Manuel Montero

Gustavo Eydallin

Alejandro M Viale

Goizeder Almagro

Francisco J Muñoz

Mehdi Rahimpour

María T Sesma

Edurne Baroja-Fernández

Javier Pozueta-Romero

Edurne Baroja- Fernández

Javier Pozueta
email: javier.pozueta@unavarra.es.

Escherichia coli glycogen metabolism is controlled by the PhoP-PhoQ regulatory system at submillimolar environmental Mg 2+ concentrations, and is highly interconnected with a wide variety of cellular processes

Keywords: AMP, energy status, Keio collection, Mg 2+ , stringent response ADP-glucose, cAMP, cyclic AMP, COG, cluster of orthologous groups, CRP, cAMP receptor protein, GlgA, glycogen synthase, GlgB, branching enzyme, GlgP, glycogen phosphorylase, GlgX, debranching enzyme

Escherichia coli glycogen metabolism is controlled by the

PhoP-PhoQ regulatory system at submillimolar environmental Mg2+ concentrations, and is highly interconnected with a wide variety of cellular processes

INTRODUCTION

Glycogen is a branched homopolysaccharide of α-1,4-linked glucose subunits with about 5% α-1,6-linked glucose at the branching points. The exact role of this polyglucan in prokaryotes is still unknown, although several works have linked glycogen metabolism to environmental survival and even to colonization and virulence in the case of pathogens [START_REF] Bonafonte | The relationship between glycogen synthesis, biofilm formation and virulence in Salmonella enteritidis[END_REF][START_REF] Chang | Carbon nutrition of Escherichia coli in the mouse intestine[END_REF][START_REF] Jones | Glycogen and maltose utilization by Escherichia coli O157:H7 in the mouse intestine[END_REF][START_REF] Sambou | Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice[END_REF][START_REF] Bourassa | Glycogen contributes to the environmental persistence and transmission of Vibrio cholerae[END_REF]. Synthesized by glycogen synthase (GlgA) using ADPglucose (ADPG) as the glucosyl moiety donor, glycogen accumulation in Escherichia coli and Salmonella spp. occurs when cellular carbon sources are in excess under conditions of other nutrients deficiency [START_REF] Dietzler | Rates of glycogen synthesis and the cellular levels of ATP and FDP during exponential growth and the nitrogenlimited stationary phase of Escherichia coli W4957(K)[END_REF][START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF]. Under these conditions synthesis of proteins and nucleic acids is arrested, and ATP and excess carbon are diverted towards glycogen biosynthesis.

Regulation of glycogen biosynthesis in E. coli involves a complex and still not well defined assemblage of factors. At the level of enzyme activity for instance, the glycogen biosynthetic process is subjected to the allosteric regulation of GlgC, a protein that catalyzes the production of ADPG from ATP and glucose-1-phosphate [START_REF] Preiss | Molecular biology and regulatory aspects of glycogen biosynthesis in bacteria[END_REF][START_REF] Ballicora | ADP-Glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis[END_REF]. At the level of gene expression, in vitro and in vivo experimental evidences suggested that the process depends on the regulation of two contiguous operons, glgBX (encompassing the genes coding for glycogen branching (GlgB) and debranching (GlgX) enzymes), and glgCAP (encoding the GlgC and GlgA anabolic enzymes as well as the catabolic glycogen phosphorylase (GlgP)) [START_REF] Preiss | Molecular biology and regulatory aspects of glycogen biosynthesis in bacteria[END_REF][START_REF] Romeo | Genetic regulation of glycogen biosynthesis in Escherichia coli: In vitro effects of cyclic AMP and guanosine 5´-diphosphate 3´diphosphate and analysis of in vivo transcripts[END_REF][START_REF] Alonso-Casajús | Glycogen phosphorylase, the product of the glgP gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli[END_REF]]. An additional gene, glgS, regulates glycogen synthesis in a still undefined manner [START_REF] Hengge-Aronis | Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli[END_REF].

Transcriptome analyses of E. coli cells growing on either glucose-containing minimal or rich media have shown that all glg genes are upregulated when cultures enter the late logarithmic growth phase [START_REF] Tao | Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media[END_REF]. Experimental evidences provided by different authors have indicated that the glgCAP operon is positively regulated at the transcriptional level by the guanosine 5´-diphosphate 3´-diphosphate (ppGpp) stringent response regulator synthesized by the product of relA [START_REF] Romeo | Genetic regulation of glycogen biosynthesis in Escherichia coli: In vitro effects of cyclic AMP and guanosine 5´-diphosphate 3´diphosphate and analysis of in vivo transcripts[END_REF][START_REF] Bridger | relA gene control of bacterial glycogen synthesis[END_REF][START_REF] Taguchi | Augmentation of glycogen synthesis under stringent control in Escherichia coli[END_REF][START_REF] Romeo | Genetic regulation of glycogen biosynthesis in Escherichia coli: in vivo effects of the catabolite repression and stringent response systems in glg gene expression[END_REF][17], and by the cyclic AMP (cAMP)/cAMP receptor protein (CRP) complex ([18-20], however, for an opposite view see [START_REF] Hengge-Aronis | Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli[END_REF]). At the post-transcriptional level, glycogen accumulation in E. coli is negatively affected by the central carbon storage regulator CsrA, which prevents glgC translation [START_REF] Romeo | Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties[END_REF][START_REF] Yang | Coordinate genetic regulation of glycogen catabolism and biosynthesis in Escherichia coli via the CsrA gene product[END_REF][START_REF] Baker | CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli[END_REF]. In addition, glycogen accumulation is positively influenced by the general stress factor RpoS by a still undefined mechanism [START_REF] Hengge-Aronis | Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli[END_REF].

To obtain clues on the role(s) of glycogen metabolism and its connection with other biological process, we have recently carried out a genome-wide screening of the genes affecting glycogen metabolism [START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF] using a systematic and comprehensive genedisrupted E. coli mutant collection (the Keio collection [START_REF] Baba | Construction of Escherichia coli K-12 in frame, single-gene knockout mutants: the Keio collection[END_REF]). This study identified 65 genes whose mutations substantially affected cell glycogen content. Amongst different mutants with reduced glycogen content we identified those lacking phoP, phoQ and mgtA. This observation was particularly interesting since these genes are induced under low extracellular Mg 2+ concentration conditions [START_REF] Smith | Microbial magnesium transport: unusual transporters searching for identity[END_REF][START_REF] Minagawa | Identification and molecular characterization of the Mg 2+ stimulon of Escherichia coli[END_REF]. In fact, PhoP-PhoQ is a twocomponent regulatory system occurring in E. coli and Salmonella spp. that monitors the availability of extracellular Mg 2+ , and transcriptionally controls the expression of many genes, including mgtA which codes for an energy-dependent Mg 2+ import system [START_REF] Garcia-Vescovi | Mg 2+ as an extracellular signal: environmental regulation of Salmonella virulence[END_REF][START_REF] Spinelli | Regulation of magnesium homeostasis in Salmonella: Mg 2+ targets the mgtA transcript for degradation by RNase E[END_REF]. The overall data thus suggested that extracellular Mg 2+ could represent an important determinant of glycogen accumulation in E. coli.

