Chapter 67

Regulation of Glycogen Synthesis

JACK PREISS

INTRODUCTION

Glycogen is a glucose-containing polysaccharide consisting of about 95% α-1,4 linkages and about 5% α-1,6-branch glucosyl linkages. The average chain length is about 12 to 14 glucose units. Accumulation of glycogen occurs in many bacteria, including Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium), under conditions of limiting growth when an excess of a carbon source, particularly glucose, is available (reviewed in references 53 and 58). During stationary phase, the glucose will be utilized as a carbon source. The precise function of glycogen in bacteria, however, is not known. Published results suggest that it may play a role in prolonging viability by providing a source of energy (e.g., for sporogenesis in bacilli [76]).

ENZYMES INVOLVED IN SYNTHESIS OF GLYCOGEN

The reactions leading to glycogen synthesis in bacteria were first reported in 1964 (51, 71). ADP-glucose (ADPGlc), rather than UDP-glucose as demonstrated for mammalian glycogen synthesis, was shown to be the glucosyl donor for bacterial glycogen synthesis (16) and is synthesized in a reaction catalyzed by ADPGlc pyrophosphorylase (ADPGlc PPase; EC 2.7.7.27):

ATP + α-glucose 1-phosphate ↔ ADPGlc + PP_i  (1)

The glucosyl unit of the ADPGlc is then transferred, in a reaction catalyzed by an ADPGlc-specific glycogen synthase (EC 2.4.1.21),

ADPGlc + α-glucan → α-1,4-glucosyl-glucan + ADP  (2)

to either a maltodextrin or glycogen primer to form a new α-1,4-glucosidic linkage. After chain elongation by glycogen synthase catalysis, branching enzyme (BE; EC 2.4.1.18) will catalyze formation of the branched α-1,6-glucosidic linkages in glycogen from the growing polyglucose chain:

Linear α-1,4-polyglucosyl chain → branched α-1,4-α-1,6-glucan polysaccharide (i.e., glycogen)  (3)

These enzyme-catalyzed reactions have been observed in extracts from at least 46 bacterial species (55, 58).

Genetic evidence that bacterial glycogen synthesis occurs solely by the ADPGlc pathway has been obtained with mutants of E. coli or S. typhimurium devoid of or deficient in glycogen (reviewed in references 27, 51, 53, 55, 58, 59, 61, and 62). Compared with the wild-type strain, they are defective in ADPGlc PPase or glycogen synthase activity or both. Moreover, mutants containing glycogen in amounts in excess of that observed in wild-type strains have also been isolated. These mutants overexpress ADPGlc PPase, glycogen synthase, and/or BE activities. Thus, at least for E. coli and S. typhimurium, the data strongly indicate the importance of the ADPGlc pathway for synthesis of bacterial glycogen invivo.

Much information has recently been obtained with respect to genetic regulation of the expression of the biosynthetic enzymes. The structural genes for the glycogen biosynthetic enzymes of E. coli and S. typhimurium (42, 48) have been cloned, which has provided insights in the genetic regulation of glycogen synthesis and has permitted oligonucleotide-directed mutagenesis of the E. coli glgC and glgA genes, the structural genes encoding ADPGlc PPase and glycogen synthase, respectively; the analyses have helped to elucidate structure-function relationships with respect to the ADPGlc PPase and glycogen synthase catalytic activities as well as the allosteric properties of ADPGlc PPase. As will be discussed, an important aspect of regulation of glycogen synthesis is the allosteric regulation of ADPGlc PPase. The current information, views, and concepts of regulation of enzyme activity and expression of the glycogen biosynthetic enzymes are presented.

ALLOSTERIC REGULATION OF ADPGlc PPase

An important regulatory effect of bacterial glycogen synthesis is exerted through the allosteric regulation of ADPGlc PPase activity (27, 51, 53, 55, 58, 59, 61, 62).

The regulatory properties of more than 50 ADPGlc PPases (mainly bacterial [53, 58, 59] but also plant [52, 54, 56, 75]) have been studied. In almost all cases, glycolytic intermediates activate ADPGlc synthesis whereas AMP, ADP, and/or P_i are inhibitors. Glycolytic intermediates in the cell can be considered as indicators of carbon excess: therefore, under conditions of limited growth with excess carbon in the media, the accumulation of glycolytic intermediates that occurs acts as a signal for the activation of ADPGlc synthesis. For most of the ADPGlc PPases studied, the activator glycolytic intermediate increases the enzyme’s apparent affinity for the substrates, ATP and glucose 1-phosphate, and increasing concentrations of activator reverse the inhibition caused by the inhibitor, AMP, ADP, or P_i.

The activator specificity of the bacterial and plant ADPGlc PPases can be classified into seven groups on the basis of their specificity of activation by the various glycolytic intermediates (23, 27, 53, 55, 58, 59, 61, 62). The variation of activator specificity observed has been postulated to correlate with the nature of carbon assimilation dominant in the bacterium or plant tissue. This has been discussed in detail in a number of reviews (23, 27, 53, 55, 58, 59, 61, 62). E. coli and S. typhimurium obtain their energy mainly through glycolysis. The primary activator for their ADPGlc PPases is fructose 1,6-bisphosphate, and 5'-adenylate is the major inhibitor; their ADPGlc synthetic activity is regulated by the fructose 1,6-bisphosphate/AMP ratio.

