Mutational analysis of segmental stabilization of transcripts from the Zymomonas mobilis gap-pgk operon

Abstract

In Zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase are transcribed together from the gap-pgk operon. However, higher levels of the former enzyme are present in the cytoplasm because of increased stability of a 5' segment containing the gap coding region. This segment is bounded by an upstream untranslated region which can be folded into many stem-loop structures and a prominent intercistronic stem-loop. Mutations eliminating a proposed stem-loop in the untranslated region or the intercistronic stem-loop resulted in a decrease in the stability and pool size of the 5' gap segment. Site-specific mutations in the unpaired regions of both of these stems also altered the message pools. Elimination of the intercistronic stem appeared to reduce the endonucleolytic cleavage within the pgk coding region, increasing the stability and abundance of the full-length message. DNA encoding the prominent stem-loop at the 3' end of the message was shown to be a transcriptional terminator both in Z. mobilis and in Escherichia coli. This third stem-loop region (part of the transcriptional terminator) was required to stabilize the full-length gap-pgk message.In bacteria, many genes whose products function together in pathways and processes are organized into operons. While this provides an efficient means to coordinate transcription, alternative controls are needed to balance the levels of individual gene products. Much of this further control is provided by endoribonuclease processing and the selective stabilization of mRNA segments (hence increased rounds of translation) as reported for the puf operon in Rhodobacter capsulata (9, 24), the tra operon in plasmid R1-19 (25), the plasmid-encoded arsenical resistance operon (35), the pap operon in uropathogenic Escherichia coli (3), and the malEFG operon in E. coli (32), among others. The differential synthesis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) encoded by the gap-pgk operon in Zymomonas mobilis also appears to be controlled in this way.Half of the ATP synthesized in Z. mobilis is produced by the combination of GAPDH and PGK. In this obligately fermentative bacterium, both enzymes are essential and are needed at near-equivalent activities. However, the catalytic efficiency of PGK exceeds that of GAPDH by severalfold. In apparent compensation, threefold-greater amounts of the GAPDH protein than PGK protein are maintained in the cytoplasm (18,19). Neither GAP nor PGK is preferentially degraded, eliminating protein turnover as a means of maintaining differences in enzyme abundance (2). Although gap andpgk are transcribed as a single operon, measurements of functional message pools were in close agreement with the observed differences in protein abundance (30). Two stable messages are produced from this operon, a 2.7-kb message containing the gap and pgk coding regions and a more abundant 1.2-kb message (5' segment) containing only the gap coding region. The more-stable, shorter mes...

Engineering a Homo-Ethanol Pathway in Escherichia coli : Increased Glycolytic Flux and Levels of Expression of Glycolytic Genes during Xylose Fermentation

“…This coefficient averaged from 5 to 12% for the comparisons of ORFs measured on the duplicate arrays from a single filter and from 30 to 43% for multiple filters probed with cDNA from the same sample. During the active stages of growth and fermentation (6,12, and 24 h), 8% of the 4,080 ORFs were expressed at significantly higher levels (P Ͻ 0.05) in KO11 than in strain B and an equal number were at lower levels. Larger differences were observed for later time points.…”

“…Most of the genes involved in xylose metabolism were judged to be expressed at higher levels in KO11 than in strain B during active fermentation (6,12, and 24 h). Increases in expression levels of 14 genes were significant (P Ͻ 0.05) and are marked by open boxes in Fig.…”

“…Figure 5 summarizes the relative expression levels for 30 genes involved in the catabolism of xylose to pyruvate. Although results at each time point were normalized for total bound cDNA, higher values were observed for most of these genes during active growth and high glycolytic flux (6,12, and 24 h) than during the latter stages of fermentation (36,48, and 72 h). For many genes, expression levels declined to 20% of initial values during the later stages of fermentation.…”

“…It is interesting that gapA and pgk (both monocistronic in E. coli) also exhibited similar trends. In some bacteria, gap and pgk are part of a single operon (6). Cluster analysis was used to provide a more quantitative evaluation of similarities in patterns of gene expression.…”

“…In Z. mobilis, mRNA abundance and mRNA stability were directly correlated with protein levels for many glycolytic enzymes (6,21). Assuming that E. coli contains 2,350,000 protein molecules per cell (22), the lower threshold for measurements of mRNA as bound cDNA corresponds to the detection of sufficient message to permit translation equivalent to 0.001% of total protein (mass), or 24 protein molecules of average size (M w , 40,000) per cell.…”