Mg 2+ is the most abundant divalent cation within living cells, being a stabilizing factor for membranes, tRNA, ribosomes, etc. that strongly determines cell metabolic and energetic status [START_REF] Smith | Microbial magnesium transport: unusual transporters searching for identity[END_REF]. In fact, the balance between Mg 2+ -bound and Mg 2+ -free adenylates is a major determinant of the correct functioning of many enzymes. This balance depends on factors such as total content of intracellular adenylates and free A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Mg 2+ , adenylate kinase (Adk) activity, membrane potential and transport of extracellular Mg 2+ across membranes [START_REF] Smith | Microbial magnesium transport: unusual transporters searching for identity[END_REF][START_REF] Purich | Studies on factors influencing enzyme responses to adenylate energy charge[END_REF][START_REF] Igamberdiev | Membrane potential, adenylate levels and Mg 2+ are interconnected via adenylate kinase equilibrium in plant cells[END_REF]. To investigate the influence of extracellular Mg 2+ on E. coli glycogen metabolism in this work we have measured both ATP and glycogen contents, and expression of glgC in cells cultured in glucose Kornberg medium without and with Mg 2+ supplementation. We found that changes in external Mg 2+ concentrations in the submillimolar range have profound effects on the ability of E. coli to accumulate glycogen. We have therefore re-screened the Keio collection for glycogen altered phenotypes employing culture medium supplemented with Mg 2+ . With the overall data amassed in this work we conclude that synthesis and utilization of glycogen is under control of a complex and intricate network wherein cell energy and nutritional status play crucial roles. We propose an integrated metabolic model wherein glycogen (a) is highly interconnected with a number of cellular processes and (b) is finely regulated by changes in environmental Mg 2+ occurring at the submillimolar concentration range. In addition, based in our observations we propose that intracellular AMP levels are critically important in regulating glycogen biosynthesis. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

EXPERIMENTAL PROCEDURES E. coli K-12 mutants and culture conditions

We used mutants from the systematic, single-gene knockout mutant collection of the nonessential genes of the E. coli K-12 derivative BW25113 [START_REF] Baba | Construction of Escherichia coli K-12 in frame, single-gene knockout mutants: the Keio collection[END_REF]. For quantitative measurement of glycogen content, cells were grown at 37ºC with rapid gyratory shaking in liquid Kornberg medium (1.1% K 2 HPO 4 , 0.85% KH 2 PO 4 , 0.6% yeast extract from Duchefa, Haarlem, The Netherlands) containing 250 μM Mg 2+ and 50 mM glucose after inoculation with 1 volume of an overnight culture for 50 volumes of fresh medium. Cells from cultures entering the stationary phase were centrifuged at 4,400 x g for 15 min, rinsed with fresh Kornberg medium, resuspended in 40 mM Tris•HCl (pH 7.5) and disrupted by sonication prior to quantitative glycogen measurement (see below). Solid Kornberg medium was prepared by addition of 1.8% bacteriological agar to liquid Kornberg medium before autoclaving.

Screening for mutants with altered glycogen content

First screening of glycogen mutants on solid glucose Kornberg medium containing 250 μM Mg 2+ was carried out following the glycogen iodine staining method [31]. Mutants identified using this procedure were subsequently cultured in liquid glucose Kornberg medium containing 250 μM Mg 2+ , and subjected to quantitative glycogen measurement analyses using an amyloglucosidase/hexokinase/glucose-6P dehydrogenase-based test kit from Sigma. Intracellular glycogen content was referred to protein, which was measured using a Bio-Rad prepared reagent. Mutants with altered glycogen content were further confirmed for the indicated deletions by PCR using specific primers. The gene mutations promoting altered glycogen accumulation described in this work imply that both insertional mutants for each individual gene provided in the Keio collection displayed the indicated phenotype. The function of each gene whose deletion affects glycogen accumulation was assigned by referring to the EchoBASE (http://ecoliyork.org/) and EcoCyc (http://www.ecocyc.org/) databases.

LacZY transcriptional fusions

The kanamycin resistance cassette inserted at the deletion point of E. coli genes from Keio collection was removed by using temperature-sensitive plasmid pCP20 carrying the FLP recombinase [START_REF] Cherepanov | Gene disruption in Escherichia coli: Tc R and Km R cassettes with the option of Flp-catalized excision of the antibioticresistance determinant[END_REF]. The scar sequence left after removal of the resistance cassette included a 34-nucleotide FRT site [START_REF] Baba | Construction of Escherichia coli K-12 in frame, single-gene knockout mutants: the Keio collection[END_REF], which was used to build lacZY transcriptional fusions as reported in [34]. Briefly, Keio collection E. coli mutants carrying pCP20 plasmid were transformed with pKG137, which has functional lacZY and Km R cassette that integrated in the proper orientation at the FRT site by the action of the FLP recombinase, yielding to lacZY transcriptional fusions where the original resistance cassette of the Keio collection was previously placed.

Western blot analyses

Bacterial extracts were separated by 10% SDS-PAGE, transferred to nitrocellulose filters, and immunodecorated by using the antisera raised against GlgC [START_REF] Eydallin | An Escherichia coli mutant producing a truncated inactive form of GlgC synthesizes glycogen: further evidences for the occurrence of various important sources of ADPglucose in enterobacteria[END_REF] and a goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma).