There is now evidence suggesting strongly that the kinetic allosteric activation and inhibitor effects observed in vitro also occur in vivo in bacterial cells. There is a class of mutants of E. coli and of S. typhimurium LT2 affected in the ability to accumulate glycogen. This mutant class has ADPGlc PPases with altered regulatory properties. Generally, those mutants with ADPGlc PPases with higher affinity for the activator, fructose 1,6-bisphosphate, and/or a lower affinity for the allosteric inhibitor, AMP, accumulate glycogen at a faster rate than the parent wild-type strain. Mutants with enzymes with a lower affinity for the activator accumulate glycogen at a slower rate than the parent strain.

Table 1 summarizes the allosteric properties of the mutant ADPGlc PPases that have been studied and their ability to accumulate glycogen in the stationary phase. With respect to E. coli, there is a direct relationship between the affinity of the enzyme for the activator and the ability of the mutant to accumulate glycogen. If the apparent affinity for the activator, fructose 1,6-bisphosphate, is higher, glycogen accumulation is higher in the mutant than in the parent strain. If the apparent affinity for the activator is lower, as seen for mutant SG14 enzyme (60), glycogen accumulation is lower in the mutant than in the parent strain. The two S. typhimurium mutant strains have ADPGlc PPases that are more affected in the affinity of the inhibitor (77). Both JP23 and JP51 enzymes have less affinity for the inhibitor, and these mutants accumulate higher amounts of glycogen than does the parent strain (77).

Table 1Comparison of allosteric kinetic constants and glycogen accumulation rates of E. coli and S. typhimurium LT2 allosteric mutant ADPGlc PPases and the ADPGlc PPases obtained from wild-type bacteria

The studies discussed above plus one showing a direct relationship between the fructose 1,6-bisphosphate concentration in E. coli cells and the rate of glycogen accumulation (8) clearly demonstrate that fructose 1,6-bisphosphate is an allosteric activator of ADPGlc PPase and a physiological activator of glycogen synthesis in E. coli and in S. typhimurium.

Chemical modification and site-directed mutagenesis studies of the E. coli ADPGlc PPase have provided evidence for the location of the activator binding site (49, 50), the inhibitor binding site (31, 32), and the substrate binding sites (21, 50). These experiments have used pyridoxal phosphate as an analog either for the activator, fructose 1,6-bisphosphate (49, 50) or, as subsequently shown, for the substrate, glucose 1-phosphate (37, 50). For an ATP analog, the photoaffinity reagent 8-azido-ATP proved to be a substrate for the E. coli enzyme (37), whereas 8-azido AMP was an effective inhibitor analog (31, 32)/. Since the E. coli ADPGlc PPase gene, glgC, had been cloned and its sequence had been determined (3, 48), the identification of the amino acid sequence around the modified residue enabled the location of the modified residue in the primary structure of the enzyme to be determined. The amino acid residue involved in binding the activator was Lys-39, and the amino acid involved in binding the adenine portion of the substrates (ADPGlc and ATP) was Tyr-114. Tyr-114 was also the major binding site for the adenine ring of the inhibitor, AMP. Lys-195 is protected from reductive phosphopyridoxylation by the substrate, ADPGlc; thus, it was proposed that it is also a part of the substrate binding site.

Figure 1 shows the amino acid sequence of the E. coli ADPGlc PPase, the substrate and allosteric sites identified via chemical modification, and the deduced amino acid sequence of the S. typhimurium enzyme (41). There is about 80% nucleotide sequence identity between the E. coli and S. typhimurium glgC genes and 90% identity in amino acid sequence. Most of the changes are conservative. However, the amino acids that have been shown to be involved in substrate and allosteric effector binding as well as those involved in maintaining allosteric function for the E. coli enzyme are all conserved.

Fig. 1Comparison of amino acid sequences of ADPGlc PPase of E. coli (EC; first row) and S. typhimurium (ST; second row). Amino acids are represented by the one-letter amino acid code. Only those amino acids that are different in the Salmonella enzyme are shown. The amino acids underlined in the E. coli sequence either have been shown to be involved in substrate or allosteric effector binding or are amino acids replaced by others in allosteric mutant E. coli enzymes. K-39 is involved in binding of the activator, fructose 1,6-bisphosphate; A-44 is substituted by Thr in the allosteric mutant SG14 enzyme (45); R-67 is substituted by Cys in allosteric mutant CL1136 enzyme (14); Y-114 has been shown to be involved in binding of the substrate, ATP (37), and the inhibitor, AMP (31, 32); K-195 has been shown to be involved in the binding of the substrate, glucose 1-phosphate (21); P-295 is substituted by Ser in the allosteric mutant SG5 enzyme (46); and G336 is substituted by Asp in the allosteric mutant 618 enzyme (28, 36, 40). All of these amino acids are conserved in the Salmonella ADPGlc PPase.

