Molecular cloning and sequencing of the glycogen phosphorylase gene from <i>Escherichia coli</i>

Choi, Yong-Lark; Kawamukai, Makoto; Utsumi, Ryutaro; Sakai, Hiroshi; Komano, Tohru

doi:10.1016/0014-5793(89)80128-0

Cited by 32 publications

(30 citation statements)

References 25 publications

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“…A potential transcription terminator for glpD is indicated by the short dashed arrows (nucleotides 1713 to 1728). The reported rho-independent transcription terminator (dashed arrows) and translation termination codon (underlined) for the convergent glgP gene (4,39) are centered at bases 2136 and 2164, respectively. (5) reports an open reading frame of 504 codons encoding a protein of molecular weight 56,131.…”

Section: Resultsmentioning

confidence: 99%

“…(5) reports an open reading frame of 504 codons encoding a protein of molecular weight 56,131. The published sequences of the glpD (5) and glgP (4,39) genes were compared with the nucleotide sequence presented in this report. The glgP gene, encoding a-glucan phosphorylase, is located downstream of glpD and is transcribed convergently toward glpD.…”

Section: Resultsmentioning

confidence: 99%

“…In many cases, REP sequences are located between genes within polycistronic operons and mediate differential expression of the genes by stabilizing upstream mRNA, possibly by protecting against 3'-5' exonuclease activity (12 (4,39). Furthermore, the 3' end of glpD was localized to within 100 bp of the end of the REP sequences, as discussed below.…”

Section: Resultsmentioning

confidence: 99%

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Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12

Austin

Larson

1991

J Bacteriol

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Aerobic sn-glycerol 3-phosphate dehydrogenase, encoded by the glpD gene of Escherichia coli, is a cytoplasmic membrane-associated respiratory enzyme. The nucleotide sequence of glpD was determined. An open reading frame of 501 codons was preceded by a consensus Shine-Dalgarno sequence. The proposed translational start and reading frame of glpD were confirmed by determining the nucleotide sequence across the fusion joint of a glpD-lacZ translational fusion. The predicted molecular weight, 56,750, corresponds well with the reported value of 58,000 for purified sn-glycerol 3-phosphate dehydrogenase. The flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins from E. coli. Repetitive extragenic palindromic sequences were identified downstream of the glpD coding region. The site for transcription termination was located between 87 and 216 bp downstream of the translation stop codon.Escherichia coli is capable of utilizing glycerol, sn-glycerol 3-phosphate (glycerol-P), and glycerophosphodiesters as carbon sources and as precursors for phospholipid biosynthesis. The dissimilation of glycerol-P and its precursors is catalyzed by proteins encoded by the genes of the glp regulon (17,18). This metabolic system acts essentially as a salvage pathway for utilization of glycerol and glycerol-P derived from the breakdown of phospholipids and triacylglycerol. The regulon is induced by glycerol-P. The glp genes, under negative genetic regulation by the glpR-encoded repressor protein (16,17), have been mapped to three separate regions on the E. coli chromosome and are arranged in at least five different operons (17).The glpQ gene encodes a periplasmic glycerophosphodiesterase that catalyzes hydrolysis of glycerophosphodiesters to glycerol-P and an alcohol (17). Glycerol-P is actively transported into the cytoplasm by the glpT-encoded permease (17). The glpTQ operon is located at min 49 on the linkage map of E. coli. It is adjacent to, and is transcribed divergently from, the glpACB (also called glpABC [7]) operon encoding the three subunits of anaerobic glycerol-P dehydrogenase (8,17,18). The glpFK operon (19, 35) is located near min 88. The glpF gene encodes a cytoplasmic membrane protein responsible for facilitating the diffusion of glycerol across the membrane (17). The glpK gene codes for glycerol kinase (21). Phosphorylation of glycerol traps glycerol-P in the cytoplasm.The glpD gene encodes aerobic glycerol-P dehydrogenase and maps near min 75 (17). The glpD gene is transcribed divergently from the adjacent gipE, glpG, and glpR genes (6,(31)(32)(33). Aerobic glycerol-P dehydrogenase is expressed maximally under aerobic growth conditions (17). Anaerobic repression of glpD expression is mediated by the twocomponent regulatory system encoded by arcA and arcB (13,14). Glycerol-P dehydrogenase is associated with the * Corresponding author. cytoplasmic membrane and is a primary dehydrogenase in the respiratory chain. It catalyzes the oxidation of glyc...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12

