Exponentially growing cells of Zymomonas mobilis normally exhibit a lag period of up to 3 h when they are transferred from a liquid medium containing 2% glucose to a liquid medium containing 10% glucose. A mutant of Z. mobilis (CU1) exhibited a lag period of more than 20 h when it was grown under the same conditions, whereas it failed to grow on a solid medium containing 10% glucose. The glucose-defective phenotype of mutant CU1 was due to a spontaneous insertion in a putative gene (ORF4) identified as part of an operon (glc) which includes three additional putative genes (ORF1, ORF2, and ORF3) with no obvious involvement in the glucose tolerance mechanism. The common promoter controlling glc operon transcription, designated P glc , was found to be osmoregulated and stimulated by the putative product of ORF4 in an autoregulated fashion, as indicated by expression of the gfp reporter gene. Additionally, reverse transcriptase PCR analysis showed that the gene cluster produces a single mRNA, which verified the operon organization of this transcription unit. Further transcriptional analysis demonstrated that glc operon expression is regulated by the concentration of glucose, which supported the hypothesis that this operon is directly involved in the uncharacterized glucose tolerance mechanism of Z. mobilis.Zymomonas mobilis is a strictly fermentative gram-negative ethanologenic bacterium with industrial importance that produces ethanol from simple hexoses at high rates and yields (11). It also has an unusual tolerance to high concentrations of ethanol (up to 13%, wt/vol) and glucose (over 30% for most strains) (47, 48). Therefore, Z. mobilis, a typical saccharophilic organism, is ideal for studying glucose tolerance and osmoregulation mechanisms. Exponentially growing cells of Z. mobilis normally exhibit a lag period of up to 3 h when they are transferred from a liquid medium containing 0.11 M (2%) glucose to a liquid medium containing 0.55 M (10%) glucose. A mutant of Z. mobilis (CU1) (14) and a rifampin-resistant derivative of this strain (CU1Rif2) (1) exhibited a lag period of more than 20 h and were unable to grow on a solid medium containing 0.55 M glucose when they were grown under the same conditions.In an effort to better understand this unusual glucose tolerance trait, we described in a previous paper isolation of a DNA fragment (4.5 kb) which complemented the glucose-defective phenotype of Z. mobilis mutant strains CU1 and CU1Rif2 (12). This fragment consists of four open reading frames (ORFs) coding for four putative polypeptides that are 167, 167, 145, and 220 amino acids long. These ORFs exhibit the typical Z. mobilis codon usage and have individual Shine-Dalgarno consensus sites under the control of a common Ϫ35 and Ϫ10 promoter element (Fig. 1). Interestingly, a protease-sensitive diffusible factor in the medium of a wild-type culture grown in medium containing 10% glucose could correct the defect in the CU1 mutant and its derivative (12).In the present work we focused on complete genetic analysis of ...