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CLONING OF ADPGlc PPases WITH ALTERED ALLOSTERIC PROPERTIES FROM E.COLI MUTANTS AFFECTED IN GLYCOGEN SYNTHESIS

As discussed above (Table 1), a class of mutants of E. coli and of S. typhimurium with altered rates of glycogen accumulation was found to have ADPGlc PPases that were affected in their allosteric properties. To gain insight with respect to amino acid residues or domains involved in maintaining allosteric function, the allosteric mutant ADPGlc PPases were cloned (14, 40, 45, 46).

Table 2 shows the various amino acid substitutions in the allosteric mutants that have been cloned and analyzed. Of interest is that the mutations causing large changes in the allosteric properties of the enzyme occur throughout the ADPGlc PPase sequence. These changes affect not only the affinities of the allosteric effectors (Table 1) but also the apparent affinities for the substrates ATP and Mg²⁺ (15, 53, 58, 60). Therefore, many domains are affected by the mutations.

Table 2Amino acid substitutions in the E. coli ADPGlc PPase allosteric mutants

Thus, site-directed mutagenesis of the ADPGlc PPase gene and analysis of various allosteric mutant genes have provided much information on the structure-function relationships of the substrate and catalytic sites. What is needed for greater clarification is knowledge of the three-dimensional structure of the enzyme. Unfortunately, no crystals of ADPGlc PPase are yet available for this type of analysis. The E. coli enzyme has been crystallized (47), but the crystals are of poor diffraction quality and are sensitive to X-ray exposure damage.

GLYCOGEN SYNTHASE

The E. coli and S. typhimurium glycogen synthases are specific for the sugar nucleotide ADPGlc. Some chemical modification studies (22) have been done to show that two distinct sulfydryl groups are important for enzyme activity and are protected by the primer, glycogen, and the substrate, ADPGlc, respectively. The reactive sulfhydryl residues are probably located at or near the binding sites for the substrates, glycogen and ADPGlc.

An affinity analog of ADPGlc, adenosine diphosphopyridoxal (ADP-pyridoxal), was used to identify the ADPGlc binding site (12). Incubation of the enzyme with the analog plus sodium borohydride led to an inactivated enzyme. The degree of inactivation correlated with the incorporation of about 1 mol of analog per mol of enzyme subunit for 100% inactivation. After tryptic hydrolysis, one labeled peptide was isolated, and the modified Lys residue was identified as Lys-15 (12). The sequence Lys-X-Gly-Gly, where lysine is the amino acid modified by ADP-pyridoxal, has been found to be conserved in the mammalian glycogen synthase (43, 79) and the plant starch synthases (75).

The structural gene for glycogen synthase, glgA, has been cloned from both E. coli and S. typhimurium (42, 48), and the nucleotide sequence of the E. coli glgA gene has been determined (29). It consists of 1,431 bp specifying a protein of 477 amino acids with a molecular weight of 52,412. Its availability has enabled Furakawa et al. (12, 13) to perform site-directed mutagenesis experiments to determine structure-function relationships for a number of amino acids in the E. coli glycogen synthase. Substitution of other amino acids for Lys at residue 15 suggested that the Lys residue is involved mainly in binding the phosphate residue adjacent to the glycosidic linkage of the ADPGlc and not in catalysis. The major effect on the kinetics of the mutations at residue 15 was an elevation of the K_m of ADPGlc by about 30- to 50-fold when either Gln or Glu was the substituted amino acid. Substitution of Ala for Gly at residue 17 decreased the catalytic rate constant, k _cat, about 3 orders of magnitude compared with the wild-type enzyme. Substitution of Ala for Gly-18 decreased the rate constant only 3.2-fold. The K_m effects on the substrates, glycogen and ADPGlc, were minimal. The researchers postulated (12) that the two glycyl residues in the conserved Lys-X-Gly-Gly sequence participated in the catalysis by assisting in maintaining the correct conformational change of the active site or by stabilizing the transition state.

Since there is still binding of the ADPGlc and appreciable catalytic activity of the Lys-15 → Gln mutant, the ADP-pyridoxal modification was repeated; in this instance, a ca. 30-times-higher concentration was needed for inactivation of the enzyme (13). The enzyme was maximally inhibited about 80%, and tryptic analysis of the modified enzyme yielded one peptide containing the affinity analog and with the sequence Ala-Glu-Asn-modified Lys-Arg. The modified Lys was identified as Lys-277. Site-directed mutagenesis of Lys-277 to form a Gln mutant was done, and the K_m for ADPGlc was essentially unchanged but the k _cat was decreased 140-fold. It was concluded that Lys-277 was involved primarily in the catalytic reaction rather than in substrate binding.

BE

Structure

The structural gene of the E. coli BE, glgB, has been cloned (4); its open reading frame (ORF) is about 200 bp downstream from the asd gene.

The complete nucleotide and deduced amino acid sequences were determined and found to be consistent with the amino acid analysis of the pure protein, with the molecular weight determined by sodium dodecyl sulfate-gel electrophoresis, and with the amino acid sequence analyses of the amino terminus and of the various peptides obtained via CNBr degradation (4). The gene consisted of 2,181 bp specifying a protein of 727 amino acids and with a molecular weight of 84,231.

