional to the increased sugarconcentrations (2, 6, 11, 30-32). It was suggested (2, 31) thatsorbitol formation resulted from an inhibition of fructokinase

* Corresponding author. Mailing address: Institut fur Biotechnolo-gie 1, Forschungszentrum Julich GmbH, P.O. Box 1913, D-52425Jilich, Germany. Phone: 49-2461-616205. Fax: 49-2461-612710.

by glucose. This would lead to fructose accumulation and favorsorbitol formation (17) by glucose-fructose oxidoreductase(GFOR; EC 1.1.99.-), an enzyme which constitutes up to 1% ofthe soluble protein from Z. mobilis (34) and is located asmature protein in the periplasm of the bacterium (1, 15, 19,20). As the physiological function of sorbitol formation byGFOR remained unknown (34), we assayed whether sorbitolhad a protective function for Z. mobilis in the presence of highsugar concentrations.From the known habitats (fruit saps, honey, etc.) of Z.

mobilis (26, 28), it may be inferred that the microorganism isfrequently challenged by high-sugar media or by periods ofdesiccation (drought). Here, we present data that Z. mobilisforms sorbitol and accumulates it as a compatible solute tocounteract detrimental osmotic effects.(A part of these results has been presented as a poster

contribution at the 7th International Congress of Bacteriologyand Applied Microbiology Division, International Union ofMicrobiological Societies, at Prague, Czech Republic, 3 to 8July 1994).

MATERIALS AND METHODS

Bacterial strains and culture conditions. Strain Z. mobilisZM6 (ATCC 29191) (28) was used. The complex mediumconsisted of (per liter) 5 g of yeast extract, 1 g of KH2PO4, 0.5g of Mg2SO4, 1 g of (NH4)2SO4, distilled water, and variousamounts of glucose, fructose, or sucrose at pH 5.0 (5). Toachieve sugar concentrations higher than 50 g/liter, sugar stocksolutions were autoclaved separately and mixed with the otheringredients afterwards. Media were inoculated with 1 to 20%of a preculture. Cells were grown anaerobically at 30'C inBellco-Glass tubes (10 ml) or in 300-ml glass vessels filled with200 ml of medium. Anaerobiosis was achieved by indigenousCO2 formation, and degassing was performed through a sterile

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FUNCTION OF SORBITOL IN Z. MOBILIS 7689

filter. Growth was recorded turbidometrically in a ShimadzuUV-160 spectrophotometer at a wavelength of 550 nm; anoptical density at 550 nm (OD550) of 1 corresponded to 0.2 mgof dry cell mass * ml-1.

For determinations of wet weight, samples of 2 ml werecollected in an Eppendorf refrigerated centrifuge (5 min at13,000 rpm). Supernatants were removed with a Pasteurpipette, the vessel walls were wiped dry, and the pellets wereweighed. Dry weights were determined by drying the pellets inthe Eppendorf vessels for 12 h at 1050C in a dry chamber untilstable values were obtained. After growth in media with highsugar concentrations (1.27 M or 23% glucose, 1.39 M or 50%sucrose), the ratio of wet weight to dry weight decreasedcompared with that in low-sugar media. For the determinationof intracellular sorbitol concentrations, the true cytosolic vol-ume was determined by silicone oil centrifugation with 3H20as permeant label (for the total volume) and [14C]taurine asimpermeant label (excluded from the cytoplasmic space) (25).The internal volume amounted to 2.3 to 1.5 RId * mg-- (drymass), depending on the external sugar concentration (25, 27).

Preparation of cell extracts and sorbitol determinations. Todetermine intracellular sorbitol concentrations, cells from agrowing culture were harvested at different incubation times bycentrifugation in an Eppendorf refrigerated centrifuge (5 min,40C, 13,000 rpm). Wet weights were determined, and to 4 mg(wet weight) of cells, 50 RI of perchloric acid (20% [wtlvol])was added and the mixture was vortexed and sonicated for 5minutes in a Branson 2200 sonification bath to obtain homog-enization. After a 2-min centrifugation, supernatants weretransferred into a new cup and neutralized by addition of 40 RIof KOH (5 M) plus triethanolamine (1 M) and kept at -20°Cfor at least 30 min. The potassium perchlorate precipitate andcell debris were removed by an additional centrifugation. Thesupernatants were used directly for sorbitol determination.Alternatively, cells were rapidly collected by filtration on glassfiber filters (Whatman GF-F; 0.2-,um pore size, 25-mm diam-eter) and washed once with incubation buffer with sorbitolomitted. The filters were dried at 90°C and transferred into testtubes, and 1 ml of 0.1% cetyl trimethyl-ammonium bromide(CTAB) was added. After gentle shaking at room temperaturefor 15 min, an aliquot of the supernatant was used for sorbitoldetermination.

