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

Abstract

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...

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

“…Two messages, estimated to be 2.6 and 1.2 kb in length, were observed from this operon (18). The longer transcript contained both genes, and the shorter transcript contained gap alone.…”

“…Expression from these genes must be coordinated in some way to provide the nearly stoichiometric activities required for efficient glycolysis. Previous studies have hypothesized the presence of transactive factors that coordinate the expression of adhB with other glycolytic genes (18,29,34). Global glycolytic controls have been reported to coordinate glycolytic enzyme expression in fermenting Saccharomyces cerevisiae (22), and analogous controls may be needed by Z. mobilis.…”

“…Both coding regions are preceded by canonical ribosomal binding sites (38). The presence of gap and pgk within the same operon presents a special regulational problem for Z. mobilis (18). The catalytic efficiency of GAP (20) is approximately one-fourth that of PGK (37), although nearly equal activities are required for glycolysis.…”

“…The longer transcript contained both genes, and the shorter transcript contained gap alone. The ratio between transcripts encoding gap and pgk was estimated to be 5:1 based upon Northern RNA hybridization (18), sufficient to account for the observed differences in enzyme biosynthesis although the possibility of the involvement of additional controls during translation has not been eliminated.…”

“…The catalytic efficiency of GAP (20) is approximately one-fourth that of PGK (37), although nearly equal activities are required for glycolysis. Previous estimates of these enzymes based upon in vitro measurements of activity indicated that the GAP protein was approximately four times as abundant as the PGK protein (18,29).…”