Classification as a Member of the α-Amylase Family

The relationship in amino acid sequence between BE and amylolytic enzymes such as α-amylase, pullulanase, glucosyltransferase, and cyclodextrin glucanotransferase was reported by Romeo et al. (68), particularly at those sequences believed to be contacts between the substrate and the enzyme. Baba et al. (2) reported that there was a marked conservation in the amino acid sequences of the four catalytic regions of amylolytic enzymes in maize endosperm BE I. As shown in Table 3, four regions that putatively constitute the catalytic regions of the amylolytic enzymes are conserved in the starch branching isoenzymes of maize endosperm, rice seed, and potato tuber and the glycogen BE of E. coli. Analysis of this high conservation in the α-amylase family has been pointed out and greatly expanded by Svensson (78) and by Jesperson et al. (24) with respect both to sequence homology and to the prediction of (β/α)₈-barrel structural domains with a highly symmetrical fold of eight inner, parallel β strands, surrounded by eight helices, in the various groups of enzymes in the family. The (β/α)₈-barrel structural domain was determined from the crystal structure of some α-amylases and cyclodextrin glucanotransferases.

Table 3Comparison of primary structures of various BEs with the four best-conserved regions of the ?-amylase family

The conservation of the putative catalytic sites of the α-amylase family in the glycogen and starch BE would be expected, as the BE catalyzes two consecutive reactions in synthesizing α-1,6-glucosidic linkages by cleavage of an α-1,4-glucosidic linkage in an 1,4-α-d-glucan to form a nonreducing-end oligosaccharide chain that is transferred to a C-6 hydroxyl group of the same or other 1,4-α-d-glucan. It would be of interest to know whether the eight highly conserved amino acid residues of the α-amylase family are also functional in BE catalysis. Further experiments such as chemical modification and analysis of the three-dimensional structure of the BE are needed to determine the precise functions and nature of its catalytic residues and mechanism. Of interest also would be determination of the regions of the C and N termini that are dissimilar in sequence and in size in the various branching isoenzymes. It may be that these amino acid sequence regions are important with respect to BE substrate specificity, the size of the chain transferred, and the extent of branching.

GENETIC REGULATION OF THE GLYCOGEN SYNTHESIS IN E.COLI

The enzymes of the glycogen biosynthetic pathway are induced in the stationary phase. The rate of glycogen synthesis is inversely related to the growth rate when growth is limited for certain nutrients, e.g., nitrogen. Consistent with this are the findings that the levels of glycogen biosynthetic enzymes in E. coli increase as cultures enter the stationary phase (27, 51, 53, 58, 59, 61, 62).

When E. coli cells are grown in an enriched medium containing yeast extract and 1% glucose, the specific activities of ADPGlc PPase and glycogen synthase increase 11- to 12-fold, while that of glycogen BE increases 5-fold, as cultures enter stationary phase (reviewed in references 27, 59, and 61). However, when cells are grown in a defined medium, the ADPGlc PPase and glycogen synthase activities are elevated in the exponential phase. BE in defined media is fully induced in the exponential phase, with only about a twofold increase in specific activity of the ADPGlc PPase and glycogen synthase when cells grown in a defined medium reach the stationary phase. The same phenomena are also seen with the glycogen biosynthetic enzyme levels in S. typhimurium (77).

These experiments suggest that the gene encoding the BE is regulated differently from the genes for ADPGlc PPase and glycogen synthase. As would be expected for a pathway that is under transcriptional control, the addition of inhibitors of RNA or protein synthesis to pre-stationary-phase cultures prevents the enhancement of glycogen synthesis in the stationary phase (6).

LOCATIONS OF THE STRUCTURAL GENES FOR GLYCOGEN BIOSYNTHESIS

The structural genes for glycogen biosynthesis are clustered in two adjacent operons, which also contain genes for glycogen catabolism. The structural genes for glycogen synthesis were shown to be located at approximately 75 min on the E. coli K-12 chromosome, and the gene order at this location was subsequently established by transduction to be glgA-glgC-glgB-asd (33). These genes encode the enzymes glycogen synthase, ADPGlc PPase, and glycogen BE and are close to asd, the structural gene for the enzyme aspartate semialdehyde dehydrogenase (EC 1.2.1.11).

The molecular cloning of the E. coli glg structural genes (48) greatly facilitated subsequent study of the genetic regulation of bacterial glycogen biosynthesis. The glg genes are dispensable for growth and are not amenable to direct selection. They were cloned into pBR322 via selection with the closely linked essential gene asd. Among several asd ⁺ plasmid clones that were isolated, pOP12 was found to contain a 10.5-kb PstI fragment which encoded the structural genes glgC, glgA, and glgB. A generally applicable method for cloning α-1,4-glucan biosynthetic genes, based on screening of clones with iodine vapor, has recently been developed (70). This approach does not require coselection via an essential gene and in principle should allow direct cloning of structural glg genes from any bacterium into E. coli, as well as the cloning of regulatory genes that affect glycogen synthesis.

