Purification of Mlc and Analysis of Its Effects on the pts Expression in Escherichia coli

Kim, Soon Young; Nam, Tae‐Wook; Shin, Dongwoo; Koo, Bon Chul; Seok, Yeong‐Jae; Ryu, Sangryeol

doi:10.1074/jbc.274.36.25398

Cited by 55 publications

(70 citation statements)

References 27 publications

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“…CRP concentrations seem to be less important during such dynamic processes and might be more important for long term adaptation to different growth substrates. It has been reported that the pts operon is regulated by cAMP⅐CRP as well as by Mlc (30,31). This regulation is weak (factor of 2-3) (32) and hence has often been neglected.…”

Section: Discussionmentioning

confidence: 99%

A Quantitative Approach to Catabolite Repression in Escherichia coli

Bettenbrock

Fischer

Kremling

et al. 2006

Journal of Biological Chemistry

133

156

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A dynamic mathematical model was developed to describe the uptake of various carbohydrates (glucose, lactose, glycerol, sucrose, and galactose) in Escherichia coli. For validation a number of isogenic strains with defined mutations were used. By considering metabolic reactions as well as signal transduction processes influencing the relevant pathways, we were able to describe quantitatively the phenomenon of catabolite repression in E. coli. We verified model predictions by measuring time courses of several extraand intracellular components such as glycolytic intermediates, EII-A Crr phosphorylation level, both LacZ and PtsG concentrations, and total cAMP concentrations under various growth conditions. The entire data base consists of 18 experiments performed with nine different strains. The model describes the expression of 17 key enzymes, 38 enzymatic reactions, and the dynamic behavior of more than 50 metabolites. The different phenomena affecting the phosphorylation level of EIIA Crr , the key regulation molecule for inducer exclusion and catabolite repression in enteric bacteria, can now be explained quantitatively.Catabolite repression in Escherichia coli designates the observation that if different carbohydrates are present in a medium under unlimited conditions, one of them is usually taken up preferentially. Although the fundamental biochemical principles of the regulatory network have been revealed, a quantitative description of this growth behavior is still missing. The center of the regulatory network is formed by the phosphoenolpyruvate (PEP) 4 :carbohydrate phosphotransferase systems (PTS). These systems are involved in both transport and phosphorylation of a large number of carbohydrates, in movement toward these carbon sources (chemotaxis), and in regulation of a number of metabolic pathways (1-3). The PTS in E. coli consist of two common cytoplasmatic proteins, EI (enzyme I) and HPr (histidine-containing protein), as well as an array of carbohydrate-specific EII (enzyme II) complexes. Because all components of the PTS, depending on their phosphorylation status, can interact with various key regulator proteins, the output of the PTS is represented by the degree of phosphorylation of the proteins involved in phosphoryl group transfer, e.g. unphosphorylated EIIA Crr inhibits the uptake of other non-PTS carbohydrates by a process called inducer exclusion. Phosphorylated EIIA Crr activates the adenylate cyclase (CyaA) and leads to an increase in the intracellular cAMP level.Understanding the regulation of carbohydrate uptake requires a quantitative description of the PTS. In this context it is important that the degree of phosphorylation of EIIA Crr is proportional to the PEP/ pyruvate ratio, when no carbohydrates are transported (4) and the respective equilibrium constant is an upper boundary when the PTS is active (5). The PTS should therefore not be regarded as a measure for the transport of PTS substrates but more as a general measure for carbohydrate availability. One feature of our contribution...