RESULTS AND DISCUSSION

Extracellular Mg 2+ is an important determinant of E. coli glycogen accumulation

Among different mutants of the Keio collection with altered glycogen content identified in our previous work [START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF], ΔmgtA, ΔphoP and ΔphoQ cells displayed glycogen-deficient phenotypes. Because the expression of mgtA, phoP and phoQ is a PhoP-PhoQdependent process triggered under conditions of low extracellular Mg 2+ , our previous observations suggested that (a) the glucose Kornberg culture medium employed in our previous work contained limiting Mg 2+ concentrations, and (b) environmental Mg 2+ may represent an important determinant of glycogen metabolism in E. coli. To test these possibilities we measured the Mg 2+ content in the glucose Kornberg culture medium employed in our previous work. Furthermore, because mgtA is a reporter gene of low external and cytoplasmic Mg 2+ concentrations [START_REF] Spinelli | Regulation of magnesium homeostasis in Salmonella: Mg 2+ targets the mgtA transcript for degradation by RNase E[END_REF], we also analyzed the expression of chromosomal mgtA::lacZY transcriptional fusion constructed on BW25113 wild type (WT) cells when cultured in glucose Kornberg medium without and with Mg 2+ supplementation. Finally, we measured the glycogen content in WT, ΔmgtA, ΔphoP, and ΔphoQ cells cultured under the above conditions . Determination of different metal contents in the yeast extract used for the preparation of the glucose Kornberg medium used in this and in our previous work [START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF] indicated the presence of around 50 μM Mg 2+ . Although this intrinsic concentration would be expected to suffice for an active bacterial growth [START_REF] Smith | Microbial magnesium transport: unusual transporters searching for identity[END_REF], it was actually limiting for growth of the BW25113 strain used here, as judged by comparing both the growth rates and yields obtained with and without supplementation of submillimolar Mg 2+ concentrations (Supplemental Figure 1). Limitations in bacterial growth due to deficient Mg 2+ contents are most probably due to the complexing effect exerted by the high phosphate concentration (ca. 120 mM) of the Kornberg medium [START_REF] Govons | Biosynthesis of bacterial glycogen: kinetic characterization of an altered adenosine diphosphate-glucose synthase from a "glycogen-excess" mutant of Escherichia coli B[END_REF]. Analyses of the expression of a chromosomal mgtA::lacZY transcriptional fusion on WT cells at the onset of the stationary phase revealed that mgtA was highly expressed at the onset of the stationary phase in medium lacking Mg 2+ supplementation (starting Mg 2+ concentration of 50 μM), while its expression was strongly repressed when the starting Mg 2+ concentration reached 250 μM or higher (Supplemental Figure 2). The overall data thus showed that the culture medium employed in our previous work [START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF] reached a threshold limiting Mg 2+ concentration during bacterial growth.

Supplementation of the culture medium with submillimolar concentrations of Mg 2+ substantially increased glycogen content in cells at the onset of the stationary phase, almost doubling the amount of this polyglucan when the starting concentration of Mg 2+ reached 250 μM or higher (not shown). This enhancing effect of Mg 2+ on glycogen accumulation was even more marked in ΔphoP, ΔphoQ, and ΔmgtA cells (and in particular for the first two mutants, Figure 1), the final amount of glycogen accumulated at the onset of the stationary phase being similar for all cell types when cultured under non-limiting Mg 2+ concentrations. The overall data thus showed that environmental Mg 2+ concentration changes in the submillimolar range may represent important determinants of glycogen accumulation in E. coli.

Extracellular Mg 2+ highly determines the intracellular levels of ATP in E. coli

Mg 2+ is the most abundant divalent cation within cells, its intracellular concentration strongly determining the cell metabolic and energy status [START_REF] Smith | Microbial magnesium transport: unusual transporters searching for identity[END_REF]. Since ATP acts as a substrate for reactions directly and indirectly involved in glycogen biosynthesis, we found of interest to analyze the intracellular ATP content in WT bacteria entering the stationary phase either in limiting and non-limiting Mg 2+ concentrations (50 μM Mg 2+ A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. and 250 μM Mg 2+ , respectively). As shown in Figure 2, these analyses revealed that ATP content in cells cultured under non-limiting Mg 2+ conditions was, in average, 2.5fold higher than that observed for cells cultured under limiting Mg 2+ conditions. The overall data thus indicate that the reduced glycogen contents observed in E. coli cells subjected to limiting Mg 2+ concentrations (cf. Figure 1) could be ascribed, at least in part, to reduced intracellular levels of ATP.

Expression of E. coli glgCAP operon is positively controlled by the PhoP-PhoQ regulatory system under limiting Mg 2+ conditions

We next investigated whether expression of the E. coli glgCAP operon is subjected to extracellular Mg 2+ regulation. For this purpose we constructed chromosomal glgC::lacZY gene transcriptional fusions in WT, ΔmgtA, ΔphoQ and ΔphoP cells, and compared the β-galactosidase activity levels reached at different growth times of these bacteria when cultured under both limiting and non-limiting Mg 2+ conditions (50 μM and 1 mM Mg 2+ , respectively). We also determined by western blot analyses the contents of GlgC at the onset of the stationary phase in WT, ΔmgtA, ΔphoQ and ΔphoP cells when growing under the two different Mg 2+ concentration regimes.

Growth of ΔmgtA, ΔphoQ and ΔphoP cells, all impaired in the MgtA high affinity Mg 2+ import system induced under low environmental Mg 2+ concentrations [START_REF] Smith | Microbial magnesium transport: unusual transporters searching for identity[END_REF][START_REF] Spinelli | Regulation of magnesium homeostasis in Salmonella: Mg 2+ targets the mgtA transcript for degradation by RNase E[END_REF], was reduced under limiting Mg 2+ concentration conditions when compared with WT cells (Figure 3A). Supplementation of the culture medium with 1 mM Mg 2+ restored growth of the above mutants to WT levels (Figure 3C). Analysis of βgalactosidase activity levels derived from glgC::lacZY transcriptional fusions indicated that, under limiting Mg 2+ concentration conditions, glgC expression was significantly lower in both ΔphoP cells and ΔphoQ cells than in both WT and ΔmgtA cells (Figure 3B). Moreover, supplementation of the culture medium with 1 mM Mg 2+ largely restored glgC::lacZY expression in ΔphoP and ΔphoQ cells (Figure 3D), the overall data indicating that expression of the E. coli glgCAP operon is under control of the PhoP-PhoQ system under low environmental Mg 2+ conditions. In agreement with this proposal, western blot analysis of GlgC showed that both ΔphoP and ΔphoQ cells accumulated lower levels of this protein than WT cells when cultured under limiting Mg 2+ concentrations (Figure 3E), whereas GlgC levels in ΔphoP and ΔphoQ cells were comparable to those of WT cells when cultured in non-limiting Mg 2+ conditions (Figure 3E). Furthermore, expression of phoQ from a plasmid vector in ΔphoQ cells cultured under limiting Mg 2+ concentrations resulted in WT levels of GlgC (Figure 3E). GlgC levels in ΔmgtA cells impaired in the MgtA high affinity Mg 2+ import system were similar to those of WT cells when cultured in both limiting and non-limiting Mg 2+ concentration conditions (not shown). These observations, together with those showing WT expression levels of glgC::lacZY in ΔmgtA cells (cf. Figure 3B), rule out a significant role of low intracellular Mg 2+ concentrations in the induction of glgCAP expression, and reinforce the notion that this operon is under positive control of the PhoP-PhoQ regulatory system under limiting environmental Mg 2+ concentrations. Noteworthy, sequence analyses of the 1000-bp-long promoter region upstream from the ATG initiation codon of glgC did not reveal the presence of a putative PhoP box, defined in E. coli as (T)G(T)TT(AA) [START_REF] Yamamoto | Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of a two-component system sensing external magnesium availability[END_REF] or (T/G)GTTTA [START_REF] Minagawa | Identification and molecular characterization of the Mg 2+ stimulon of Escherichia coli[END_REF] tandem direct repeats. It is thus possible that the PhoP-PhoQ mediated Mg 2+ regulation of glgCAP is indirect, as has been described for the PmrA-regulated genes [START_REF] Kox | A small protein that mediates the activation of a two-component system by another two-component system[END_REF].