The arrangement of genes encoded by pOP12 was also determined by deletion mapping experiments (48), and the nucleotide sequence of the entire glg gene cluster was determined (3, 4, 29, 68, 83). The genetic and physical map of the E. coli K-12 glg gene cluster is shown in Fig. 2. The continuous nucleotide sequence of over 15 kb of this region of the genome has been determined and includes the sequences of the flanking genes asd (18) and glpD (encoding glycerol phosphate dehydrogenase; EC 1.1.99.5, EC 1.1.1.8) (1). This region of the E. coli K-12 chromosome is centered at kb 4140 on the physical map of Kohara et al. (26) and is encompassed within kb 3584 to 3594 on version 6 of the physical map of Rudd et al. (original version [72] and version 6 [K. Rudd, personal communication]).

Fig. 2The E. coli K-12 glycogen gene cluster. This map was constructed from the sequences obtained from references 1, 3, 4, 18, 29, 68, and 83.

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Nucleotide sequence analysis indicated that in addition to the glgC, glgA, and glgB genes, pOP12 contains one ORF, glgX, located between glgB and glgC, and a second ORF, originally termed glgY, located downstream from glgA (68). The function of glgX is not known, but its deduced amino acid sequence is significantly related to those of α-glucanases and transferases, including α-amylases, pullulanase, cyclodextrin glucanotransferase, the glycogen BE, and others. The homologous regions include residues that have been reported to be involved in substrate binding and cleavage by α-amylases and the amylase family (24, 68, 78).

The glgY gene, now designated glgP, was identified by homology with the rabbit muscle glycogen phosphorylase gene (68, 83). This gene encodes glycogen phosphorylase via the expression and characterization of its gene product (83). Neither glgX or glgP is needed for glycogen or α-1,4-glucan synthesis, suggesting that both may be primarily involved in glycogen catabolism (68).

Inspection of the organization of the gene cluster suggests that the glg genes may be transcribed as two tandomly arranged operons, glgBX and glgCAP (Fig. 2). The coding regions of glgB and glgX overlap by 1 bp, glgC and glgA are separated by 2 bp, and glgA and glgP are separated by 18 bp. The close proximity of these genes suggests translational coupling within the two proposed operons. However, a noncoding region of approximately 500 bp separates glgB and glgC. As discussed below, studies of the regulation of the glg structural genes, with ' lacZ translational fusions and other approaches, indicate a two-operon arrangement for the glg gene cluster, in which the glgCAP and glgBX operons may be preceded by growth phase-regulated promoters. Transcriptions initiating upstream of glgC have been analyzed by S1 nuclease mapping (71) and will be discussed later.

GENETIC REGULATION OF GLYCOGEN SYNTHESIS BY cAMP AND GUANOSINE 5'-DIPHOSPHATE 3'-DIPHOSPHATE

The first evidence that cyclic AMP (cAMP) affects bacterial glycogen synthesis was that addition of exogenous cAMP to E. coli W4597(K) results in a modest enhancement in the rate of in vivo glycogen biosynthesis (9, 10). It was later observed that the genes cya, encoding adenylate cyclase (EC 4.6.1.1), and crp, encoding cAMP receptor protein (CRP), are required for optimal synthesis of glycogen and that exogenous cAMP can restore glycogen synthesis in a cya strain but not in a crp mutant (34). Because cAMP itself is not an effector of ADPGlc PPase and addition of cAMP to cultures does not affect the intracellular concentrations of the allosteric effectors of ADPGlc PPase, it was proposed that cAMP may affect the synthesis of an enzyme that metabolizes an unknown effector of ADPGlc PPase.

It was shown later that cAMP and CRP are strong positive regulators of expression of the glgC and glgA genes but do not affect glgB expression (71). The addition of cAMP and CRP to S-30 extracts during in vitro coupled transcription-translation reactions with pOP12 as the genetic template resulted in up to 25- and 10-fold increases in the expression of glgC and glgA, respectively, but did not affect glgB expression (71). cAMP and CRP also enhanced the expression of glgC and glgA encoded by either plasmids or restriction fragments in reaction mixtures of completely defined composition, i.e., the dipeptide synthesis assay (81). In these reactions, the formation of the first dipeptide of a specified gene product, directed by a DNA template, is quantified (71, 81). A restriction fragment that contained glgC and 0.5 kb of DNA from the upstream noncoding region of glgC was sufficient to permit cAMP-CRP-regulated expression in the dipeptide synthesis assay (81), suggesting that the glgC gene contains its own cAMP-regulated promoter(s).

Evidence for a CRP binding site on a 243-bp restriction fragment from the upstream region of glgC was obtained by using gel retardation analysis (71). There are also potential consensus CRP binding sequences within the glgC upstream region preceding both the E. coli (71) and S. typhimurium (69) glgC genes. These are shown in Fig. 3.

Fig. 3cAMP-CRP binding sites and sequences upstream of the glgC gene of S. typhimurium and E. coli. The E. coli site was detected by DNase I protection analysis (71), and that of S. typhimurium was derived by sequence homology to the E. coli sequence (69).

