regulation of glycogen metabolism in bacteria

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REGULATION OF GLYCOGEN METABOLISM IN BACTERIA k.Kiran kumar I.D-5011 CHAIRMAN- DR.Annapurna DIV. OF MICROBIOLOGY

INTRODUCTION:

INTRODUCTION Microorganisms have the capacity to utilise a variety of nutrients and adapt to continuously changing environments Mainly , bacteria accumulate carbon and energy reserves to cope up with the starvation conditions temporarily present in the environment. Glycogen is one of the major intracellular reserve polymer in bacteria.

Cont..:

Cont.. Glycogen biosynthesis is the main strategy for such metabolic storage. A variety of sensing and signalling mechanisms have evolved to ensure the production of this homo polysaccharide. Regulation of glycogen metabolism in bacteria occurs at both transcriptional and translational level.

what is glycogen ?:

what is glycogen ? It is a polyglucan consisting of α -1,4- linked glucose subunits with α -1,6- linked glucose at the branching points. In bacteria, the average length of chain is 8-12 glucose units , and the molecular size of glycogen is estimated to be about 10 7 -10 8 Da.

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Glycogen is a polymer of glucose residues linked by a (1  4) glycosidic bonds, mainly a (1  6) glycosidic bonds, at branch points. Glycogen chains & branches are longer than shown.

CONT…:

CONT… In E.coli glycogen accumulates under conditions of limited growth & when excess carbon is present .( lillie 1980) But in streptococcus mitis & Rhodopsuedomonas capsulata glycogen accumulates to optimal level during exponential phase.

Why only glycogen is stored ? Why not others?:

Why only glycogen is stored ? Why not others? Glycogen as a macromolecule & has a little effect on the internal osmotic pressure of the cell. Here osmotic pressure depends on the colligative properties .

Bacterial glycogen metabolism:

Bacterial glycogen metabolism Bacteria requires glycogen for environmental survival, symbiotic performance , colonization ,maintenance in non-growing conditions & virulence & biofilm formation . Bacterial glycogen metabolism is also influenced by allosteric properties & stucture -function relationships of enzymes.( preiss 2009)

Allosteric regulation of glycogen biosynthesis :

Allosteric regulation of glycogen biosynthesis Activation of glycogen synthesis occurs when high amounts of fructose 1,6-diphosphate is in cell Inhibition of glycogen synthesis occurs when high amounts of AMP is in cell or by 2,4-DNP

Physiology of the biofilm formation process:

Physiology of the biofilm formation process The levels of both cytoplasmic glycogen and biofilm rose correlatively when S. enteritidis was pre-incubated in media containing increasing levels of glucose concentration

Glycogen synthesis-biofilm formation:

Glycogen synthesis- biofilm formation Biofilm formation by S. enteritidis SE3934 in: (A) exponentia l phase; (B – D) stationary phase

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TEM of the glycogen granules in S. enteritidis SE3934 cells in the exponential phase TEM of the glycogen granules in stationary phase after being incubated in TSB+2% glucose

Glycogen vs glucose concentration:

Glycogen vs glucose concentration The amount of glycogen produced was increased with increasing glucose concentration up to 2% Streptococcus mutans >2% glucose led to decreasing glycogen levels due to the saturation of the uptake or accumulating systems

Glycogen synthesis vs virulence:

Glycogen synthesis vs virulence The virulence of the biofilm -producing strain in infected chickens increased proportionally to the amount of stored glycogen , suggesting a possible role of the glycogen deposit in the virulence of S. enteritidis

The regulation of bacterial glycogen metabolism:

The regulation of bacterial glycogen metabolism

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Glucose Glc-6-p ADPG GLYCOGEN Glg C Asp P Glg A Glc-1-P GlgP , GlgX PGM Glg B PTS Schematic Model Of Glycogen Metabolism In E.Coli

Enzymology and spatial aspects of the process:

Enzymology and spatial aspects of the process Exception is Prevotella bryantii bacterium that lacks GlgC , andwhose GlgA exclusively recognizes UDPG as a glucosyldonor . Unlike yeast and mammalian cells where glycogenin participates in the initiation of glycogen synthesis, no glycogenin analog has been described in bacteria.

