Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pif). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

The efficient diversion of pyruvate from normal fermentative pathways to ethanol production in Klebsiella oxytoca M5A1 requires the expression of Zymomonas mobilis genes encoding both pyruvate decarboxylase and alcohol dehydrogenase. Final ethanol concentrations obtained with the best recombinant, strain M5A1 (pLOI555), were in excess of 40 g/liter with an efficiency of 0.48 g of ethanol (xylose) and 0.50 g of ethanol (glucose) per g of sugar, as compared with a theoretical maximum of 0.51 g of ethanol per g of sugar. The maximal volumetric productivity per hour for both sugars was 2.0 g/liter. This volumetric productivity with xylose is almost twice that previously obtained with ethanologenic Escherichia coli. Succinate was also produced as a minor product during fermentation. * Corresponding author. t Florida Agricultural Experiment Station publication no. R01797.

In Zymomonas mobilis, three-to fourfold more glyceraldehyde-3-phosphate dehydrogenase protein than phosphoglycerate kinase is needed for glycolysis because of differences in catalytic efficiency. Consistent with this requirement, higher levels of glyceraldehyde-3-phosphate dehydrogenase were observed with twodimensional polyacrylamide gel electrophoresis. The genes encoding these enzymes (gap and pgk, respectively) form a bicistronic operon, and some form of regulation is required to provide this differential expression. Two transcripts were observed in Northern RNA analyses with segments of gap as a probe: a more abundant 1.2-kb transcript that contained gap alone and a 2.7-kb transcript that contained both genes. Based on the relative amounts of these transcripts, the coding regions for glyceraldehyde-3-phosphate dehydrogenase were calculated to be fivefold more abundant than those for phosphoglycerate kinase. Assuming equal translational efficiency, this is sufficient to provide the observed differences in expression. Operon fusions with lacZ provided no evidence for intercistronic terminators or attenuation mechanisms. Both gap operon messages were very stable, with half-lives of approximately 16 min (1.2-kb transcript) and 7 min (2.7-kb transcript). Transcript mapping and turnover studies indicated that the shorter gap message was a stable degradation product of the full-length message. Thus differential expression of gap and pgk results primarily from increased translation of the more stable 5' segment of the transcript containing gap. The slow turnover of the messages encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase is proposed as a major feature contributing to the high level of expression of these essential enzymes.

In Zymomonas mobUis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAP) and phosphoglycerate kinase (PGK) are encoded in an operon that is transcribed from tandem promoters. The promoter-proximal gap gene is expressed at six-to ninefold higher levels than the pgk gene from chromosomal genes and from multiple copies of plasmid-borne genes. Two dominant transcripts were identified. The smaller, most abundant transcript contained primarily the gap message, whereas the larger, less abundant message contained both genes. The ratio of message levels for gap and pgk was calculated to be 5:1 and is sufficient to account for the observed differences in levels of GAP and PGK. The differences in message abundance are proposed to result from either transcriptional attenuation or preferential degradation of the 3' region encoding pgk. Increases in gene dosage were accompanied by one-third the expected increase in enzymatic activity on the basis of estimates of copy number, consistent with the presence of a limiting, positive regulatory factor. However, GAP and PGK expressions were not reduced from the chromosome in recombinants that contained multiple copies of the gap operon with inactive genes.Glyceraldehyde-3-phosphate dehydrogenase (GAP) and phosphoglycerate kinase (PGK) catalyze the synthesis of a high-energy phosphate bond and its transfer to ADP during glycolysis. This enzyme system and pyruvate kinase represent primary routes for ATP synthesis in Zymomonas mobilis (16, 24). The genes encoding GAP and PGK have been cloned and sequenced (7, 9). These two genes were found to be adjacent in several independent library clones containing Z. mobilis DNA, separated by 221 base pairs (bp) of DNA. Primer extension analysis identified tandem promoter regions for the gap gene (9). No promoter regions were found immediately upstream of the pgk gene by primer extension analysis, and the two genes were proposed to constitute a single operon (7). The pgk gene is followed by an unusual 7-bp sequence (CCTGCA) that is repeated 52 times and could serve as a transcriptional terminator.The GAP and PGK proteins are approximately equal in size and catalyze sequential reactions in glycolysis. Approximately equal activities are needed. However, the catalytic rate of PGK is four times that of GAP (18), and four times more GAP than PGK is required. The separation of gap and pgk genes in Escherichia coli (1) and Saccharomyces cerevisiae (11) provides a simple means to independently control the synthesis of these proteins that is not available in Z. mobilis. On the basis of activities reported previously (13, 18), GAP can be calculated to be four-to eightfold more abundant than PGK in disrupted-cell preparations. The two genes have similar patterns of codon usage and canonical ribosomal-binding sites for efficient translation. Such differential expression would not be expected from adjacent genes in an operon in the absence of additional regulatory features.In this study, we have begun to address possible reasons for the differenti...

Background People living with HIV in the USA smoke at a rate nearly three times that of the general population, and Black women are disproportionately affected by HIV infection. Purpose This study was conducted to test the preliminary efficacy of a digital storytelling intervention for smoking cessation in U.S. women living with HIV. Methods Participants in the treatment arm viewed a film in which women living with HIV talk about quitting smoking, and those in the control arm viewed an attention-control film in which women talk about living with HIV infection. Participants in both arms received eight weekly video-call counseling sessions focused on smoking cessation and nicotine patches or gum during the same period. Participants were followed on a monthly basis from quit day for 3 months. Results Of the 53 participants randomized, four withdrew before receiving any intervention, one dropped out during the intervention, and 48 (90.6%) completed the study. No difference was found in the baseline characteristics between the two arms with the exception that the treatment arm had higher nicotine dependence scores [t(1.51) = 2.30, p = .03] than the control arm. Seven day point-prevalence abstinence rates at 3 month follow-up were not found to differ between the two arms. However, the odds of achieving 3 month prolonged abstinence were four times greater (odds ratio = 4.23, 95% confidence interval = 1.10, 16.23) in the treatment arm than the control arm when the analysis was performed with those (n = 49, 92.5%) who received any part of the allotted intervention. Conclusions A digital storytelling intervention seems to be a valuable strategy to enhance the effect of conventional tobacco dependence treatment for women living with HIV. However, the underlying mechanism of the effect of digital storytelling necessitates further investigations in a large RCT. Clinical Trials Registration No. NCT03289676