Gene expression in Zymomonas mobilis: promoter structure and identification of membrane anchor sequences forming functional lacZ' fusion proteins

Conway, Tyrrell; Osman, Yehia A.; Ingram, L. O.

doi:10.1128/jb.169.6.2327-2335.1987

Cited by 26 publications

(20 citation statements)

References 39 publications

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“…Methods for plasmid purification, digestion with restriction enzymes, ligation, transformation of E. coli, and conjugation of plasmids into Z. mobilis CP4 have been described elsewhere (7,8 (9) was used as a probe for gap, and a 0.51-kbp fragment (PstI to EcoRI) from pLOI322 (7) was used as a probe for pgk. Northern hybridization.…”

Section: Methodsmentioning

confidence: 99%

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Differential expression of gap and pgk genes within the gap operon of Zymomonas mobilis

et al. 1989

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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...

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Section: Methodsmentioning

confidence: 99%

“…The organisms and plasmids used are shown in Table 1. Recombinants of Z. mobilis CP4 were maintained and grown as described previously (8,13) in complex medium containing 100 g of glucose and 120 mg of chloramphenicol per liter. Cultures were incubated in a water bath maintained at 30°C and agitated with a magnetic stirrer (60 rpm).…”

Section: Methodsmentioning

confidence: 99%

Differential expression of gap and pgk genes within the gap operon of Zymomonas mobilis

et al. 1989

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“…Computer searches extending from -1 to -100 bps upstream of the mRNA start site, also revealed repetitive and palindromic A + T-rich regions preceding and extending into the -35 region of Z 1 , Z4 and Z12. As proposed by Conway et al (5), such regions may possibly be involved in the regulation of gene expression. Of the 14 different DNA fragments sequenced, 12 carried two promoter consensus matches.…”

Section: Discussionmentioning

confidence: 94%

Isolation and characterization of Zymomonas mobilis DNA fragments acting as promoter transcriptional elements in Escherichia coli.

Vancov

Dunn

1994

J. Gen. Appl. Microbiol.

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Promoter libraries of Zymomonas mobilis were generated in Escherichia coli using the CAT promoterless vector, pKK232-8. Among the 15 clones retained for further study, quantitative analysis of promoter strength revealed a 140-fold difference between the weakest and strongest promoter fragment. Nucleotide sequence determination and primer extension analysis were used to locate possible promoter-like sequences. Inspection of the DNA sequence surrounding the mRNA start-site, revealed regions similar to the E. coli sigma 70 consensus sequence (TTGACA-17 bps-TATAAT), approximately 4-8 bps upstream. Analogous to E. coli upstream activators, regions high in AT content (70-80%) and rich in static DNA bands, were found preceding the -35 hexamer. Only one promoter fragment clone (Zi) was found to carry an open reading frame preceded by a sequence resembling a ribosome-binding site with a free energy of SD-anti-SD binding (dG°) of -12.4 kcal/mol.The general features of gene regulation in Zymomonas mobilis are not well established, nor are those affecting the expression of the Z. mobilis genes after transfer into Escherichia coll. According to the literature, there appears to be two types of promoter classes in Z. mobilis: those that function well in Z. mobilis but poorly in E. coli, and those that initiate transcription in both Z. mobilis and E. coil at relatively similar frequencies. The latter group of transcriptional elements are less well studied. Conway et al. (5) isolated DNA fragments from Z. mobilis which contained promoter activity and amino-terminal sequences. Although both E, coil and Z. mobilis recognized similar regions of DNA for transcriptional initiation, comparisons revealed that two of the three fragments did not contain sequences

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“…RNA was isolated from Z. mobilis as previously described (13). In RNA turnover experiments, rifampin (200 mg per liter) was added to 100-ml cultures (optical density at 550 nm, 0.5) and samples (10 ml) were removed at appropriate times for processing.…”

Section: Methodsmentioning

confidence: 99%

“…Genetic methods. Methods for plasmid isolation, digestion with restriction enzymes, and conjugation from E. coli to Z. mobilis have been described (13,18,29). DNA fragments used in subcloning, Northern hybridization, and S1 mapping were isolated after purification from agarose gels.…”

Section: Methodsmentioning

confidence: 99%

Segmental message stabilization as a mechanism for differential expression from the Zymomonas mobilis gap operon

Eddy

Keshav

et al. 1991

J Bacteriol

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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.

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Gene expression in Zymomonas mobilis: promoter structure and identification of membrane anchor sequences forming functional lacZ' fusion proteins

Cited by 26 publications

References 39 publications

Differential expression of gap and pgk genes within the gap operon of Zymomonas mobilis

Differential expression of gap and pgk genes within the gap operon of Zymomonas mobilis

Isolation and characterization of Zymomonas mobilis DNA fragments acting as promoter transcriptional elements in Escherichia coli.

Segmental message stabilization as a mechanism for differential expression from the Zymomonas mobilis gap operon

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