Sorbitol was determined spectrophotometrically at 492 nmby use of sorbitol dehydrogenase, NADH, diaphorase, andiodonitrotetrazolium chloride (3). The internal sorbitol con-centrations were calculated on the basis of the measured truecytoplasmic volumes in the corresponding experiments (seeabove). Protein concentrations were determined by a dye-binding method (4) with bovine serum albumin as a standard.

RESULTS

Sorbitol promotes growth of Z. mobilis at high glucose orfructose concentrations. Strain ZM6 (ATCC 29191) was grownin complex media with various glucose concentrations andincubated anaerobically at 30°C in the presence or absence of50 mM sorbitol. No significant influences of added sorbitolwere seen at 0.55 M (10%) or 0.83 M (15%) glucose (data notshown). At 1.11 M (20%), however, a pronounced lag of up to20 h was observed when cells were not supplemented withsorbitol, whereas cells grew without a detectable lag in mediumwith 1.11 M glucose plus sorbitol (data not shown). The evenmore pronounced differences between cultures with or withoutsorbitol at 1.39 M (25%) and 1.66 M (30%) glucose are shownin Fig. 1A. Cells in the presence of sorbitol grew nearly withoutlag at 1.39 M glucose, but a severe growth lag of more than 72

C)

6ui

to

ci6

1UA

0 480 48 96 144

0 48 96 144 192Time (h)

FIG. 1. Growth of Z. mobilis on sugar-rich media. Cells wereinoculated after preculture (complex medium plus 0.55 M [10%]glucose) into complex medium containing 1.39 M (25%) glucose (0,0) or 1.66 M (30%) glucose (O, *) (A) or 1.11 M (20%) fructose (0,0) or 1.39 M fructose (O, *) (B). Growth was monitored by readingthe OD550. Open symbols represent cultures without sorbitol addition,and closed symbols represent cultures in the presence of 50 mMsorbitol. Data are the means of three independent determinations.

h occurred when sorbitol was omitted. At 1.66 M glucose, nogrowth occurred within 20 days of incubation without sorbitol(data not shown); with 50 mM sorbitol, growth started after alag of about 90 h.When cells were grown in complex media with fructose,

sorbitol did not exert such a significant promoting effect withthe carbon source at 0.55 or 0.83 M (data not shown). Aclear-cut influence was observed at 1.39 M (25%) fructose, asnonsupplemented cultures would not grow at this concentra-tion (Fig. 1B), and cultures with added sorbitol grew with a lagof about 90 h at a growth rate of 0.03 h-1 (data not shown).Growth rates on high-fructose media were generally lowerthan those on glucose, with prolonged lag phases in thebeginning. This may be caused by formation of differentby-products during growth on fructose (32). The specificGFOR activities were similar at increased fructose concentra-tions (data not shown), so no specific induction took place. Atthe end of the fermentations, sorbitol contents in the mediawere assayed and found to be virtually unaltered. Therefore,no significant sorbitol catabolism had occurred.

Sorbitol is accumulated in the bacterial cells depending on

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7690 LOOS ET AL.

TABLE 1. Internal and external sorbitol concentrations dependingon the presence of sorbitol or fructose and various

glucose concentrations

Wet wt of cells Sorbitol concn (mM)bGlucose (M)a Additive per ml of

medium (mg) External Intracellular

0.55 None (control) 3.8 0.3 1.40.83 None (control) 2.8 0.2 3.00.55 1 mM sorbitol 3.2 1.1 1.60.83 1 mM sorbitol 3.2 1.1 8.30.55 10 mM sorbitol 4.0 9.5 8.20.83 10 mM sorbitol 3.5 8.8 43.31.11 10 mM sorbitol 3.5 8.6 138.01.39 10 mM sorbitol 2.9 8.5 183.00.55 280 mM fructose 4.7 65.0 47.00.83 280 mM fructose 2.4 39.9 137.01.11 280 mM fructose 1.9 32.3 301.0

a Z. mobilis ZM6 was grown in 10-ml batch cultures in complex medium plusglucose at different concentrations (0.55, 0.83, 1.11, or 1.39 M) to an OD550 ofabout 3.0.

b Sorbitol concentrations were determined in the culture supernatant (exter-nal) or from the cell internal volume (intracellular). The problem of the origin ofsorbitol in the zero controls has not yet been solved.