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Experiments in which proteins encoded by the glg structural genes were expressed from plasmid pOP12 in maxicells showed that exogenously added cAMP stimulated the expression of glgC and glgA but did not affect glgB expression (65). Evidence that glgC is regulated by cAMP in vivo was obtained by constructing an in-frame plasmid-encoded glgC'-'lacZ translational fusion, designated pCZ3–3, which contained 0.5 kb of the upstream noncoding region of glgC (65). This gene fusion was expressed approximately fivefold better in a cya ⁺ strain than in an isogenic Δ cya strain, and expression was stimulated by exogenous cAMP in both strains.

The first suggestion that glycogen biosynthesis in E. coli is positively regulated by ppGpp was that relA strains are glycogen deficient (5, 35, 80). It is now well established that expression of the glgC and glgA genes is stimulated by ppGpp (65, 71). Expression of glgC in transcription-translation reactions was increased three- to fourfold in the presence of ppGpp; glgA expression exhibited approximately a twofold enhancement. The expression of glgB was not affected by ppGpp.

cAMP and ppGpp also have independent effects on glgCA expression in in vitro transcription-translation experiments (71). Actually, their combined effects on glgC expression in transcription-translation experiments can be synergistic (71). The addition of cAMP-CRP or ppGpp results in an increase of 6.3- or 1.6-fold, respectively, in the expression of glgC over the basal or unactivated level of expression, whereas the addition of the two together leads to an 18.8-fold stimulation.

Evidence for positive regulation of glgC expression in vivo by ppGpp was obtained using the glgC'-'lacZ translational fusion in pCZ3–3 (65). This gene fusion was introduced into strains comprising an isogenic series that varied in basal levels of ppGpp as a result of increasingly severe mutations in spoT (73). The spoT gene affects the levels of ppGpp in the cell. The expression of the glgC'-'lacZ gene fusion was exponentially correlated with ppGpp levels in this series of strains (65).

NEGATIVE GENETIC REGULATION OF GLYCOGEN SYNTHESIS

Studies of glycogen-excess E. coli B mutants SG3 and AC70R1, which exhibit enhanced levels of the enzymes in the glycogen synthesis pathway (i.e., are derepressed mutants), suggested that glycogen synthesis is under negative genetic regulation (27, 48, 53, 61). The mutations in these strains, glgR and glgQ, respectively, affect glg transcription (71), although these mutated genes have not been cloned and subjected to sequence analysis.

The 5' termini of four in vivo transcripts in the upstream region were identified within 0.5 kb of the glgC coding region by S1 nuclease protection analyses (71) (Fig. 4 and 5).

Fig. 4The glgC transcripts A, B, and C were mapped by S1 nuclease protection analysis (71) and are located at -60, -130, and -245, respectively, relative to the glgC initiation codon. The 5? termini of glgC transcripts are indicated by asterisks, and the best ?10 and ?35 regions are underlined.

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Fig. 5Regulatory sites for glgC transcription. The CRP binding region was located by mobility shift analysis (71). Transcripts from wild-type strains of E. coli B and E. coli K-12 and isogenic glycogen synthesis mutants were analyzed by S1 nuclease protection analysis (71).

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The glgR mutation is closely linked to the glycogen structural genes by P1 transduction analysis; the mutation results in 8- to 10-fold-higher levels of ADPGlc PPase and 3- to 4-fold-higher levels of glycogen synthase in exponential phase but does not alter the level of BE in minimal media (55). Analysis of RNA transcripts for glgC in strain SG3 having the glgR mutation reveals an increase in transcript B only (71) (Fig. 4 and 5). Therefore, it appears that the glgR mutation may alter a cis-acting site involved in the regulation of transcript B. This effect might be mediated via a negative regulatory site, but the current experimental evidence is also consistent with an overexpressed phenotype or a higher-affinity CRP binding site.

The glgQ mutation is not linked to the glycogen gene cluster in P1 transduction and results in 11-, 5.5-, and 2-fold increases in ADPGlc PPase, glycogen synthase, and glycogen BE, respectively (59). Therefore, glgQ appears to affect one or more trans-acting factors for the expression of the genes in the two glycogen operons. Levels of the four transcripts for the glgC gene are elevated in the glgQ mutant, AC70R1 (71) (Fig. 4 and 5). Transcript A was affected the most dramatically, with approximately 25-fold-higher levels being present in AC70R1 than in the wild-type strain E. coli B or strain SG3 (71). Since the levels of BE are also elevated in AC70R1, it was not considered likely that glgQ was a mutation in the cAMP-CRP or ppGpp regulatory system, neither of which affects glgB expression. The levels of expression of the chromosomal lacZ gene in AC70R1 (glgQ mutant) and in the wild-type strain E. coli B were also similar, providing further evidence for the idea that glgQ affects a different regulatory system for the glg genes (69). In summary, glgQ affects mainly transcript A and glgR affects mainly transcript B.

THE csrA GENE PRODUCT IS A NEGATIVE EFFECTOR OF THE glg OPERONS

Recent experiments have resulted in the identification and characterization of an E. coli K-12 gene, csrA, that encodes a negative factor for glg transcription (67). The relationship of csrA to the E. coli B mutations glgR and glgQ is not yet known.

