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

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Glucose-fructose oxidoreductase (E.C. 1. I .99.-) from the ethanol-producing Gram-negative bacterium Zyrnomonas mobilis is a periplasmic, soluble enzyme that forms a homotetramer of 1 6 0 kDa with one NADP(H) cofactor per subunit that is tightly, but noncovalently, bound. The enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. The obtained crystals belong to the space group P21212, with unit cell constants of 84.6 A, 94.1 A, and 117.OA, consistent with two monomers in the asymmetric unit.They diffract to a resolution of about 2 A and are suitable for X-ray structure determination.

The enzyme glucose‐fructose oxidoreductase (GFOR) from the Gram‐negative ethanologenic bacterium Zymomonas mobilis was purified to homogeneity and was shown to be a tetrameric protein with a subunit size of Mr 42 500. Using immunogold‐labelling in combination with electron microscopy, ultrathin sections of Z. mobilis wild type cells showed that the enzyme GFOR is located in the periplasm off the bacterial cells. Z. mobilis strains which carried the cloned gfo gene on plasmid pSUP104, had 5–6‐fold increased GFOR enzyme activities. Moreover, these cells accumulated large amounts of a presumable unprocessed pre‐GFOR protein (Mr 48 000).

Glucose-fructose oxidoreductase (GFOR) is a periplasmic enzyme of the ethanologenic, Gram-negative bacterium Zymomonas mobilis. It contains tightly bound NADP+ as cofactor. In Z. mobilis GFOR-recombinant strains, a precursor form of GFOR was accumulated. To assay the preGFOR for its NADP(H) content and enzymatic activity, it was purified from an overproducing strain. Using SDS-PAGE, the precursor subunit size was determined to approximately 45 kDa (compared with a 40 kDa subunit size for the mature GFOR subunit). The N-terminal amino acid sequence of the precursor was determined. The N-terminal residues of the GFOR matched with the signal sequence from the DNA sequence of the gene gfo. The precursor form of GFOR was enzymatically active and contained the cofactor NADP(H).