external glucose stress. As sorbitol promoted growth on mediawith high sugar concentrations, we assayed whether this solutewas accumulated intracellularly. This would be a prerequisitefor a compatible solute (7). Cells were grown under a variety ofglucose concentrations, with or without the addition of sorbitolor fructose (Table 1). Almost no sorbitol was found in themedium or in the cells when growth took place at 0.55 M(10%) or 0.83 M (15%) glucose alone. When sorbitol wasadded to the growth medium (1 or 10 mM), intracellularsorbitol accumulation increased as much as 18-fold (at 1.39 Mglucose and 10 mM sorbitol in the medium). The presence of0.28 M (5%) fructose, in addition to glucose, led both to theformation of sorbitol in the medium and to its accumulation inthe cells.

Kinetics of sorbitol accumulation. These results indicatedthat sorbitol was effectively accumulated only if high sugarconcentrations were present. Therefore, the sorbitol accumu-lation after a glucose upshift was analyzed further. Cells froma preculture at 0.55 M glucose (0.33 M residual glucose) wereinoculated in parallel into a 0.55 M glucose medium or wereshifted (without prior washing) to a 1.11 M glucose medium,both containing 10 mM sorbitol. The initial glucose concentra-tions then were 0.77 M (14%) and 1.27 M (23%), respectively.After 2 h of incubation at 0.77 M glucose, less than 40 mMsorbitol was found intracellularly (about a fourfold accumula-tion), whereas at 1.27 M more than 270 mM (27-fold) wasdetected (data not shown). The glucose upshift thereforeresulted in a massive sorbitol accumulation. The accumulationreached its maximum (at 10 mM external sorbitol after about5 h of incubation) (Fig. 2) when cells had entered theexponential-growth phase. Intracellular sorbitol concentra-tions decreased thereafter concomitantly with the extracellularglucose concentration (Fig. 2) when cells had entered thestationary-growth phase.For a detailed investigation of sorbitol uptake, we analyzed

the basic kinetic parameters of sorbitol transport after glucoseupshift from 0.33 to 1.27 M under various external sorbitolconcentrations. Cells were inoculated from the late-exponen-tial-growth phase (OD550, 3.0). Sorbitol concentrations from 2to 100 mM were applied. As seen in Fig. 2 and 3A, sorbitol waseffectively taken up, reaching peak values of up to 1 M

600-

mI o400

.0

0

0 2 4 6 8 10 12Time (h)

FIG. 2. Kinetics of sorbitol uptake following a glucose upshift.Cells were inoculated from a preculture with 0.55 M glucose intomedia with 1.11 M glucose (final glucose concentration, 1.3 M) in thepresence of different sorbitol concentrations. At the indicated times,samples of 10 ml were withdrawn and analyzed for intracellularsorbitol concentrations (sorbitol int.). The symbols indicate 2 mM (V),10 mM (V), 50 mM (*), and 100 mM (0) sorbitol.

(cytosolic concentration) when external sorbitol was added athigh concentrations. The carrier-mediated sorbitol uptakefollowed saturation kinetics of a Michaelis-Menten type (Fig.3B) with an apparent Km of 34 mM and a Vm. of 11.2nmol - min- 1 * mg- 1 (dry mass).The observation of high internal accumulation and the fact

that sorbitol uptake was saturable (Fig. 3A and B) indicatedthe presence of an energy-dependent transport system. Inorder to define the type of energy coupling, we assayedwhether sorbitol accumulation was affected by changes in theelectrochemical potential of the plasma membrane. Since ithas previously been shown that several commonly used iono-phors and uncouplers are not effective in Z. mobilis (25), weused CCCP (carbonyl cyanide m-chlorophenylhydrazone), aprotonophoric uncoupler, at the high concentration of 100p.M. Under glucose upshift conditions, CCCP completelyprevented accumulation of sorbitol which was added at 10 or50 mM external concentrations (data not shown). We haveshown previously that addition of CCCP up to 100 p.M in thepresence of excess glucose does not result in the reduction ofthe cytosolic ATP level (25). We thus conclude that the drivingforce for accumulative sorbitol uptake is related to the elec-trochemical potential or a component of it.