Transposon mutants in which glycogen biosynthesis in E. coli K-12 had been affected were isolated (67). The transposon approach used facilitated identification, molecular cloning, and mapping of trans-acting regulatory genes for glycogen biosynthesis. Mutations were introduced into a strain that contained pCZ3-3 (plasmid having a glgC'-'lacZ translational fusion), and the resulting mutants were stained with I₂ vapor to detect cell colony glycogen. The plasmid-encoded β-galactosidase (EC 3.2.1.23) was also determined in glycogen-excess mutants (67), and several glycogen-excess mutants overexpressing the plasmid-borne glgC'-'lacZ fusion were isolated. One mutant, TR1-5, accumulated about 24-fold more glycogen than an isogenic wild-type strain (67). The gene affected by the mutation in TR1-5, csrA (for "carbon storage regulator"), has been cloned, sequenced, and mapped on the E. coli genome, and some of its regulatory effects have been studied (66, 67).

The mutation in TR1-5 was also shown to affect glycogen levels by causing elevated expression of genes representative of both glycogen operons, glgC and glgB. Levels of ADPGlc PPase expressed from the chromosome were approximately 10-fold higher in mutant TR1-5 than in an isogenic csrA ⁺ strain in the stationary phase. The β-galactosidase activities expressed from the glgC'-'lacZ and the glgB'-'lacZ translational fusions were approximately 7-fold and 2- to 3-fold higher, respectively, in mutant TR1-5 than in an isogenic csrA ⁺ strain. The mutation in TR1-5 affects glycogen levels and expression of the glgB and glgC genes in both the exponential and stationary phases.

The csrA gene also appears to regulate the expression of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (EC 4.1.1.38). Expression of a phosphoenolpyruvate carboxykinase operon fusion (pckA'-'lacZ) was increased about twofold in exponential and stationary phases in mutant TR1-5, suggesting that gluconeogenesis may also be under negative control by csrA (67). When several isogenic strains were grown on synthetic media, it was found that csrA ⁺ and csrA::Kan^r strains (transductants with the mutation in TR1-5) were capable of growth on a wide variety of carbon sources. However, a strain that contained the functional csrA gene encoded on a multicopy (pUC19-based) plasmid, pCSR10 (67), could grow on glucose and fructose but not on any of the gluconeogenic substrates (succinate, glycerol, pyruvate, and l-lactate). When strains were plated on a richer defined medium, growth of a pCSR10-containing strain was supported by some gluconeogenic substrates, including acetate, as major carbon sources. However, the pCSR10-containing strain formed only pinpoint colonies on succinate, whereas each of the other strains grew well. Perhaps the csrA gene affects succinate utilization independently of its effect on gluconeogenesis, possibly at the level of succinate transport into the cell.

The csrA gene, mapped by both genetic and physical approaches (66), is located at 58 min or kb 2830 on the physical map of the E. coli K-12 genome (26). csrA lies between alaS, which encodes alanyl-tRNA synthetase (EC 6.1.1.7), and the serV operon of tRNA genes and is transcribed counterclockwise on the chromosome (66).

Nucleotide sequence analysis revealed that the largest ORF, located between alaS and serV and present in pCSR10, exhibited an upstream sequence typical of E. coli ribosome binding sites (59, 66). Sequence analysis of the csrA mutant allele in strain TR1-5 showed that this ORF is disrupted by the Kan^r marker in this mutant (59, 66).

The csrA ORF encodes a 61-amino-acid polypeptide, which was strongly expressed from plasmid pCSR10 in S-30 transcription-translation experiments (67). Deletion mapping experiments of the plasmid-encoded csrA gene demonstrated that the ORF is required to mediate the inhibitory effects on the glycogen synthesis phenotype in vivo. Computer-assisted database searches failed to reveal substantial homology of csrA or its proposed polypeptide product, CsrA, with any existing genes, proteins, or prokaryotic sequence motifs. Hence, csrA may encode a previously unknown kind of genetic regulatory factor.

Analysis of glgC transcripts by S1 nuclease protection mapping showed that the steady-state levels of all four glgC transcripts are elevated in mutant TR1-5 and are severely depressed in a pCSR10-containing strain, indicating that csrA affects the transcriptional regulation of glgC (59, 67). The molecular mechanism that could account for the simultaneous effects of the factor CsrA on four glgC transcripts is unknown.

SIGMA FACTOR Eσ70 TRANSCRIBES glgCAP

Analysis of transcripts originating upstream from the glgCAP operon by S1 nuclease protection mapping revealed four transcripts. Three of these transcripts were mapped to high resolution (71). The nucleotide sequences immediately preceding the 5' ends of these transcripts were weakly related to consensus sequences for E. coli promoters (71) (Fig. 4). Although positively regulated promoters typically show weak similarity to the consensus sequence (17), it is also possible that one or more of the glg promoters are recognized via an alternative sigma factor. Therefore, the dependence of glgC expression on three sigma factors was tested in coupled transcription-translation, using monoclonal antibodies that selectively inhibit transcription by specific and selective recognition of the sigma factors (25, 39, 64).