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Section: Discussionmentioning

confidence: 99%

A Quantitative Approach to Catabolite Repression in Escherichia coli

Bettenbrock

Fischer

Kremling

et al. 2006

Journal of Biological Chemistry

133

156

View full text Add to dashboard Cite

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“…Cloning is described in SI Materials and Methods. Expression and purification of EIIB proteins and wild-type and mutant Mlc proteins with a single amino acid change were performed as described previously (7,12). Whereas wild-type and mutant Mlc proteins with a single amino acid change were insoluble in 10 mM Tris⅐HCl buffer (pH 7.5) containing 1 mM DTT, Mlc harboring two point mutations (Arg306Gly and Leu310Gly) at O-domain was soluble at neutral pH.…”

Section: Methodsmentioning

confidence: 99%

“…Glucose, a representative PTS sugar, mediates transcriptional activation of several genes for PTS-related sugar transporters and some enzymes involved in glycolysis (1, 6). Mlc, a tetrameric protein (7), is a signaling mediator for glucose induction of several PTS operons and related genes (8)(9)(10)(11)(12)(13)(14).A unique feature of induction of the Mlc regulon by glucose is that the effector molecule modulating Mlc activity is a membranebound protein, EIICB Glc (7,15,16), in which cytosolic EIIB protein is attached to membrane-embedded EIIC protein through a linker of Ͼ20 aa. In the absence of glucose, EIICB Glc mainly exists in the phosphorylated form (pEIICB Glc ).…”

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Analyses of Mlc–IIB ^Glc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration

Nam

Jung

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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In Escherichia coli, glucose-dependent transcriptional induction of genes encoding a variety of sugar-metabolizing enzymes and transport systems is mediated by the phosphorylation state-dependent interaction of membrane-bound enzyme IICB Glc (EIICB Glc ) with the global repressor Mlc. Here we report the crystal structure of a tetrameric Mlc in a complex with four molecules of enzyme IIB Glc (EIIB), the cytoplasmic domain of EIICB Glc . Each monomer of Mlc has one bound EIIB molecule, indicating the 1:1 stoichiometry. The detailed view of the interface, along with the high-resolution structure of EIIB containing a sulfate ion at the phosphorylation site, suggests that the phosphorylation-induced steric hindrance and disturbance of polar intermolecular interactions impede complex formation. Furthermore, we reveal that Mlc possesses a built-in flexibility for the structural adaptation to its target DNA and that interaction of Mlc with EIIB fused only to dimeric proteins resulted in the loss of its DNA binding ability, suggesting that flexibility of the Mlc structure is indispensable for its DNA binding.enzyme IICB Glc ͉ glucose signaling ͉ protein-protein interaction ͉ transcription regulation B acteria sense continuous changes in their environment and adapt metabolically to compete effectively with other organisms for limiting nutrients. One sensory transduction system monitoring availability of a certain group of carbon sources is the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (1). In addition to concomitant transport and phosphorylation of sugars, PTSs take part in a variety of physiological processes through direct interactions with their target proteins (2-5).It is a normal occurrence that cells make more proteins necessary to transport and metabolize PTS sugars when they encounter an environment where PTS sugars are available. Glucose, a representative PTS sugar, mediates transcriptional activation of several genes for PTS-related sugar transporters and some enzymes involved in glycolysis (1, 6). Mlc, a tetrameric protein (7), is a signaling mediator for glucose induction of several PTS operons and related genes (8)(9)(10)(11)(12)(13)(14).A unique feature of induction of the Mlc regulon by glucose is that the effector molecule modulating Mlc activity is a membranebound protein, EIICB Glc (7,15,16), in which cytosolic EIIB protein is attached to membrane-embedded EIIC protein through a linker of Ͼ20 aa. In the absence of glucose, EIICB Glc mainly exists in the phosphorylated form (pEIICB Glc ). Because Mlc cannot interact with pEIICB Glc , Mlc dissociates from pEIICB Glc to bind to target promoters and repress their transcription. When glucose is available, transported glucose takes the phospho-groups away from pEIICB Glc and the dephosphorylated EIICB Glc recruits Mlc to sequester it from its target promoters. This leads to increased synthesis of EIICB Glc and other PTS proteins necessary to take up and metabolize glucose more efficiently.The physiological importance of the glucose-specific enz...