Because Mg 2+ is a determinant of both intracellular ATP levels and correct functioning of many enzymes including those involved in glycogen biosynthesis, the moderate reduction of glycogen levels occurring in ΔmgtA cells cultured under limiting (50 μM) Mg 2+ concentration (60% of the glycogen accumulated by WT cells, cf. Figure 1 and Figure 2 in [START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF]) can be ascribed to both low ATP production and reduced activity of enzymes directly or indirectly involved in glycogen metabolism. In turn, the more drastically reduced glycogen levels of ΔphoP cells cultured with 50 μM Mg 2+ (10% of the levels occurring in WT cells, cf. Figure 1 in this work and Figure 2 in [START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF]) can be ascribed to a combination of factors including reduced ATP levels, low activity of enzymes involved in glycogen metabolism, and low expression of glgCAP operon.

Screening, identification and classification of E. coli genes whose deletions affect glycogen accumulation under non-limiting Mg 2+ conditions

Using the iodine staining technique we screened the Keio mutant collection for altered glycogen content when cells were cultured in glucose Kornberg medium containing a starting Mg 2+ concentration of 250 μM. In the presence of iodine vapors, "glycogenexcess" mutants stain darker than their brownish parental cells, whereas "glycogendeficient" mutants stain yellow (Supplemental Figure 3). On inspecting the mutant library, 92 mutants (2.3 % of the library) showed increased levels of glycogen (Figure 4), while 91 mutants (2.3 % of the library) had reduced levels of glycogen when compared to WT cells (Figure 5). The 183 genes now identified whose deletions affect glycogen accumulation were classified into clusters of orthologous groups (COGs) categories (Tables 1 and2). Supplemental Table 1 shows the function of each gene product. Notably, a large group of 31 mutants, representing 17 % of the genes identified, corresponded to genes of unknown function, about which little or nothing is known.

As summarized in Figure 6, the general trend indicates that glycogen metabolism of E. coli cells cultured under non-limiting Mg 2+ conditions is affected by proteins that can be embodied in the following groups:

• Stringent response,

• General stress response,

• Amino acid metabolism, The asterisk (*) indicates categories which were not identified in our previous screening [START_REF] Eydallin | Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12[END_REF].

In the following sections, the possible roles of some of the identified components are discussed.

The stringent response

During nutrient starvation E. coli elicits the so-called stringent response that switches the cell from a growth-related mode to a maintenance/survival/biosynthesis mode. The hallmark of this pleiotropic response is the accumulation of the alarmone (p)ppGpp, which is synthesized by the relA product [39. When (p)ppGpp accumulates, growth is arrested and it is expected that an important pool of ATP will be diverted towards glycogen biosynthesis under conditions of carbon source excess. Consistent with this view, ΔrelA cells of the Keio collection impaired in (p)ppGpp synthesis displayed a glycogen-deficient phenotype (Figure 5).

Although it is widely accepted that glycogen accumulation is positively influenced by relA [START_REF] Bridger | relA gene control of bacterial glycogen synthesis[END_REF][START_REF] Romeo | Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties[END_REF], available data on the regulatory action of this gene on the expression of glg genes are contradictory. Whereas transcription profiling analyses of the stringent response failed to show that glg genes belong to the RelA regulon [START_REF] Durfee | Transcription profiling of the stringent response in Escherichia coli[END_REF], similar type of analyses showed that relA positively regulates their expression [17]. Our own analyses of the expression of a chromosomal glgC::lacZY gene fusion in both WT cells and ΔrelA cells of the Keio collection indicated that glgCAP expression was largely abolished in cells lacking relA (Figure 7), strongly indicating that the E. coli glgCAP operon belongs to the RelA regulon.

E. coli glycogen accumulation could be subjected also to different levels of posttranscriptional regulation by (p)ppGpp. In addition to its role as a global regulator of gene expression in bacteria, (p)ppGpp potently inhibits PurA [START_REF] Hou | Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthatase[END_REF], which catalyzes the first committed step in de novo biosynthesis of the main GlgC inhibitor, AMP [START_REF] Ballicora | ADP-Glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis[END_REF].

Noteworthy, ΔrelA cells accumulated high levels of AMP when compared to WT cells (Figure 8). It is thus conceivable that reduction of glycogen levels in ΔrelA cells is due, at least in part, to the inhibitory effect exerted by high cellular levels of AMP on GlgC. This view is also consistent with our observations that ΔpurA cells accumulated low levels of AMP (Figure 8) and high levels of glycogen (Figure 4) when compared to WT cells, and with previous reports showing that cells bearing a mutated GlgC form that is insensitive to AMP allosteric regulation displayed glycogen-excess phenotypes [START_REF] Govons | Biosynthesis of bacterial glycogen: kinetic characterization of an altered adenosine diphosphate-glucose synthase from a "glycogen-excess" mutant of Escherichia coli B[END_REF].

The overall data thus indicate that relA control on E. coli glycogen levels may result from an interplay of its (p)ppGpp-mediated positive effect on glgCAP transcription, deviation of surplus ATP flux from nucleic acid and protein biosynthetic pathways to glycogen production, and its negative effect on intracellular levels of the GlgC negative effector AMP.

The general stress response

RpoS is an alternative sigma factor of the RNA polymerase for the general stress response, which has been proposed to be absolutely required for normal glycogen biosynthesis [START_REF] Lange | Identification of a central regulator of stationary-phase gene expression in Escherichia coli[END_REF]. glgA::lacZ and glgC::lacZ fusions analyses have shown that RpoS does not regulate glgCAP transcription in the MC4100 strain [START_REF] Hengge-Aronis | Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli[END_REF], but positively controls the expression of glgS, a gene whose product exerts a positive but still unknown effect on glycogen accumulation [START_REF] Hengge-Aronis | Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli[END_REF]. Our analysis using glgC::lacZY fusions constructed on ΔrpoS mutants of the Keio collection also indicated no significant effect of this mutation on the expression of the glgCAP operon in cells grown in glucose Kornberg medium (Figure 7). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RpoS is regulated in

Amino acid metabolism

Yeast extract (the amino acid source of the Kornberg medium employed in this work) is deficient in various amino acids [START_REF] Reitzer | Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, asparagines, L-alanine, and D-alanine, In: Escherichia coli and Salmonella cellular and molecular biology[END_REF]. Amino acid starvation elicits the stringent response mediated by (p)ppGpp [40]. It is thus highly conceivable that mutations impairing amino acid synthesis and/or provision will lead to glycogen overaccumulation. Consistent with this view, several mutants impaired in the synthesis of tyrosine, phenylalanine, tryptophan and arginine (ΔaroA, ΔaroC, ΔaroF, ΔaroK, ΔaroL, ΔargH, ΔcarA, ΔcarB, and ΔtyrA) or in proline import (ΔproW) accumulated higher levels of glycogen than the WT strain (Figure 4). In addition, several mutants impaired in either the import of sulfur sources (ΔcysA, ΔcysP, ΔcysU and ΔcysW) or enzymatic activities required for cysteine biosynthesis (ΔcysC, ΔcysD, ΔcysE, ΔcysG, ΔcysH, ΔcysI, ΔcysJ, ΔcysM, ΔcysN, ΔserA, ΔserB and ΔserC) also displayed glycogen-excess phenotypes (Figure 4). Because cysteine constitutes the almost exclusive metabolic entrance of reduced sulfur into cell metabolism, it is likely that the glycogen-excess phenotype of mutants impaired in cysteine biosynthesis is the result of the stringent response elicited by both nitrogen (amino acid) and sulfur starvation.

It is important to note that, consistent with the view that the stringent response downregulates the production of AMP (see above), glycogen-excess mutants impaired in amino acid synthesis such as ΔserA and ΔcysI accumulated low AMP levels (Figure 8). It is thus conceivable that, as discussed above, increase of glycogen levels in these mutants may also be ascribed to high GlgC activity occurring as a consequence of the low intracellular concentrations of the GlgC negative effector AMP.

Proteases

Lon and the two-component Clp ATP-dependent proteases play a major role in the degradation of damaged polypeptides and in the recycling of amino acids in response to nutritional downshift, which is a process involving a major portion of the maintenance energy requirement [48]. Therefore, it is expected that impairments in major cell protein degradation machineries will elicit the stringent response that, as discussed above, lead to enhancement of glycogen content. Consistent with this presumption, Δlon, ΔclpP or ΔclpA cells accumulated higher levels of glycogen than WT cells (Figure 4).

Carbon sensing, transport and metabolism

As expected, Δpgm, ΔglgC and ΔglgA cells (all lacking enzymes directly implicated in glycogen synthesis) displayed glycogen-less phenotypes (Figure 5). Because E. coli possesses sources, other than GlgC, of ADPG linked to glycogen biosynthesis [31,[START_REF] Eydallin | An Escherichia coli mutant producing a truncated inactive form of GlgC synthesizes glycogen: further evidences for the occurrence of various important sources of ADPglucose in enterobacteria[END_REF], the total absence of glycogen in ΔglgC cells is ascribed to the polar effect of glgC deletion on the downstream glgA gene. This presumption was confirmed using ΔglgC cells ectopically expressing glgA since, as shown in Supplemental Figure 4, these cells accumulated high glycogen levels. ΔglgB cells accumulated very low levels of glycogen (Figure 5) and, due to their disability to produce branched α-1,6-glucosidic linkages, they stained blue when exposed to iodine vapors (Supplemental Figure 3) [START_REF] Bailey | Physical properties of starch. Relationship between iodine stain and chain length[END_REF]. The very low glycogen content occurring in ΔglgB cells is ascribed to the fact that (a) GlgB is likely required for the initiation of the glycogen granule [50] and/or (b) formation of α-1,6-glucosidic may be absolutely required for maximal glycogen synthesis. ΔglgX and ΔglgP cells showed a marked increase of glycogen levels, which is in agreement with the role of GlgX and GlgP in glycogen breakdown [START_REF] Alonso-Casajús | Glycogen phosphorylase, the product of the glgP gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli[END_REF]51]. ΔptsI, ΔptsH and ΔptsG cells displayed glycogen-deficient phenotypes (Figure 5), which is consistent with the overriding importance of the phosphoenolpyruvate dependent phosphotransferase system (PTS) in the sensing and uptake of extracellular glucose, and in reflecting the energy status of the cell [START_REF] Kotrba | Bacterial phosphotransferase system (PTS) in carbohydrate uptake and control of metabolism[END_REF].

Both cAMP produced by the membrane-bound adenylate cyclase (the product of cya), and CRP are required for expression of glgS and PTS-related genes required for nomal glycogen production [START_REF] Hengge-Aronis | Identification and molecular analysis of glgS, a novel growth-phase-regulated and rpoS-dependent gene involved in glycogen synthesis in Escherichia coli[END_REF]53]. Consistent with previous reports showing that cya and crp are important regulators of glycogen metabolism [START_REF] Leckie | Regulation of bacterial glycogen synthesis. Stimulation of glycogen synthesis by endogenous and exogenous cyclic adenosine 3':5'-monophosphate in Escherichia coli and the requirement for a functional CRP gene[END_REF], Δcya and Δcrp cells of the Keio collection displayed marked glycogen-deficient phenotypes (Figure 5).

Therefore, the glycogen-deficient phenotype of Δcya and Δcrp cells could be ascribed, at least in part, to downregulation of glgS and/or PTS-related genes. As to the possible regulation of glg genes by cya and crp, both in vitro and in vivo experiments have shown that cAMP/CRP positively regulates glgC and glgA expression in E. coli [START_REF] Romeo | Genetic regulation of glycogen biosynthesis in Escherichia coli: In vitro effects of cyclic AMP and guanosine 5´-diphosphate 3´diphosphate and analysis of in vivo transcripts[END_REF][START_REF] Romeo | Genetic regulation of glycogen biosynthesis in Escherichia coli: in vivo effects of the catabolite repression and stringent response systems in glg gene expression[END_REF]20]. However, recent transcriptome analyses failed to indicate that glg genes belong to the cAMP regulon [53]. Our own analysis using a chromosomal glgC::lacZY fusion constructed on the Δcya mutant of the Keio collection (Figure 7) revealed that expression of glgCAP operon was not affected in this mutant, reinforcing the idea that E. coli glycogen metabolism is not regulated at the level of glgCAP expression by cAMP. We must emphasize that, by still unidentified mechanisms and reasons, Δcya and Δcrp cells accumulated high levels of AMP when compared to WT cells (Figure 8). The overall data thus indicate that control of E. coli glycogen metabolism by cAMP is the result of an interplay between the positive effect of this cyclic nucleotide on expression of glgS and PTS-related genes, and its negative effect on the intracellular levels of the GlgC negative effector AMP.