A monoclonal antibody directed against E. coli σ ⁷⁰ inhibited up to 85% of glgC expression (59), while an antibody directed against σ ⁵⁴ or σ ³² did not inhibit expression of glgC, relative to glnAP2 (σ ⁵⁴-dependent) and dnaK (σ ³²-dependent) controls. Therefore, even though nitrogen limitation enhances glycogen synthesis in vivo, the expression of glgC is not regulated by the nitrogen starvation, σ ⁵⁴-dependent transcriptional controls. This result agrees with the finding that NtrC and NtrA (σ ⁵⁴) did not enhance expression of glg genes in S-30 experiments (71). The heat shock regulatory system (σ ³² dependent) also appears to have no involvement in the control of glgC expression. Therefore, the major active form of RNA polymerase (Eσ ⁷⁰) is apparently utilized for glg expression in the S-30 transcription-translation system. The S-30 extracts used in the experiments were prepared from exponential-phase cells to obtain optimal translational activity and would not have contained endogenous activity from a sigma factor or an accessory factor synthesized in stationary phase. Therefore, expression via one or more glgC transcripts could have been undetected in these experiments.

POTENTIAL REGULATION OF GLYCOGEN SYNTHESIS BY OTHER GENES

Interruption of the gene for a putative stationary-phase sigma factor, katF, also leads to decreased levels of glycogen (30). However, experiments by others indicate that katF does not regulate glgC gene expression in vivo (T. Romeo, personal communication; 20).

Studies from several laboratories provide evidence for a class of stationary-phase-induced genes depending on a specific sigma factor for expression (30, 44, 74). The gene for this sigma factor is referred to as katF or rpoS. Iodine staining of katF ⁺ and katF strains suggests that a functional katF allele is required for optimum accumulation of glycogen in E. coli MC4100 (30). As indicated above, the effect of katF on glycogen synthesis is not exerted via regulation of the structural genes for the enzymes of the glycogen pathway.

Hengge-Aronis and Fischer have isolated, cloned, and sequenced glgS, an E. coli K-12 gene that stimulates glycogen synthesis and requires katF for optimum expression (20). The glgS gene appears to be transcribed via a cAMP-dependent promoter and a katF-requiring promoter. Iodine staining of colonies indicates that a null glgS mutant accumulates more glycogen than a katF mutant, indicating that katF may have effects on glycogen synthesis in addition to glgS induction. The glgS mutation did not affect the expression of glgC or glgA gene fusions.

The function of glgS and the role of katF in glycogen synthesis remain to be elucidated, even though several mechanisms as to how glgS may affect glycogen biosynthesis have been suggested. These factors may regulate a process or metabolic step prior to the glycogen biosynthesis pathway per se that affects the ultimate level of accumulated glycogen.

A PROPOSED INTEGRATED MODEL FOR GENETIC REGULATION OF THE GLYCOGEN BIOSYNTHETIC PATHWAY IN E. COLI

Regulation of glycogen metabolism involves a multitude of factors coordinating the glycogen synthetic rate with the physiology of the cell. The genetic regulation of the glycogen biosynthesis pathway by cAMP and ppGpp allows E. coli to adjust its metabolic capacity for converting the available carbon substrate into glycogen in response to the availability of carbon or energy or amino acids, respectively. When cells are rapidly multiplying, the levels of the enzymes are repressed, and although the energy and carbon are available for glycogen synthesis, the rate of glycogen synthesis is low. Upon nutrient deficiency, syntheses of ADPGlc PPase and glycogen synthase are induced and the capacity for glycogen synthesis is greater. The level of glycogen that is ultimately accumulated will depend on substrate availability and is subject to allosteric regulation of the ADPGlc PPase activity.

Genetic regulation thus determines the capacity for glycogen synthesis, which should be distinguished from regulation of the absolute glycogen levels. As an example, the glycogen biosynthetic enzymes are induced in stationary phase when cells are grown on enriched medium lacking glucose, yet glycogen synthesis does not occur because of insufficient carbon source. However, in media with excess glucose, in which nitrogen is limiting, expression of the genes for the biosynthetic enzymes is somewhat weaker, as cAMP is at a low level. However, the glycogen synthetic rate is greater because of the carbon availability and because the conditions are such that allosteric activation occurs.

Mutants that are affected in either negative (glgR, glgQ, and csrA) or positive (cya, crp, relA, and spoT) control systems for glgCA gene expression clearly demonstrate that genetic regulation of the levels of glycogen biosynthetic enzymes is most important in determining the ultimate level of glycogen synthesized and accumulated under any given physiological condition.

The structural and regulatory genes involved in glycogen metabolism in E. coli and the effects of both positive and negative regulatory factors which control the expression of the glg genes of the glycogen biosynthetic pathway are listed in Table 4. Many important questions remain to be solved, particularly the regulatory role of the factor CsrA (66, 67). The physiological states that the CsrA regulatory system responds to remain to be determined. Moreover, the functions of glgS, katF, and many carbon starvation-induced genes (e.g., csi [19, 82]) in glycogen synthesis at the biochemical and molecular levels remain to be established.

Table 4Genes involved in glycogen metabolism in E. coli