Austin

Larson

1991

J Bacteriol

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“…An E. coli protein with a-glucan phosphorylase activity was reported by Chen and Segel (5). Subsequently, two groups reported a gene, glgP (mapping adjacent to glgA in the glg cluster), that encodes an a-glucan phosphorylase (6,39); its activity at high glucan concentrations was comparable to those of other wellstudied phosphorylases. A debranching enzyme that was believed to be cytoplasmic was reported for E. coli (15), but its activity on glycogen as a substrate was low, and the gene encoding the enzyme was not identified.…”

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confidence: 98%

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA

et al. 1992

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In the gap between two closely linked flagellar gene clusters on the Escherichia coi and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coi map), we found an open reading frame whose sequence suggested that it encoded an a-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other ct-amylases were to the excreted liquefying a-amylases of bacilli, with >40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coil gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the ac-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an a-amylase rather than a 1-amylase. The newly discovered gene has been named amyA.The natural physiological role of the AmyA protein is not yet evident.During an investigation of the flagellar genes ofEscherichia coli and Salmonella typhimuium, we encountered a nearby open reading frame whose deduced product sequence suggested that it was that of a cytoplasmic a-amylase.The only major cytoplasmic polysaccharide in these enteric bacteria is glycogen, which is laid down as an energy and carbon reserve, especially under conditions in which carbon is abundant but another major essential element, such as nitrogen, is limiting (27). Under normal growth conditions, glycogen represents only about 2.5% of the dry weight of the cell (26), but it can reach as high as 30% under conditions of carbon abundance and deprivation of another essential element, such as nitrogen (33). A possible role for a cytoplasmic a-amylase might therefore be in glycogen metabolism.The enzymes responsible for glycogen synthesis in E. coli have been studied extensively; they are glucose-i-phosphate adenyltransferase, glycogen synthase, and 1,4-a-glucan branching enzyme. The corresponding structural genes (glgC, glgA, and glgB) are clustered at 76 min on the map (1,2,18).Less is known about the genetics and enzymology of glycogen breakdown in E. coli. By analogy with mammalian systems, one might expect at least an a-glucan phosphorylase and a debranching enzyme. An E. coli protein with a-glucan phosphorylase activity was reported by Chen a...

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“…Glycogen degradation requires the activity of glycogen phosphorylase [EC 2.4.1.1], which is encoded by the glgP gene (Choi et al, 1989;Romeo et al, 1988;Yu et al, 1988). The glgC and glgA genes, along with the glycogen degradation enzyme glgP, are apparently cotranscribed in an operon glgCAP.…”

Section: Introductionmentioning

confidence: 97%

Design of expression systems for metabolic engineering: Coordinated synthesis and degradation of glycogen

Dedhia

Chen

Bailey

1997

Biotechnol. Bioeng.

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In metabolic engineering, systems which allow coordinated control of two metabolic pathways can be useful. We designed two expression systems and demonstrated their application by coordinating glycogen synthesis and degradation. The first expression vector pMSW2 expressed the glycogen synthesis genes in one operon and the glycogen degradation gene in a separate, coordinately regulated operon. The plasmid was designed to switch off expression of the first operon and activate expression of the second operon on addition of IPTG. As an alternative means to control glycogen synthesis and degradation pathways, we constructed expression vector pGTSD100, which contains the native Escherichia coli glycogen synthesis and degradation operon under control of the tac promoter. Both expression vectors work successfully to control the net synthesis and degradation of glycogen. In cultures of the E. coli strain TA3476 carrying the plasmid pMSW2, before the addition of IPTG, glycogen continued to accumulate in the culture. About three hours after IPTG was added, glycogen levels began to decrease. When no IPTG was added to cultures of TA3476:pMSW2, glycogen accumulated in the cells as before but the rate of degradation of glycogen was much lower. When IPTG was added to TA3476:pMSW2, the total cell protein at the end of batch cultivation was approximately 15% higher compared to cultures without IPTG addition. The extra biomass was formed during the glycogen degradation phase.

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Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli

Cited by 32 publications

References 25 publications

Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12

Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA

Design of expression systems for metabolic engineering: Coordinated synthesis and degradation of glycogen

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