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“…It was shown that the ptsH P1 promoter is regulated by the repressor of the fructose PTS, FruR (12). Moreover, both the ptsH P0 promoter and the ptsG P1 promoter have been shown to be subject to activation by the cAMP⅐CRP complex and repression by Mlc (13)(14)(15)(16)(17)(18). Subsequently, it was demonstrated that the unphosphorylated form of EIICB Glc can relieve the repression of the Mlc regulon by sequestering Mlc through the direct protein-protein interaction to induce the expression of the Mlc regulon including PTS proteins (19 -21).…”

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Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

Jeong

Kim

Cho

et al. 2004

Journal of Biological Chemistry

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Because the phosphoenolpyruvate:sugar phosphotransferase system plays multiple regulatory roles in addition to the phosphorylation-coupled transport of many sugars in bacteria, synthesis of its protein components is regulated in a highly sophisticated way. Thus far, the cAMP receptor protein (CRP) complex and Mlc are known to be the major regulators of ptsHIcrr and ptsG expression in response to the availability of carbon sources. In this report, we performed ligand fishing experiments by using the promoters of ptsHIcrr and ptsG as bait to find out new factors involved in the transcriptional regulation of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, and we found that the anaerobic regulator ArcA specifically binds to the promoters. Deletion of the arcA gene caused about a 2-fold increase in the ptsG expression, and overexpression of ArcA significantly decreased glucose consumption. In vitro transcription assays showed that phospho-ArcA (ArcA-P) represses ptsG P1 transcription. DNase I footprinting experiments revealed that ArcA-P binds to three sites upstream of the ptsG P1 promoter, two of which overlap the CRP-binding sites, and the ArcA-P binding decreases the CRP binding that is essential for the ptsG P1 transcription. These results suggest that the response regulator ArcA regulates expression of enzyme IICB Glc mediating the first step of glucose metabolism in response to the redox conditions of growth in E. coli.The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) 1 consists of two general cytoplasmic proteins, enzyme I and histidine phosphocarrier protein HPr, and some sugar-specific components collectively known as enzyme II (1, 2). Glucose-specific enzyme II of Escherichia coli consists of two subunits, soluble enzyme IIA Glc (EIIA Glc ) and membrane-bound enzyme IICB Glc . Thus, glucose transport in E. coli involves three soluble PTS components (enzyme I, HPr, and EIIA Glc , encoded by the ptsHIcrr operon) and one membranebound protein, enzyme IICB Glc (EIICB Glc ), encoded by the ptsG gene. During translocation of glucose, a phosphoryl group derived from phosphoenolpyruvate is transferred sequentially along a series of proteins to the transported glucose molecule, eventually converting it into glucose 6-phosphate. The sequence of phosphotransfer is from phosphoenolpyruvate to the general PTS proteins enzyme I and HPr and further to the carbohydrate-specific cytoplasmic EIIA Glc , membrane-bound EIICB Glc , and glucose.The PTS takes an important part in metabolic adaptation to environmental changes to compete effectively with ambient organisms by sensing the availability of nutrients in the environment. In addition to sugar transport, multiple roles are exerted by the PTS and these include chemoreception (3), catabolite repression (4), carbohydrate transport and metabolism (1, 5, 6), carbon storage (7,8), and the coordination of carbon and nitrogen metabolism (9). More recently, we found that EIIA Glc of the PTS also regulates the flux between respirat...

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Purification of Mlc and Analysis of Its Effects on the pts Expression in Escherichia coli

Cited by 55 publications

References 27 publications

A Quantitative Approach to Catabolite Repression in Escherichia coli

A Quantitative Approach to Catabolite Repression in Escherichia coli

Analyses of Mlc–IIB ^Glc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

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Purification of Mlc and Analysis of Its Effects on the pts Expression in Escherichia coli

Cited by 55 publications

References 27 publications

A Quantitative Approach to Catabolite Repression in Escherichia coli

A Quantitative Approach to Catabolite Repression in Escherichia coli

Analyses of Mlc–IIB Glc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration

Expression of ptsG Encoding the Major Glucose Transporter Is Regulated by ArcA in Escherichia coli

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Analyses of Mlc–IIB ^Glc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration