We investigated the applicability of the green fluorescent protein of Aequorea victoria as a reporter for gene expression in the strictly fermentative Gram-negative ethanologenic bacterium Zymomonas mobilis and in the moderately halophilic bacterium Halomonas elongata. We have succeeded to express a mutated gene of green fluorescent protein under the control of different promoters in Z. mobilis and H. elongata grown under various glucose or salt concentrations, respectively. Our results demonstrate that gfp can serve as an easily assayable reporter gene in both organisms. Maximum fluorescence was obtained in Z. mobilis grown aerobically and in H. elongata grown under elevated salt concentration in solid medium. For both bacteria the fluorescence obtained was higher when the gfp gene was placed under the control of a native promoter.
Twelve polycyclic triterpenic hydrocarbons (alpha- and gamma-polypodatetraenes, dammara-20(21),24-diene, 17-isodammara-12,24-diene, eupha-7,24-diene, hop-17(21)-ene, neohop-13(18)-ene, 17-isodammara-20(21),24-diene, neohop-12-ene, fern-8-ene, diploptene and hop-21-ene) were detected in the hydrocarbon fraction from the bacterium Zymomonas mobilis. Some of them have never been reported from bacteria. These triterpenes were present in Z. mobilis in significant amounts, comparable to those of diploptene, which is usually the major triterpenic hydrocarbon in hopanoid-producing bacteria. The occurrence of such compounds confirms the lack of specificity of bacterial squalene cyclases and the possibility of alternative cyclization routes induced by the existence in the cyclization process of intermediate carbocations of sufficient lifetime.
Exponentially growing cells of Zymomonas mobilisnormally exhibit a lag period of up to 3 h when transferred from 0.11 M (2%) to 0.55 M (10%) glucose liquid medium. A mutant ofZ. mobilis (CU1Rif2), fortuitously isolated, showed more than a 20-h lag period when grown under the same conditions, whereas on 0.55 M glucose solid medium, it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable (0.55 M sucrose or fructose) or nonfermentable (0.11 M glucose plus 0.44 M maltose or xylose) sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew without any delay on 0.55 M glucose on which wild-type cells had been incubated for 3 h and removed at the beginning of their exponential phase. This apparent preconditioning was not observed with medium obtained from wild-type cells grown on 0.11 M glucose and supplemented to 0.55 M after removal of the wild-type cells. Undelayed growth of CU1Rif2 on 0.55 M glucose previously conditioned by the wild type was impaired by heating or protease treatment. It is suggested that in Z. mobilis, a diffusible proteinaceous heat-labile factor, transitionally not present in 0.55 M glucose CU1Rif2 cultures, triggers growth on 0.55 M glucose. Biochemical analysis of glucose uptake and glycolytic enzymes implied that glucose assimilation was not directly involved in the phenomenon. By use of a wild-type Z. mobilis genomic library, a 4.5-kb DNA fragment which complemented in low copy number the glucose-defective phenotype as well as glucokinase and glucose uptake of CU1Rif2 was isolated. This fragment carries a gene cluster consisting of four putative coding regions, encoding 167, 167, 145, and 220 amino acids with typical Z. mobilis codon usage, −35 and −10 promoter elements, and individual Shine-Dalgarno consensus sites. However, strong homologies were not detected in a BLAST2 (EMBL-Heidelberg) computer search with known protein sequences.
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 ...
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