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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. Anaerobiosis was achieved by indigenous CO2 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 Shimadzu UV-160 spectrophotometer at a wavelength of 550 nm; an optical density at 550 nm (OD550) of 1 corresponded to 0.2 mg of dry cell mass * ml-1.

    For determinations of wet weight, samples of 2 ml were collected in an Eppendorf refrigerated centrifuge (5 min at 13,000 rpm). Supernatants were removed with a Pasteur pipette, the vessel walls were wiped dry, and the pellets were weighed. Dry weights were determined by drying the pellets in the Eppendorf vessels for 12 h at 1050C in a dry chamber until stable values were obtained. After growth in media with high sugar concentrations (1.27 M or 23% glucose, 1.39 M or 50% sucrose), the ratio of wet weight to dry weight decreased compared with that in low-sugar media. For the determination of intracellular sorbitol concentrations, the true cytosolic vol- ume was determined by silicone oil centrifugation with 3H20 as permeant label (for the total volume) and [14C]taurine as impermeant label (excluded from the cytoplasmic space) (25). The internal volume amounted to 2.3 to 1.5 RId * mg-- (dry mass), depending on the external sugar concentration (25, 27).

    Preparation of cell extracts and sorbitol determinations. To determine intracellular sorbitol concentrations, cells from a growing culture were harvested at different incubation times by centrifugation 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 5 minutes in a Branson 2200 sonification bath to obtain homog- enization. After a 2-min centrifugation, supernatants were transferred into a new cup and neutralized by addition of 40 RI of KOH (5 M) plus triethanolamine (1 M) and kept at -20°C for at least 30 min. The potassium perchlorate precipitate and cell debris were removed by an additional centrifugation. The supernatants were used directly for sorbitol determination. Alternatively, cells were rapidly collected by filtration on glass fiber filters (Whatman GF-F; 0.2-,um pore size, 25-mm diam- eter) and washed once with incubation buffer with sorbitol omitted. The filters were dried at 90°C and transferred into test tubes, and 1 ml of 0.1% cetyl trimethyl-ammonium bromide (CTAB) was added. After gentle shaking at room temperature for 15 min, an aliquot of the supernatant was used for sorbitol determination.

    Sorbitol was determined spectrophotometrically at 492 nm by use of sorbitol dehydrogenase, NADH, diaphorase, and iodonitrotetrazolium chloride (3). The internal sorbitol con- centrations were calculated on the basis of the measured true cytoplasmic volumes in the corresponding experiments (see above). 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 or fructose concentrations. Strain ZM6 (ATCC 29191) was grown in complex media with various glucose concentrations and incubated anaerobically at 30°C in the presence or absence of 50 mM sorbitol. No significant influences of added sorbitol were seen at 0.55 M (10%) or 0.83 M (15%) glucose (data not shown). At 1.11 M (20%), however, a pronounced lag of up to 20 h was observed when cells were not supplemented with sorbitol, whereas cells grew without a detectable lag in medium with 1.11 M glucose plus sorbitol (data not shown). The even more pronounced differences between cultures with or without sorbitol at 1.39 M (25%) and 1.66 M (30%) glucose are shown in Fig. 1A. Cells in the presence of sorbitol grew nearly without lag at 1.39 M glucose, but a severe growth lag of more than 72

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    FIG. 1. Growth of Z. mobilis on sugar-rich media. Cells were inoculated after preculture (complex medium p

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