Different regions of Mlc and NagC, homologous transcriptional repressors controlling expression of the glucose and <i>N</i>‐acetylglucosamine phosphotransferase systems in <i>Escherichia coli</i>, are required for inducer signal recognition

Pennetier, Carole; Domínguez-Ramírez, Lenin; Plumbridge, Jacqueline

doi:10.1111/j.1365-2958.2007.06041.x

Cited by 32 publications

(58 citation statements)

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“…The nagB gene was amplified using the following pairs of oligonucleotides, Nag70-Nag72 (NagB-L1), Nag71-Nag72 (NagB-L2), Nag70-Nag73 (NagB-L3), and Nag71-Nag73 (NagB-L4) (Table 2), and inserted as XbaI-EcoRI fragments into pXE1 to give pXE(NagB-L1), pXE(NagB-L2), pXE(NagB-L3), and pXE(NagB-L4). pXE1 is a single-copy R1 replicon plasmid in which expression of the cloned gene is from a promoter within the DNA-derived region of the plasmid expressing the cI857 repressor (16). The whole insert of each NagB plasmid was sequenced with oligonucleotides XE1 and RBP22 (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Allosteric Regulation of Glucosamine-6-Phosphate Deaminase (NagB) and Growth ofEscherichia colion Glucosamine

et al. 2009

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Growth on N-acetylglucosamine (GlcNAc) produces intracellular N-acetylglucosamine-6-phosphate (GlcNAc6P), which affects the regulation of the catabolism of amino sugars in Escherichia coli in two ways. First, GlcNAc6P is the inducing signal for the NagC repressor, and thus it increases the expression of the enzymes of the nagE-nagBACD operon. Second, it is the allosteric activator of glucosamine-6P (GlcN6P) deaminase, NagB, and thus increases the catalytic capacity of this key enzyme in the metabolism of amino sugars. We showed previously that both the level of expression of the nagB gene and the transport of glucosamine were limiting the growth rate on GlcN (L. I. Álvarez-Añorve et al., J. Bacteriol. 187:2974-2982, 2005). We were unable to conclude if the lack of allosteric activation of wild-type NagB was also contributing to the slower growth rate on GlcN. Using a single-copy plasmid, with a constitutive promoter, we have separated the effects of GlcNAc6P on the NagB protein level and on deaminase activity. We show that over a range of intracellular NagB concentrations it is the quantity of the substrate, GlcN6P, which is limiting growth rather than the concentration of the allosteric activator, GlcNAc6P. On the other hand, the F174A mutant of NagB, which requires higher concentrations of GlcNAc6P for activity in vitro, grew better on GlcN in the presence of GlcNAc6P. However, wild-type NagB behaves as if it is already fully allosterically activated during growth on GlcN, and we present evidence suggesting that sufficient GlcNAc6P for allosteric activation is derived from the recycling of peptidoglycan.Amino sugars are widely distributed in nature and are valuable nutrients to most organisms. The most commonly found amino sugars are glucosamine (GlcN) and N-acetylglucosamine (GlcNAc). Their most abundant source is chitin, the high-molecular-weight polymer composed of 1-4 ␤-linked GlcNAc residues found in the cell walls of fungi and the exoskeletons of crustaceans and other arthropods. Amino sugars are both carbon and nitrogen sources but are also essential components of subcellular structures like the peptidoglycan (PG) of bacterial cell walls and the high-molecular-weight glycosaminoglycans and glycoproteins of the extracellular matrix of higher eukaryotes. Enteric Escherichia coli bacteria are likely to encounter host-derived amino sugars during their normal life cycle. The amino sugars, once taken up by the bacteria, can be used both to synthesize the PG and lipopolysaccharides of its cell wall and as an energy source.We previously investigated why E. coli grows more slowly on glucosamine than on N-acetylglucosamine. Growth on GlcNAc requires the nagE-or manXYZ-encoded phosphotransferase (PTS) transporters, which produce N-acetylglucosamine-6-phosphate (GlcNAc6P), and the two genes nagA and nagB, which encode GlcNAc6P deacetylase and GlcN6P deaminase, respectively, needed for the metabolism of GlcNAc6P (Fig. 1). Growth on GlcN requires the manXYZ-encoded transporter, producing GlcN6P, and the nagB gene pro...

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Section: Methodsmentioning

confidence: 99%

Allosteric Regulation of Glucosamine-6-Phosphate Deaminase (NagB) and Growth ofEscherichia colion Glucosamine

et al. 2009

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“…EIIB Glc -mediated membrane sequestration was proposed to cause structural restrictions leading to reduced flexibility and therefore to lower affinity for its DNA targets (142). Arg-424 located close to the phosphorylatable Cys-421 in PtsG makes polar contacts with the carboxylate of the C-terminal glycine of Mlc (136,144). Unphosphorylated EIIB Glc prevails when the cells take up glucose.…”

Section: Fig 5 Pts-catalyzed Glucose Uptake and The Eiiamentioning

confidence: 99%

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Deutscher

Aké

Derkaoui

et al. 2014

Microbiol Mol Biol Rev

352

374

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SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

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“…We have recently shown, by the use of chimeric proteins, that the H-T-H regions of Mlc and NagC, by themselves, are not sufficient for normal specific Mlc or NagC binding and that the rest of the native protein is necessary for full DNA binding specificity. These studies suggested that the linker between the H-T-H domain and the rest of the protein could be implicated in operator specificity (22). By analogy with the structures of DNA bound to LacI or PurR, we could propose that binding of Mlc or NagC involves amino acid contacts from outside the H-T-H domain, possibly from this unstructured linker, which could form the equivalent of the hinge helix of the LacI-or PurR-operator complex.…”

Section: Discussionmentioning

confidence: 99%

“…However, their C-terminal domains, involved in signal transduction, are quite different, so that the proteins are active under different conditions, and it is thought that amino acids of the ␤-loop wing, adjacent to the H-T-H motif, contribute to the DNA binding site specificity of these two transcription factors (6,48). Mlc and NagC seem to resemble a diverging isorepressor pair, where operator specificity has been achieved without altering the canonical H-T-H interaction, and specific inducing signal recognition occurs, even though the C-terminal domains are still homologous (22). It is interesting that Mlc and NagC control the expression of two PTS transporters, PtsG and NagE, which are themselves homologues.…”

Section: Discussionmentioning

confidence: 99%

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Switching Control of Expression ofptsGfrom the Mlc Regulon to the NagC Regulon

Qaidi

Plumbridge

2008

J Bacteriol

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The Mlc and NagC transcriptional repressors bind to similar 23-bp operators. The sequences are weakly palindromic, with just four positions totally conserved. There is no cross regulation observed between the repressors in vivo, but there are no obvious bases which could be responsible for operator site discrimination. To investigate the basis for operator recognition and to try to understand what differentiates NagC sites from Mlc sites, we have undertaken mutagenesis experiments to convert ptsG from a gene regulated by Mlc into a gene regulated by NagC. There are two Mlc operators upstream of ptsG, and to switch ptsG to the NagC regulon, it was necessary to change two different characteristics of both operators. Firstly, we replaced the AT base pair at position ؉/؊11 from the center of symmetry of the operators with a GC base pair. Secondly, we changed the sequence of the CG base pairs in the central region of the operator (positions ؊4 to ؉4 around the center of symmetry). Our results show that changes at either of these locations are sufficient to lose regulation by Mlc but that both types of changes in both operators are necessary to convert ptsG to a gene regulated by NagC. In addition, these experiments confirmed that two operators are necessary for regulation by NagC. We also show that regulation of ptsG by Mlc involves some cooperative binding of Mlc to the two operators.Mlc and NagC are homologous proteins, and both act as transcriptional repressors in Escherichia coli. Mlc represses genes involved in the uptake of glucose, while NagC controls the use of N-acetylglucosamine (GlcNAc). Both glucose and GlcNAc are transported into E. coli via the phosphotransferase system (PTS). Mlc represses ptsG, the gene for the major glucose transporter; the ptsHI-crr genes, which encode the soluble components of the PTS; the manXYZ genes, which encode an alternative transporter for glucose, as well as other hexoses; and also malT, the positive transcriptional regulator of the mal regulon (4,11,12,24,26,27). NagC represses the divergent nagE-nagBACD operons for the uptake and degradation of GlcNAc and the chb operon, which contains genes for the transport and degradation of chitobiose (a dimer of GlcNAc) (28, 31). In addition, NagC activates the expression of the glmUS operon, which contains genes of the biosynthetic pathway for UDP-GlcNAc (23), and also the expression of the fimB recombinase necessary for the off-to-on switching of the fim operon for type I fimbriae (39, 40).The Mlc and NagC proteins are 40% identical, with 70% similarity, and are members of the repressor subgroup of the ROK (repressors, open reading frames, kinases) family (44). Although Mlc and NagC have clearly defined and different functions in E. coli, it is surprising that the sequence of the helix-turn-helix (H-T-H) motifs in the N-terminal DNA binding domain are unexpectedly similar (Fig. 1B). The C-terminal domains of Mlc and NagC are also homologous, but the inducing signals which displace Mlc and NagC from their DNA binding sites are very ...

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Different regions of Mlc and NagC, homologous transcriptional repressors controlling expression of the glucose and N‐acetylglucosamine phosphotransferase systems in Escherichia coli, are required for inducer signal recognition

Cited by 32 publications

References 58 publications

Allosteric Regulation of Glucosamine-6-Phosphate Deaminase (NagB) and Growth ofEscherichia colion Glucosamine

Allosteric Regulation of Glucosamine-6-Phosphate Deaminase (NagB) and Growth ofEscherichia colion Glucosamine

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Switching Control of Expression ofptsGfrom the Mlc Regulon to the NagC Regulon

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