Δpgi cells lack phosphoglucose isomerase, the enzyme that catalyzes the conversion of glucose-6-phosphate (G6P) into fructose-6-phosphate. As shown in Figure 5 Δpgi cells accumulated low glycogen levels. In glucose-containing media Δpgi cells accumulate high levels of G6P [55]. This hexose-P strongly inhibits the glucose transporter and destabilizes ptsG mRNA [55,[START_REF] Dietzler | Evidence for the coordinate control of glycogen synthesis, glucose utilization, and glycolysis in Escherichia coli[END_REF]. Therefore, the glycogen-deficient phenotype of Δpgi cells may be ascribed to impairments in the incorporation of glucose linked to glycogen biosynthesis as a consequence of the high intracellular G6P levels.

ΔybhE and Δzwf cells lack the two enzymes of the pentose phosphate pathway 6phosphogluconololactonase and G6P dehydrogenase, respectively. Metabolic intermediates of this pathway are necessary for the production of aromatic amino acids. It is therefore conceivable that, as discussed above, the glycogen-excess phenotype displayed by these mutants (Figure 4) is the consequence of the stringent response triggered by the lack of internal amino-acid supply.

Iron metabolism

Iron is needed as a cofactor for a large number of enzymes. In aerobic conditions E. coli utilizes high-affinity extracellular siderophores that solubilize and capture Fe(III) prior to transport and metabolism [START_REF] Mchugh | Global iron-dependent gene regulation in Escherichia coli[END_REF]. Fur, a dominant sensor of iron availability, generally represses iron siderophore biosynthetic and transport genes such as fepB, fepD and fepG [START_REF] Mchugh | Global iron-dependent gene regulation in Escherichia coli[END_REF]. Under iron limiting conditions, iron dissociates from Fur, and increased transcription of genes ensues. Iron limitation causes the stringent response due to a SpoT-dependent increase of the ppGpp pool [START_REF] Vinella | Iron limitation induces SpoT-dependent accumulation of ppGpp in Escherichia coli[END_REF]. It is therefore expected that impairment in iron supply will lead to a stringent response induced glycogen overaccumulation (see above). Consistent with this presumption, ΔfepB, ΔfepD and ΔfepG cells defective in the transport of ferric-siderophore displayed glycogen-excess phenotypes (Figure 4). By contrast, Δfur cells accumulated low levels of glycogen when compared with WT cells (Figure 5). In addition to its role as sensor of iron availability, Fur can be considered to be a global regulator [START_REF] Mchugh | Global iron-dependent gene regulation in Escherichia coli[END_REF]. To investigate the possible Fur-dependent transcriptional regulation of glycogen metabolism we analyzed glgCAP expression in Δfur cells of the Keio collection by means of a chromosomal glgC::lacZY fusion. As shown in Figure 7, glgC::lacZY derived β-galactosidase activity was not substantially modified in the Δfur mutants as compared to WT cells, indicating that E. coli glg genes are not members of the Fur regulon.

Energy production and cellular redox status

ATP is a primary signal in regulating glycogen biosynthesis, and acts as substrate for the ADPG-producing reaction catalyzed by GlgC [START_REF] Dietzler | Evidence for the coordinate control of glycogen synthesis, glucose utilization, and glycolysis in Escherichia coli[END_REF]. It is therefore conceivable that mutations in components required for the proper functioning of the aerobic electron transport chain and ATP generation will negatively affect glycogen accumulation.

Consistent with this view, ΔubiG and ΔubiH cells (deficient in ubiquinone production) and ΔiscU, ΔiscS, Δfdx and ΔhscB cells impaired in the machinery for the assembly/maintenance of Fe-S clusters (components required for the proper functioning of the aerobic electron transport chain and ATP generation [START_REF] Johnson | Structure, function and formation of biological iron-sulfur clusters[END_REF]) showed reduced glycogen levels (Figure 5).

Glutathione is a major determinant of the redox status of the cell, playing a prime role in maintaining the correct assembly of electron transport chain components [60]. ΔgshB and Δgor cells (lacking the machinery necessary to produce and reduce glutathione, respectively) displayed a glycogen-deficient phenotype (Figure 5), indicating that some components involved in glycogen metabolism are strongly affected by the cellular redox status.

Envelope composition and integrity

RpoE is an essential transcription initiation factor that governs the response to envelope stress and the expression of genes that are needed to heal envelope damage. The major point of regulation of RpoE is at the level of its interaction with the anti-sigma RseA factor [START_REF] Alba | Regulation of the Escherichia coli sigma-E dependent envelope stress response[END_REF]. When E. coli is subjected to extra-cytoplasmic stresses, RseA degrades and RpoE activity is induced. Another major point of regulation of RpoE takes place at the post-transcriptional level, since it has been shown that Hfq interaction with rseA mRNA downregulates rseA expression [START_REF] Ding | Hfq is essential for Vibrio cholerae virulence and downregulates sigma-E expression[END_REF]. Remarkably both ΔrseA and Δhfq cells accumulated low glycogen levels (Figure 5), suggesting that RpoE-mediated envelope stress response may to some extent negatively affect glycogen accumulation.

Mutants of genes coding for proteins involved in the maintenance of the cell envelope integrity such as rfaE, galU, tolB, tolR, tolQ, pal, and ponB, displayed glycogen-deficient phenotypes (Figure 5). All these mutants are likely to promote both envelope stress and deformation of membrane that causes inhibition of the electron transport chain, energy production and formation of membrane potential necessary for nutrient import [START_REF] Houssin | Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli[END_REF][START_REF] Andersen | Bilayer thickness and membrane protein function: an energetic perspective[END_REF]. The ΔrfaE mutant for instance lacks an enzyme required for E. coli lipopolysaccharide biosynthesis [START_REF] Valvano | The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-Dmanno-heptose[END_REF]. ΔgalU lacks the enzyme that catalyses the synthesis of UDP-glucose necessary for the synthesis of cell envelope components [START_REF] Genevaux | Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion[END_REF].

