The complete nucleotide sequence of the ilvGMEDA cluster of Escherichia coli K-12

Cox, James L.; Cox, Betty Jo; Fidanza, Vincenzo; Calhoun, David H.

doi:10.1016/0378-1119(87)90136-3

Cited by 36 publications

(23 citation statements)

References 25 publications

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“…4). Like EDD from E. coli and Z. mobilis, P. aeruginosa EDD exhibited close resemblance (51% similarity and 30% identity) to the product of E. coli ilvD, dihydroxy-acid dehydratase (13). The most striking conservation among all four peptide sequences consisted of the two segments ALVTDGRMSG (residues 507 to 516) and HVSPEA (residues 526 to 531).…”

Section: Resultsmentioning

confidence: 95%

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1

et al. 1994

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The hexC locus ofPseudomonas aeruginosa PAO1 was localized to a 247-bp segment of chromosomal DNA on the multicopy broad-host-range vector pRO1614. The presence of this plasmid (pPZ196) in strain PAO1 produced the so-called "hexC effect," a two-to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. The extent of the hexC effect was restricted, since three independently regulated metabolic enzymes were not affected by the presence of the hexC plasmid. Furthermore, the hexC-containing plasmid did not suppress catabolite repression control. Nucleotide sequence analysis of the segment of DNA encompassing hexC revealed a 128-bp region rich in adenosine-plus-thymine (AT) content separating two divergent open reading frames (ORFs). Transcriptional start sites for these two genes were mapped to the intergenic region, demonstrating that this sequence contained overlapping divergent promoters. The intergenic region contained potential regulatory sequences such as dyad symmetry motifs, polydeoxyadenosine tracts, and a sequence matching the integration host factor recognition site in Escherichia coli. One of the ORFs encoded a 610-amino-acid protein with 55 to 60%Yo identity to 6-phosphogluconate dehydratase from E. coli and Zymomonas mobiis. The second ORF coded for a protein of 335 amino acids that displayed 45 to 60% identity to the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) family of enzymes. The NAD-dependent GAP gene on the P. aeruginosa chromosome was previously unmapped. GAP was found to exhibit the hexC-dependent increase in its basal activity, establishing it as a fifth catabolic enzyme in the multioperonic hex regulon.Pseudomonads utilize a number of alternative pathways for the catabolism of carbohydrates to carbon and energy (reviewed in reference 37). In Pseudomonas aeruginosa, the coordinately regulated enzymes glucokinase (GLK), glucose-6-phosphate dehydrogenase (ZWF), and the Entner-Doudoroff pathway enzymes 6-phosphogluconate dehydratase (EDD) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) play a central role in the dissimilation of glucose to pyruvate and glyceraldehyde-3-phosphate (Fig. 1). These enzymes are coinduced by growth of P. aeruginosa on such diverse carbohydrates as glucose, gluconate, mannitol, and glycerol (33, 37).Also, their expression is subject to catabolite repression control (CRC) by succinate or other intermediates of the tricarboxylic acid (TCA) cycle (40,58).The genes encoding these four enzymes (glk, zwf, edd, and eda) were mapped by transductional analysis to 39 min on the PAO1 chromosome (14,30,47). Cloning and complementation analyses revealed, however, that the four genes were expressed from separate promoters (15,57), indicating that the genes were not contained in a single operon. Two regulatory effects further defined this group of genes. The first was the hexR mutation, thus far inseparable from edd,...

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Section: Resultsmentioning

confidence: 95%

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1

et al. 1994

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“…The two dehydratase enzymes are quite similar, exhibiting 57.7% identity, and 75.3% of the residues are chemically conserved. These enzymes are both related by primary sequence to the E. coli dihydroxy-acid dehydratase that is encoded by the ilvD gene (8). An alignment of the two E. coli enzymes shows them to be 30.3% identical and 54.2% conserved (Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of promoters for the edd-eda operon

