Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2

Abstract

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the Sail site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in X1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the Sall site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coil K-12 RR1(pPL301) were increased 6-to 7-fold and 13-to 15-fold, respectively.Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.The biosynthesis of bacterial glycogen involves three enzymes: ADPglucose pyrophosphorylase (EC 2.7.7.27), glycogen synthase (EC 2.4.1.21), and branching enzyme (EC 2.4.1.18), which are encoded by glgC, -A, and -B genes, respectively, in Escherichia coli (12,(27)(28)(29)(30). In this biosynthetic pathway, allosteric regulation has been shown to occur at the ADPglucose pyrophosphorylase level (12,(27)(28)(29)(30). ADPglucose pyrophosphorylase has been isolated and characterized from a number of bacteria, and the nature of the activator has been shown to be related to the major route of carbon metabolism of the particular organism (12,(27)(28)(29)(30).Among the enteric bacteria, ADPglucose pyrophosphorylase is activated by fructose 1,6-bisphosphate (27), whereas ADP, AMP, and Pi are allosteric inhibitors (29, 30).Okita et al. (25) have cloned the gigA, -B, and -C genes from E. coli K-12 into the PstI site of pBR322. The nucleotide sequence of the structural genes glgA, -B, and -C have been reported (2, 3, 13). The allosteric properties of ADPglucose pyrophosphorylase from E. coli K-12 were also studied in great detail with respect to its activator binding site, inhibitor binding site, and substrate binding site (14, 16) by means of a photoaffinity labeling technique with azido adenine nucleotides. The combination of nucleotide sequencing and active site labeling has enabled us to determine the location of the activator, inhibitor, and substrate binding sites.As might be expected, the ADPglucose pyrophosphorylase of Salmonella typhimurium shows the same spectrum of allosteric activators and inhibitors as the E. coli ADPglucose pyrophosphorylase (31). Therefore, it was of interest to clone the glycogen b...

Glycogen production by different Salmonella enterica serotypes: contribution of functional glgC to virulence, intestinal colonization and environmental survival

“…It was initially tempting to attribute the absence of ADPglucose pyrophosphorylase activity to the major sequence change that was found in the glgC gene of the three strains of S. Gallinarum, which had been isolated from different parts of the world, particularly since the product of the glgC gene is responsible for considerable regulation of glycogen synthesis through allosteric competition by fructose 1,6-bisphosphate (Leung & Preiss, 1987a). However, the absence of this change in S. Pullorum suggested that this was not the sole reason.…”

Toll-Like Receptor 2 Activation by Chlamydia trachomatis Is Plasmid Dependent, and Plasmid-Responsive Chromosomal Loci Are Coordinately Regulated in Response to Glucose Limitation by C. trachomatis but Not by C. muridarum

“…4C) and CM3.1 (data not shown), although the strains are unable to accumulate glycogen within intracytoplasmic inclusions. Glycogen synthase, the product of the glgA gene, is required for the formation of the ␣-1-4-glucosidic linkage characteristic of the main polyglucose chains of glycogen, and bacteria with glgA deleted or mutated are unable to synthesize glycogen (23). Transcription of this gene was ϳ6-fold reduced…”

Biosynthesis of bacterial glycogen: primary structure of Salmonella typhimurium ADPglucose synthetase as deduced from the nucleotide sequence of the glgC gene

“…The bacteria, phage strains, and plasmids used in this study are as follows: E. coli K-12 JM101 [supE thi A(lac-proAB) F' traD36 proAB lacIq AM1S], E. coli K-12 JM103 [supE thi A(lac-proAB) strA endA sbcA hsdR F' traD36 proAB lacIq AM15]; bacteriophages M13 mp8, 9, 10, and 11 (17); and plasmid pPL301, which contains the S. typhimurium glgC and glgA genes on a 5.8-kilobase-pair insert on the SalII site of pBR322 (14).…”

“…The ADPglucose synthetases of E. coli and S. typhimurium are similar in that (i) they have similar subunit and native molecular weights; (ii) they have the same spectrum of activators and inhibitors; (iii) they have immunological cross-reactivity; (iv) of the first 27 amino acids in the N terminus 25 are identical; (e) genetically, the glg genes of both are clustered around 75 units on their genetic maps and are cotransducible with asd and glpD genes (28). Recently, we have cloned the glgC and glgA genes from S. typhimurium (14). This paper is a report of the nucleotide sequence, the deduced amino acid sequence, and codon usage pattern of ADPglucose synthetase from S. typhimurium.…”