Osmotic stress

Osmotic upshock results in a large decrease in the cytoplasmic volume and deformation of membrane that causes inhibition of electron transport system, respiration, sugar uptake and glycogen accumulation [START_REF] Houssin | Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli[END_REF][START_REF] Roth | Restoration of cell volume and the reversal of carbohydrate transport and growth inhibition of osmotically upshocked Escherichia coli[END_REF][START_REF] Seibold | The glgX gene product of Corynebacterium glutamicum is required for glycogen degradation and for fast adaptation to hyperosmotic stress[END_REF]. In addition, osmotic stress induces the expression of RpoE [START_REF] Bianchi | Hyperosmotic shock induces the sigma 32 and sigma E stress regulons of Escherichia coli[END_REF]. Consistent with the view that the energy status of the cell and glycogen metabolism are strongly determined by the cell osmotic conditions, ΔkdpB cells impaired in the high affinity potassium influx system that maintains the turgor pressure across the inner membrane [72] displayed a glycogen-deficient phenotype (Figure 5).

Nucleotide metabolism

AMP is the natural negative regulator of GlgC [START_REF] Govons | Biosynthesis of bacterial glycogen: kinetic characterization of an altered adenosine diphosphate-glucose synthase from a "glycogen-excess" mutant of Escherichia coli B[END_REF]. It is thus expected that mutants impaired in the synthesis of this purine nucleotide will display AMP-deficient and glycogen-excess phenotypes. Consistent with this presumption, mutants of genes that code for enzymes involved in the de novo synthesis of AMP such as ΔpurA, ΔpurC, ΔpurD, ΔpurE, ΔpurF, ΔpurH, ΔpurL, and ΔpurM displayed glycogen-excess phenotypes and accumulated low AMP levels when compared to WT cells (Figure 4 and Figure 8, respectively). Furthermore, cells ectopically expressing purB displayed AMP-excess and glycogen-deficient phenotypes (Figure 9).

End-turnover of tRNA

End-turnover of tRNA consists of the removal and readdition of the 3´terminal AMP residues to uncharged tRNA. RNase T (the product of rnt) is a nuclease highly specific for uncharged tRNA-C-C-A that releases AMP and tRNA-C-C, and that highly controls tRNA turnover in E. coli [START_REF] Deutscher | Rnase T is responsible for the end-turnover of tRNA in Escherichia coli[END_REF]. Cells impaired in tRNA turnover accumulate defective tRNA molecules, leading to arrest of protein biosynthesis as a consequence of elevation of ppGpp levels [START_REF] Deutscher | relA overcomes the slow growth of cca mutants[END_REF]. In agreement with the idea that the stringent response plays a pivotal role in glycogen metabolism, Δrnt mutants displayed a glycogen excess phenotype (Figure 4).

Additional remarks

Results presented in this work show that glycogen metabolism is a highly regulated process that is connected with a wide variety of cellular functions. In addition, we show that extracellular Mg 2+ is an important determinant of both energy status of the cell and glycogen accumulation. Noteworthy, analyses of the intracellular AMP levels in mutants representing all categories of both glycogen-excess and glycogen-deficient mutants revealed a trend-wise reverse correlation between AMP and glycogen contents (Figure 8). No such correlation could be found between ADP and glycogen content (Supplemental Figure 5). This observation is consistent with the view discussed above that AMP plays a major role in regulating glycogen metabolism in E. coli. To further explore this hypothesis we carried out a time-course analysis of AMP and glycogen contents in cells cultured in liquid glucose Kornberg medium. As shown in Figure 10, these analyses clearly revealed a reverse correlation between intracellular glycogen and AMP contents, the overall data further supporting the idea that AMP acts a major determinant of glycogen metabolism in E. coli.

Figure 11 illustrates a suggested model of glycogen metabolism wherein major determinants of glycogen accumulation include levels of intracellular Mg-bound ATP necessary for GlgC activity (strongly determined by extracellular Mg 2+ concentrations, transport of Mg 2+ across membranes, ATP synthesis and consumption, and possibly Adk activity), levels of AMP (the main GlgC inhibitor), levels of ppGpp (which accumulates under conditions of limited provision of nutrients such as amino acids, sulfur, Mg 2+ , iron, etc.), levels of cAMP, availability of a carbon source, redox status of the cell, and less well-defined systems sensing the cell energy status through the activity of the electron transport chain. According to this model, under conditions of limited nutrient provision, a decreased demand in ATP-dependent protein and nucleic acid synthesis will take place, and excess ATP will be diverted towards glycogen biosynthesis when an excess carbon source is present in the medium. Adk activity represents an important tool for maintaining Mg 2+ homeostasis and generating the membrane potential that drives nutrient transport and ATP synthesis. It allows amplification of extracellular Mg 2+ -dependent small changes in ATP and ADP concentrations into proportionally larger changes in AMP concentrations. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Figure 11: Integrated model of glycogen metabolism. Suggested metabolic model wherein major determinants of glycogen metabolism include the PhoP-PhoQ regulatory system activated under low extracellular Mg 2+ concentration conditions, intracellular concentration of Mg-bound ATP necessary for GlgC activity (determined by extracellular Mg 2+ concentration, transport of Mg 2+ across membranes, ATP synthesis and consumption, and Adk activity), levels of AMP (the main GlgC inhibitor), levels of ppGpp (which accumulates under conditions of limited provision of nutrients such as amino acids, sulfur, iron, etc.), levels of cAMP, availability of a carbon source, redox status of the cell, and less well-defined systems sensing the cell energy status through the activity of the electron transport chain. According to this model, under conditions of limited nutrient provision, a decreased demand in ATP-dependent protein and nucleic acid synthesis will take place, and excess ATP will be used for glycogen biosynthesis when glucose is present in the medium. [START_REF] Baba | Construction of Escherichia coli K-12 in frame, single-gene knockout mutants: the Keio collection[END_REF][START_REF] Keseler | EcoCyc: a comprehensive database resource for Escherichia coli[END_REF][START_REF] Misra | EchoBASE: an integrated post-genomic database for Escherichia coli[END_REF]. The numbers in parentheses represent the number of glycogen-related genes to the number of genes belonging to each of COG category.