et al. 1992

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The nucleotide sequence of the entire Escherichia coli edd-eda region that encodes the enzymes of the Entner-Doudoroff pathway was determined. The edd structural gene begins 236 bases downstream ofzwf. The eda structural gene begins 34 bases downstream of edd. The edd reading frame is 1,809 bases long and encodes the 602-amino-acid, 64,446-Da protein 6-phosphogluconate dehydratase. The deduced primary amino acid sequences of the E. coli and Zymomonas mobilis dehydratase enzymes are highly conserved. The eda reading frame is 642 bases long and encodes the 213-amino-acid, 22,283-Da protein 2-keto-3-deoxy-6-phosphogluconate aldolase. This enzyme had been previously purified and sequenced by others on the basis of its related enzyme activity, 2-keto-4-hydroxyglutarate aldolase. The data presented here provide proof that the two enzymes are identical. The primary amino acid sequences of the E. coli, Z. mobiis, and Pseudomonas putida aldolase enzymes are highly conserved. When E. coli is grown on gluconate, the edd and eda genes are cotranscribed.Four putative promoters within the edd-eda region were identified by transcript mapping and computer analysis. P1, located upstream of edd, appears to be the primary gluconate-responsive promoter of the edd-eda operon, responsible for induction of the Entner-Doudoroff pathway, as mediated by the gntR product. High basal expression of eda is explained by constitutive transcription from P2, P3, and/or P4 but not P1.

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“…5). A search of the GenBank database using the deduced amino acid sequence for edd as the query sequence revealed a match to the E. coli dihydroxyacid dehydratase that is encoded by the ilvD gene (17). A comparison of the putative Z. mobilis 6-phosphogluconate dehydratase and E. coli dihydroxyacid dehydratase as aligned by the UWGCG Gap program is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism

1990

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The Zymomonas mobilis genes that encode glucose-6-phosphate dehydrogenase (zwj), 6-phosphogluconate dehydratase (edd), and glucokinase (gik) were cloned independently by genetic complementation of specific defects in Escherichia coli metabolism. The identity of these cloned genes was confirmed by various biochemical means. Nucleotide sequence analysis established that these three genes are clustered on the genome and revealed an additional open reading frame in this region that has significant amino acid identity to the E. coli xylose-proton symporter and the human glucose transporter. On the basis of this evidence and structural analysis of the deduced primary amino acid sequence, this gene is believed to encode the Z. mobilis glucose-facilitated diffusion protein, gif. The four genes in the 6-kb cluster are organized in the order gif, zwf, edd, gik. The gif and zwf genes are separated by 146 bp. The zwf and edd genes overlap by 8 bp, and their expression may be translationally coupled. The edd and gik genes are separated by 203 bp. The gik gene is followed by tandem transcriptional terminators. The four genes appear to be organized in an operon. Such an arrangement of the genes that govern glucose uptake and the first three steps of the Entner-Doudoroff glycolytic pathway provides the organism with a mechanism for carefully regulating the levels of the enzymes that control carbon flux into the pathway.Zymomonas mobilis is a gram-negative bacterium which, during evolution, has become highly specialized for growth in plant saps with a high sugar content (37,51,53). This obligately fermentative organism possesses remarkably simple carbon and energy metabolism. Z. mobilis is only able to utilize glucose, fructose, and sucrose, which are converted to the sole fermentation products ethanol and carbon dioxide. Yet, in terms of biosynthetic capabilities, this organism has only two growth factor requirements. Z. mobilis uses the Entner-Doudoroff pathway exclusively for conversion of carbohydrates to pyruvate and the decarboxyclastic mechanism for ethanol production, with the key enzyme pyruvate decarboxylase. The enzymes responsible for this fermentation compose as much as 50% of the total soluble protein. Z. mobilis is totally dependent on substrate-level phosphorylation for energy production and, due to its use of the Entner-Doudoroff pathway, obtains only a single mole of ATP per mole of glucose fermented. For this organism, rapid carbon flux is necessitated by inefficient energy production and is facilitated by high levels of the pathway enzymes.Despite enormous carbon flux, Z. mobilis must keep the levels of toxic metabolic intermediates low while providing sufficient pools of precursor metabolites for biosynthetic pathways. The physiology and biochemistry of this organism are dictated by these constraints on metabolism. The glycolytic enzymes appear to operate near their maximal capacity, and there is no substantial allosteric control of physiologically irreversible enzymes (53). It can be concluded that carbo...

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The complete nucleotide sequence of the ilvGMEDA cluster of Escherichia coli K-12

Cited by 36 publications

References 25 publications

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1

Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of promoters for the edd-eda operon

Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism

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