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Schematic representation of the pathways of glycogen synthesis and degradation in yeast

Cont..:

Cont.. Bac . GlgA can not only elongate a(1,4)-linked glucans , but can also form the primer required for the elongation process by catalyzing its own glucosylation ( ugalde ) Genetic evidence says that GlgC is the sole enzyme catalyzing the production of ADPG as observed the E. coliAC70R1-504 strain.

Cont..:

Cont.. But,recently there is identification of a trehalose glucosyltransferase ( TreT ) that catalyzes the reversible conversion of trehalose and ADP into ADPG and glucose in the archeon Thermococcus litoralis is noteworthy ( Qu et al., 2004).

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FIG: 5,6,7 are the glgX mutants, 8,9,10 are WT STRAINS

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GlgP catalyzes E. coli glycogen breakdown in vivo. (A) Iodine staining of (1) WT, (2)D glgP , and (3) D glgCAP cells.

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Glycogen accumulation in1) GlgP -overproducing cells and2) glgP and3) glgCAP E. coli mutants

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(B) Time course analyses of the glycogen content in WT ( ) glgP (a)and glgCAP ( )cells

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(A) Iodine staining of WT and glg CAP mutants (Sections 1 and 2, respectively) cultured in solid Kornberg medium supplemented with 50 mM glucose.

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(B) Glycogen content in WT and glg CAP cells E.coli salmonella

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(C) ADPG contents in WT and glgCAP cells. E.COLI Salmonella

Allosteric regulation of enzymes :

Allosteric regulation of enzymes In the case of E. coli, fructose-1,6-bisphosphate activates GlgC , whereas AMP acts as an important inhibitor. E. coli cells bearing a mutated GlgC form that is insensitive to AMP allosteric modulation and purA cells impaired in the first committed step of AMP biosynthesis both display glycogen-excess phenotypes

Cont..:

Cont.. A strong and highly specific interaction between E. coli GlgP and the PTS component HPr was found. GlgP binding to HPr is maximal when HPr is totally phosphorylated . AspP activity is positively affected by both glucose-1,6-bisphosphate and nucleotide–sugars and also by macromolecular crowding

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Location of glycogen granules and enzymes

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Glycogen granules and several enzymes involved in bacterial glycogen metabolism are localized in the cell periphery In,microcompartments , wherein the enzymes are physically associated in complexes ( metabolons ) to facilitate metabolite channeling .

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(b) Green fluorescent protein (GFP fluorescence of cells expressing the indicated glycogen-related enzymes fused with GFP.

Organization of structural glycogen genes:

Organization of structural glycogen genes In bacterial gene expression is that genes of related functions are often clustered in a single operon In E. coli and Salmonella glycogen metabolism are clustered in two tandemly arranged operons : glgBX and glgCAP

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6 Schematic representation of the known bacterial glg operons .

Regulation of the expression of glycogen genes:

Regulation of the expression of glycogen genes several factors have been described to control bacterial glycogen accumulation. In E. coli, this includes negative regulation by by the carbon storage regulator CsrA positive regulation by RpoS , the PhoP / PhoQ regulatory system, the stringent response by the cAMP /CRP complex & GlgS gene

CONT..:

CONT.. RpoS is an alternative sigma factor of the RNA polymerase for the general stress response. In nutrient starvation, E. coli elicits the ‘ stringent response’ that switches the cell from growth related mode to maintenance mode The pleiotropic physiological response by alarmones pppGpp and ppGpp .

Secondary messengers:

Secondary messengers cAMP , (p) ppGpp and c- di -GMP nucleotide-based second messengers c- di -GMP is an alarmone used bacteria to orchestrate the switch between free-living and sedentary life styles.

PhoP–PhoQ regulatory system:

PhoP–PhoQ regulatory system PhoP–PhoQ is a two-component system that monitors the availability of extracellular Mg 2+ . Mg 2+. is a stabilizing factor for membranes that strongly determines cell metabolic and energetic status phoP - phoQ mutants display glycogen-deficient phenotypes when cultured under conditions of limiting concentration Mg 2+ .

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Extracellular Mg 2+ in the submillimolar range is an important determinant of intracellular glycogen content in mgtA , phoP and phoQ cells.

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Extracellular Mg2+ is an important determinant of E. Coli intracellular ATP levels

CRP regulatory proteins:

CRP regulatory proteins cAMP complexed to CRP (1) is required for normal glycogen accumulation (2) CAMP/CRP is required for the expression of glgS and PTS -related genes which in turn are required for normal glycogen production

Glycogen metabolism is highly interconnected with a wide variety of cellular processes:

Glycogen metabolism is highly interconnected with a wide variety of cellular processes (1) stringent response . (2) general stress response. (3) low extracellular Mg 2+ availability. (4) carbon sensing, transport and metabolism. (5)factors determining intercellular communication, aggregative and social behavior . (6) sulfur metabolism

Cont…:

Cont… (7) nitrogen metabolism. (8 ) iron metabolism . (9) end turnover of tRNA . (10)envelope composition and integrity, (11)energy production and cellular redox status . (12) small RNAs ( sRNAs )-binding proteins. (13) nucleotide metabolism . (14) osmotic stress.

Iron metabolism:

Iron metabolism fur mutants display a glycogen- deficient phenotype fepB , fepD and fepG mutants display glycogen- excess phenotypes Iron limitation causes the SpoT -dependent stringent response

End-turnover of tRNA:

End-turnover of tRNA rnt mutants impaired in tRNA turnover accumulate defective tRNA molecules and high levels of ppGpp thus resulting in increased glycogen content.

Envelope composition and integrity:

Envelope composition and integrity cells impaired in RseA and Hfq functions accumulate low glycogen levels, suggesting that RpoE mediated envelope stress response Mutants of genes such as rfaE , galU , tolB , tolR display glycogen-deficient phenotypes.

Energy production and redox status:

Energy production and redox status ATP is a primary signal in regulating glycogen biosynthesis, and acts as substrate for the ADPG -producing reaction catalyzed by GlgC mutations in ubiG and ubiH genes negatively affect glycogen accumulation

sRNAs-binding proteins:

sRNAs -binding proteins In prokaryotes, many sRNAs are specifically expressed during adaptation to nutritional stress The majority of these sRNAs require binding proteins for in gene regulation. (1) Hfq (2) CsrA

global regulator CsrA:

global regulator CsrA CsrA ( pleiotropic gene) prevents glycogen accumulation by both promoting glgCAP decay It affects the expression of glgS and hfqCsrA negatively csrA and csrD overexpressing E. coli cells display a glycogen-deficient phenotype

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Model of CsrA -mediated regulation of E. coli glgC .

Hfq:

Hfq Hfq is a chaperone that facilitates the base pairing between sRNAs and their target mRNAs Deletion of hfq has pleiotropic phenotypes. Mutants impaired in the hfq function glycogen-deficient phenotype.

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Glycogen content vs keio collections

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Glycogen content (referred as percentage of glycogen accumulated by WT cells) of both glycogen-deficient and glycogen-less mutants of the Keio collection. Average glycogen content in WT cells was equivalent to 147 nmol glucose/mg protein

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Glycogen content (referred as percentage of glycogen accumulated by WT cells) of glycogen-excess mutants of the Keio collection. Average glycogen content in WT cells was equivalent to 147 nmol glucose/mg protein.

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GLU Suggested integrated model of glycogen metabolism

Reasearch needs:

Reasearch needs Involvement of sRNAs in the regulation of glycogen metabolism in not completely understood The identification and characterization of sources, other than GlgC , of ADPG biosynthesis The precise molecular function of GlgS is still poorly resolved

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THANK U