Legends to figures

Table 2. E. coli genes whose deletions caused a "glycogen-deficient" or "glycogenless" phenotype. Genes are classified into COG categories [START_REF] Baba | Construction of Escherichia coli K-12 in frame, single-gene knockout mutants: the Keio collection[END_REF][START_REF] Keseler | EcoCyc: a comprehensive database resource for Escherichia coli[END_REF][START_REF] Misra | EchoBASE: an integrated post-genomic database for Escherichia coli[END_REF]. The numbers in parentheses represent the number of glycogen-related genes to the number of genes belonging to each of COG category. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980

  Biochemical Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  E.coli in response to several different signals. At the transcriptional level, (p)ppGpp positively affects rpoS transcript elongation and/or stability [44]. At the post-transcriptional level full rpoS expression requires hfq function [45], DksA [40], and DnaK [46]. Consistent with the view that RpoS exerts a positive effect on glycogen accumulation, ΔrpoS cells of the Keio collection accumulated low In addition, ΔdksA, Δhfq, ΔrelA, ΔdnaK and ΔglgS cells displayed glycogen-deficient phenotypes (Figure 5). Biochemical Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Moreover, ΔtolB, ΔtolR, ΔtolQ and Δpal cells are impaired in proteins of the Tol-Pal system essential in maintaining envelope integrity [67]. In addition, ΔponB (ΔmrcB) cells lack a bi-functional membrane-bound enzyme catalyzing transglycosylation and transpeptidation reactions, which are essential in the late stages of peptidoglycan biosynthesis [68]. Biochemical Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980

Figure 1 :

 1 Figure 1: Extracellular Mg 2+ in the submillimolar range is an important determinant of intracellular glycogen content in ΔmgtA, ΔphoP and ΔphoQ cells. (A) Glycogen content at the onset of the stationary phase in ΔmgtA, ΔphoP, ΔphoQ and WT cells cultured in glucose Kornberg liquid medium with the indicated Mg 2+ concentrations. (B) Iodine staining of WT and ΔmgtA, ΔphoP, ΔphoQ cells cultured in glucose Kornberg solid medium containing the indicated Mg 2+ concentrations.

Figure 2 :

 2 Figure 2: Extracellular Mg 2+ is an important determinant of E. coli intracellular ATP levels. ATP contents were determined at the onset of the stationary phase in WT cells cultured in glucose Kornberg liquid medium containing the indicated Mg 2+ concentrations. The mean ± SE of 4 independent experiments are shown. For details see Materials and Methods.

Figure 3 :

 3 Figure 3: glgCAP is under positive control of the PhoP-PhoQ regulatory system in E. coli under limiting Mg 2+ conditions. Comparative analysis of growth (A,C) and expression of chromosomal glgC::lacZY fusions (B,D) between WT cells ( ), ΔphoP ( ), ΔphoQ ( ) and ΔmgtA ( ) cells cultured in glucose Kornberg liquid medium either under limiting (A,B) and non-limiting (C,D) Mg 2+ conditions (50 μM and 1 mM Mg 2+ , respectively). (E) Western blot analysis of GlgC in WT cells (lanes 1 and 2), ΔphoP mutants (lanes 3 and 4) ΔphoQ mutants (lanes 5 and 6) and phoQ expressing ΔphoQ cells (lane 7) cultured in glucose Kornberg liquid medium under limiting (lanes 1, 3, 5 and 7) and non-limiting (lanes 2, 4 and 6) Mg 2+ conditions (50 μM and 1 mM Mg 2+ , respectively). The lower panel shows the Coomassie blue stained gel in the polypeptide region 40-55 kDa corresponding to GlgC migration. The gel was loaded with 30 μg per lane of total soluble proteins of each of the described cells. For details see Materials and Methods.

Figure 4 :

 4 Figure 4: Intracellular glycogen content of glycogen-excess mutants of the Keio collection. Glycogen content is referred as percentage of glycogen accumulated by WT cells. Average glycogen content in WT cells was 140 nmol glucose/mg protein. Cells were cultured in liquid glucose Kornberg medium containing 250 μM Mg 2+ and harvested at the onset of the stationary phase.

Figure 5 :

 5 Figure 5: Intracellular glycogen content of both glycogen-less and glycogendeficient mutants of the Keio collection. Glycogen content is referred as percentage of glycogen accumulated by WT cells. Average glycogen content in WT cells was 140 nmol glucose/mg protein. Cells were cultured in liquid glucose Kornberg medium containing 250 μM Mg 2+ and harvested at the onset of the stationary phase.

Figure 6 :

 6 Figure 6: Functional categorization of the genes whose expression affects glycogen content in E. coli. Genes whose expression leads to glycogen-excess phenotype are in black bars, whereas genes whose expression leads to glycogen-deficient phenotype are in white bars.

Figure 7 :

 7 Figure 7: The E. coli glgCAP operon is under control of RelA. Comparative analysis of (A) growth and (B) expression of glgC::lacZY fusions in WT cells ( ), Δcya ( ),

Figure 8 :

 8 Figure 8: AMP content in mutants representing the different glycogen-excess and glycogen-deficient categories of the Keio collection. Average AMP content in WT cells was 0.95 nmol AMP/mg protein. Cells were cultured in glucose Kornberg medium containing 250 μM Mg 2+ and harvested when entering the stationary phase. White bars correspond to glycogen-less or glycogen-deficient mutants, whereas black bars correspond to glycogen-excess mutants.

Figure 9 :

 9 Figure 9: Intracellular AMP is a major determinant of glycogen accumulation. (A) Iodine staining, (B) glycogen content and (C) AMP content in WT and purB overexpressing cells cultured in solid glucose Kornberg medium containing 250 μM Mg 2+ . The results are the mean ± SE of 4 independent experiments.

Figure 10 :

 10 Figure 10: Time-course analyses of AMP and glycogen content in E. coli. Timecourse analyses of (A) culture growth, (B) AMP content and (C) glycogen content in WT cells cultured in liquid cultures of glucose Kornberg medium containing 250 μM Mg 2+ .

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Biochemical Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

4Figure 3 E

 3 Figure 2

Biochemical 6 Biochemical

 6 Figure 6

Figure 7

 7 Figure7

Figure 8 Biochemical

 8 Figure8

Biochemical

  Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 10 ACFigure 11 Biochemical

 1011 Figure10

Table 1 . E. coli genes whose deletions caused a "glycogen-excess" phenotype.

 1 Genes are classified into COG categories

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

This research was partially supported by the grant BIO2007-63915 from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) and by Iden Biotechnology S. L.. M. M. acknowledges a post-doctoral contract from I3P program of Consejo Superior de Investigaciones Científicas. A. M. V. expresses his gratitude to the Ministerio de Educación y Cultura, the Consejo Superior de Investigaciones Científicas and the Public University of Nafarroa for their support. G. E. acknowledges a pre-doctoral fellowship from the Public University of Nafarroa. We thank Cristina Cirauqui (Institute of Agrobiotechnology) María Angeles Barado and Jessica Díaz de Cerio (Iden Biotechnology) for technical assistance and support. Biochemical Journal Immediate Publication. Published on 25 Aug 2009 as manuscript BJ20090980 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090980

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.