Negative transcriptional regulation of a positive regulator: the expression of <i>malT</i>, encoding the transcriptional activator of the maltose regulon of <i>Escherichia coli</i>, is negatively controlled by Mlc

Decker, Katja; Plumbridge, Jacqueline; Boos, Winfried

doi:10.1046/j.1365-2958.1998.00694.x

Cited by 94 publications

(131 citation statements)

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“…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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confidence: 99%

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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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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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“…Several regulatory circuits modulate the activity of MalT, e.g. MalT expression is regulated by Mlc, a glucose-inducible repressor (8). In addition, MalT is negatively controlled by at least three proteins: MalY, MalK, and Aes.…”

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confidence: 99%

The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

Mandrich¹,

Caputo²,

Martin³

et al. 2002

Journal of Biological Chemistry

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Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the ␣-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because ␣-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.The Escherichia coli aes (or ybac) gene encodes the 36-kDa cytoplasmic protein Aes (1), a carboxylesterase belonging to the hormone-sensitive lipase family (HSL) 1 (2). Although the physiological role of this enzyme is still obscure, it has recently received greater attention due to the discovery that Aes is involved in the regulation of the maltose regulon in E. coli (reviewed in Ref. 3). The E. coli maltose system consists of 10 genes encoding proteins for the uptake and metabolism of maltodextrin and maltose (3). MalT is the transcriptional activator of the maltose regulon and the prototype of a new family of transcription factors (4, 5) and integrates several signals through its amino-terminal three domains (6). To activate mal genes transcription MalT acts together with the cyclic AMPcatabolite protein complex, the inducer maltotriose, and ATP (7). Several regulatory circuits modulate the activity of MalT, e.g. MalT expression is regulated by Mlc, a glucose-inducible repressor (8). In addition, MalT is negatively controlled by at least three proteins: MalY, MalK, and Aes. MalY is an enzyme with cystathionine ␤-lyase activity (9, 10), which ...

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“…The mlc encodes a 44-kDa protein, Mlc, that has been shown to regulate the expression of ptsG encoding IICB Glc , manXYZ encoding enzyme II of the mannose PTS, malT encoding the activator of maltose regulon, and mlc itself (8 -10). Mlc is proposed to be a global regulator of carbohydrate metabolism (8,9).…”

Section: Discussionmentioning

confidence: 99%

“…Expression of the gene is autoregulated (8), and the translation efficiency of the mlc is low because the translation initiation codon of the mlc is GTG (12). We cloned the mlc under the control of P L and changed the GTG to ATG in our expression vector pNS100 for better expression of the gene.…”

Section: Discussionmentioning

confidence: 99%

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

Kim¹,

Nam²,

Shin³

et al. 1999

Journal of Biological Chemistry

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Products of the pts operon of Escherichia coli have multiple physiological roles such as sugar transport, and the operon is controlled by two promoters, P0 and P1. Expression of the pts P0 promoter that is increased during growth in the presence of glucose is also activated by cAMP receptor protein⅐cAMP. Based on the existence of a sequence that has a high similarity with the known Mlc binding site in the promoter, the effects of the Mlc protein on the pts P0 promoter expression were studied. In vivo transcription assays using wild type and mlc-negative E. coli strains grown in the presence and absence of glucose indicate that Mlc negatively regulates expression of the P0 promoter, and Mlc-dependent repression is relieved by glucose in the growth medium. In vitro transcription assay using purified recombinant Mlc showed that Mlc repressed transcription from the P0 but did not affect the activity of the P1. DNase I footprinting experiments revealed that a Mlc binding site was located around ؉1 to ؉25 of the promoter and that Mlc inhibited the binding of RNA polymerase to the P0 promoter. Cells overexpressing Mlc showed a very slow fermentation rate compared with the wild type when grown in the presence of various phosphoenolpyruvate-carbohydrate phosphotransferase system sugars but few differences in the presence of non-phosphoenolpyruvate-carbohydrate phosphotransferase system sugars except maltose. These results suggest that the pts operon is one of major targets for the negative regulation by Mlc, and thus Mlc regulates the utilization of various sugars as well as glucose in E. coli. The possibility that the inducer of Mlc may not be sugar or its derivative but an unknown factor is proposed to explain the Mlc induction mechanism by various sugars.

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Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc

Cited by 94 publications

References 57 publications

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

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

The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

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

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Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc

Cited by 94 publications

References 57 publications

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

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

The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

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

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

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