Sorbitol formation and its accumulation during growth onhigh sucrose concentrations. A prerequisite for sorbitol for-mation by the GFOR is the simultaneous presence of glucoseand fructose (34). Besides honey, which consists of mixtures ofglucose and fructose, other known habitats of Z. mobilis do notcontain enough free fructose (28). Z. mobilis, however, is ableto split sucrose into its moieties, glucose and fructose. As canbe seen from Fig. 4, when shifted to a medium containing 1 Msucrose, sorbitol was produced and accumulated inside thecells to concentrations of up to 700 mM while the externalconcentration of sorbitol at the same time did not exceed 30mM. Concomitantly with sorbitol accumulation, the onset ofexponential growth was observed. The pool of internal sorbitolstayed, however, relatively constant and did not decrease as inthe case of glucose (Fig. 4). We conclude that Z. mobilis formsthe compatible solute sorbitol from sucrose also and that itaccumulates sorbitol to counteract detrimental effects of highsugar concentrations.

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FUNCTION OF SORBITOL IN Z. MOBILIS 7691

1200

- 1000E

800

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1 400c

200

0

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-6'E10

O 2

E=L

6-

0.° 4

CL

140

0 1 2

Time (h)

B

I- 0 0.1 0.2 0.3 0.4

v/ISj (1039g dm-1min-1)FIG. 3. Kinetic analysis of the sorbitol uptake system. Cells were

grown as indicated in the legend to Fig. 2 and inoculated into mediawith 1.11 M glucose and different sorbitol concentrations. (A) Internalsorbitol concentrations were determined at the indicated time points.The symbols indicate sorbitol concentrations in the media of 2 mM(0), 5 mM (A), 12 mM (V), 25 mM (0), 50 mM (A), and 100 mM (V).The data represent the means of three independent measurements.(B) Eadie-Hofstee plot indicating the determination of Vm,. and Kmvalues for sorbitol uptake.

A

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Function of sorbitol in osmotic stress induced by nonme-tabolizable substances. In order to see whether accumulationof sorbitol is a specific response to the presence of metaboliz-able substrates of Z. mobilis or constitutes a general mecha-nism to overcome osmotic stress, we grew Z. mobilis incomplex media with 10% glucose and increased concentrationsof the nonmetabolizable disaccharide maltose, which is knownnot to enter Z. mobilis cells (27). In the absence of addedsorbitol, cells would grow normally with 0.28 M (10%) addedmaltose, whereas with increased concentrations (up to 1.1 M or40%) growth was halted. This inhibition could be overcome bythe presence of sorbitol (50 mM) (data not shown).Sodium chloride is often used to provoke osmotic stress in

bacteria (8, 9). NaCl inhibits growth of Z. mobilis completely at0.34 M (2%) in the medium (28). To see whether sorbitolhelped to withstand this NaCl inhibition, Z. mobilis was grownin medium containing 0.55 M (10%) glucose and various NaClconcentrations, i.e., from 0 to 0.34 M (2%) in steps of 0.25%.Although sorbitol enhanced growth rates at increased NaClconcentrations, it could not help to overcome growth arrest at0.34 M (2%) salt (data not shown). In addition to relativelymild osmotic stress by 0.34 M NaCl (in comparison with 1.1 Mmaltose or other sugars), the increased ionic strength may begrowth limiting.With 13C nuclear magnetic resonance spectroscopy, crude

extracts of Z. mobilis cells after a glucose upshock in thepresence of 10 mM sorbitol were analyzed for sugar speciesaccumulated in the cytosol. Besides sorbitol, glucose, andfructose, no other compatible solutes could be detected insignificant concentrations in cells grown at 1.11 M glucose. Inparticular, neither glycine betaine, glycerol, proline, glutamate,mannitol, nor trehalose was present (data not shown). Wefurthermore observed that the addition of glycine betaine (10mM) did not improve growth after a glucose upshock in theabsence of sorbitol. Apart from the metabolizable sugars,sorbitol thus seems to be the only major compatible solute inZ. mobilis.

DISCUSSION

Bacteria are able to accumulate compatible solutes likeglycine betaine in the cytoplasm to reduce adverse effects ofabiotic stress like salinity or drought (7-9, 18). Other compat-

CO0F.

0

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10

8

m6 F.

(04 5lo

2

0

Time (h)FIG. 4. Growth on sucrose and formation and intracellular accumulation of sorbitol. Cells from a preculture with 0.55 M (20%) sucrose

(residual sucrose, 0.3 M) were shifted to medium containing 1.0 M sucrose (final concentration). Growth was monitored by measurement of thewet weight (in milligrams per 100 microliters) (0). Formation of external sorbitol (sorbitol ext.) (K) and intracellular accumulation of sorbitol(sorbitol int.) (*) were measured at the indicated time points.

