Characterization of the molecular mechanisms involved in the differential production of erythrose‐4‐phosphate dehydrogenase, 3‐phosphoglycerate kinase and class II fructose‐1,6‐bisphosphate aldolase in Escherichia coli

Abstract

SummaryA gapA-pgk gene tandem coding the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase, is most frequently found in bacteria. However, in Enterobacteriaceae , gapA is replaced by an epd open reading frame (ORF) coding an erythrose-4-phosphate dehydrogenase and an fbaA ORF coding the class II fructose-1,6-bisphosphate aldolase follows pgk . Although epd expression is very low in Escherichia coli , we show that, in the presence of glucose, the 3 epd , pgk and fbaA ORFs are efficiently cotranscribed from promoter epd P0. Conservation of promoter epd P0 is likely due to its important role in modulation of the metabolic flux during glycolysis and gluconeogenesis. As a consequence, we found that the epd translation initiation region and ORF have been adapted in order to limit epd translation and to create an efficient RNase E entry site. We also show that fbaA is cotranscribed with pgk , from promoter epd P0 or an internal pgk P1 promoter of the extended -10 class. The differential expression of pgk and fbaA also depends upon an RNase E segmentation process, leading to individual mRNAs with different stabilities. The secondary structures of the RNA regions containing the RNase E sites were experimentally determined which brings important information on the structural features of RNase E ectopic sites.

Transcription Analysis of Central Metabolism Genes in Escherichia coli. Possible Roles of σ38 in Their Expression, as a Response to Carbon Limitation

“…Two promoters have been reported for this operon; one that transcribes the three genes, located upstream of epd , which initiates transcription 132 nucleotides from the ATG initiation codon and a second one, located at the 3′ end of the epd structural gene that transcribes only pgk and fbaA . This latter promoter initiates transcription 235 nucleotides from the ATG initiation codon of the pgk gene [45]. As can be seen in figures 2f-1 and S1f-1 transcription from the epd gene initiated in all strains from the same reported promoter whose expression is controlled by Crp and Cra [45].…”

“…The consensus nucleotide sequence of the −10 region for the σ 38 sigma subunit (8 nucleotides, framed in the figures) described by Weber et al [5], was utilized for detecting possible σ 38 recognition sites and a 10 nucleotides −10 σ 70 consensus sequence was utilized for detecting possible σ 70 binding sites. Numbers in parenthesis indicate references in which previously reported TSSs have been described: Meyer et al [36], Froman et al [37], Helling and Evans [39], Charpentier et al [41], Thouvenot et al [43], Bardey et al [45].…”

Complex I Dysfunction Redirects Cellular and Mitochondrial Metabolism in Arabidopsis

“…Fru-1,6-bisP aldolase (EC 4.1.2.13) and GAPDH (EC 1.2.1.12) activities were measured on isolated mitochondria and whole cell extracts as described by Bardey et al (2005). Triton X-100 (0.05%, v/v) was added to reaction media in order to solubilize membranes.…”

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

“…During incubation (0 to 100 s), aliquots were taken and the reaction was stopped by the addition of 80 mM EDTA. The amount of GAP formed was determined by coupling the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent conversion of GAP to 1-arseno-3-phosphoglycerate to the reduction of NAD ϩ as described previously (10). The assay contained 100 mM TrisHCl, pH 7.5, 3 mM NAD ϩ , 20 mM potassium arsenate, 14 U TIM, and 8 U GAPDH.…”