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7692 LOOS ET AL.

fructose ho

levan -~

sucrose -

glucoses

fructose

outermembrane

glucose

pen-plasm fruc ose

inner A Ekmembrane A

cytosol

II\\\\\

FIG. 5. Scheme of Z. mobilis peripheral met;glucose, fructose, and sorbitol. Sucrose can be splicrase (LS) or invertase B (INV) to the moieties glThe periplasmic GFOR converts the two sugars iand sorbitol. Transporters in the inner membrfacilitator (GF) for glucose and fructose, the sort

only when cells encounter high concentrations of fructose. (ii)Free fructose is less frequent in natural habitats than sucrose.Sucrose, however, is a source of fructose through the actions ofsucrases (invertases) or levansucrase. Both types of sucrose-hydrolyzing activities are present when Z. mobilis grows onsucrose (21, 23). (iii) Glucose is the preferred substrate of theglucose facilitator in Z. mobilis, outcompeting fructose (10).When growing on sucrose, the fructose moiety is not primarilytransported but can be utilized for the formation of fructo-oligomers or levan (14, 32). Also, free fructose concentrationscan rise and fructose can then serve as a substrate for the

luconolactone GFOR which is present constitutively (34). (iv) The essence ofthe periplasmic localization of GFOR could be the simulta-neous accessibility for both substrates (glucose plus fructose),

)rbitol which is hardly possible in the cytosol as high amounts of bothsugars are not to be expected inside the cells. Thus, GFORcould act as an osmosensor which reacts only at high fructoseconcentrations, indicating in an indirect manner the presenceof high sucrose. As a consequence, GFOR forms the compat-ible solute sorbitol from the moieties of sucrose. Figure 5summarizes these issues.To our knowledge, formation of sorbitol for use as a

compatible solute in bacteria has not been described before.However, in sugar-tolerant yeasts such as Hansenula anomala,sorbitol and other polyols are known as compatible solutes

abolism of sucrose, (29). Sorbitol can protect proteins during dehydration byit by either levansu- osmotic or thermal stress and is therefore used to preservelucose plus fructose. proteins during storage (33). We conclude that Z. mobilis, ininto gluconolactone response to high external sugar concentrations, accumulatesrane are a glucose sorbitol by a novel transport system and makes use of sorbitolvitol carrier (SC) of as a compatible solute.

the present work, and pOSSibly a transporter tor gluconolactone and/orgluconic acid (?).

ible solutes described for bacteria are trehalose, sucrose,proline, potassium ions and/or glutamate, and ectoine (forrecent reviews, see references 8 and 9). Here, we presentevidence that the gram-negative organism Z. mobilis accumu-lates sorbitol in response to an osmotic upshock by externalsugar. Sorbitol accumulation can also be elicited by addition ofsubstances in high concentrations like maltose, which cannotpenetrate the plasma membrane of Z. mobilis (27). As theorganism can produce sorbitol from sucrose (after splitting ofsucrose into glucose plus fructose) by action of a periplasmicGFOR, it uses sucrose both as a carbon and energy source andto generate an osmoprotective compound.We found that sorbitol uptake is a carrier-mediated process

characterized by Michaelis-Menten kinetics (Ki,, 34 mM; Vm.,11.2 nmol mmin- * mg-' [dry mass]). It is, furthermore, energydependent, as it was abolished by addition of the uncouplerCCCP, which is consistent with a secondary transport mecha-nism. Whether the sorbitol transport system is constitutivelyactive or can be induced by hyperosmotic stress remains to berevealed. Whereas sorbitol addition enabled the cells to growin the presence of up to 1.66 M external sugar, growth in thepresence of comparably low-molarity (0.34 M) NaCl ceasedeven if sorbitol was added. This makes sense if NaCl not onlyleads to osmotic stress but enters the cells and disturbs cellmetabolism, e.g., because of high ionic strength.

Z. mobilis makes use of a GFOR (34), which is located in theperiplasmic space of the organism (1, 20), to form the com-patible solute sorbitol. Some features of the GFOR restrictformation of sorbitol to conditions of hyperosmotic stress. (i)Because of its extraordinary low affinity to fructose (Km, 400mM to 1 M [12, 34]), sorbitol is formed at appreciable rates

ACKNOWLEDGMENTS

We thank Joachim Strohhacker for 3C nuclear magnetic resonancemeasurement and Camille Lambert and Christa Kamp for technicalassistance.

This work was supported by a grant from the Deutsche Forschungs-gemeinschaft (Sa-408/2-2).

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