Publications by authors named "Plumbridge J"

Glucosamine-6-phosphate (GlcN6P) deaminases from Escherichia coli (EcNagBI) and Shewanella denitrificans (SdNagBII) are special examples of what constitute nonhomologous isofunctional enzymes due to their convergence, not only in catalysis, but also in cooperativity and allosteric properties. Additionally, we found that the sigmoidal kinetics of SdNagBII cannot be explained by the existing models of homotropic activation. This study describes the regulatory mechanism of SdNagBII using enzyme kinetics, isothermal titration calorimetry (ITC), and X-ray crystallography.

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In order to respond to ever-changing environmental cues, bacteria display resilient regulatory mechanisms controlling gene expression. At the post-transcriptional level, this is achieved by a combination of RNA-binding proteins, such as ribonucleases (RNases), and regulatory RNAs, including antisense RNAs (asRNAs). Bound to their complementary mRNA, asRNAs are primary targets for the double-strand-specific endoribonuclease, RNase III.

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Rationale: Dopamine D2-like receptors (D2R) are important drug targets in schizophrenia and Parkinson's disease, but D2R ligands also cause cognitive inflexibility such as poor reversal learning. The specific role of D2R in reversal learning remains unclear.

Objectives: We tested the hypotheses that D2R agonism impairs reversal learning by blocking negative feedback and that antagonism of D1-like receptors (D1R) impairs learning from positive feedback.

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NagC is a transcription factor that represses genes involved in N-acetylglucosamine catabolism in Escherichia coli. Repression by NagC is relieved by interaction with GlcNAc6P, the product of transport of GlcNAc into the cell. The DNA-binding domain of NagC contains a classic helix-turn-helix (HTH) motif, but specific operator recognition requires, in addition, an adjacent linker sequence, which is thought to form an extended wing.

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Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures.

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Unlabelled: We have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites in Escherichia coli Growth on GlcNAc increased intracellular pools of both GlcNAc6P and GlcN6P 10- to 20-fold compared to growth on glucose. Growth on GlcN produced a 100-fold increase in GlcN6P but only a slight increase in GlcNAc6P. Changes to the amounts of downstream glycolytic intermediates were minor compared to growth on glucose.

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The Mlc transcription factor in Escherichia coli controls the expression of the phosphotransferase system genes implicated in the transport of glucose into the cell. Transport of glucose derepresses Mlc-repressed genes by provoking the sequestration of Mlc to the membrane, via an interaction with the dephosphorylated EIIB domain of the glucose transporter, PtsG. NagC, a paralogue of Mlc in E.

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Amino sugars are dual-purpose compounds in bacteria: they are essential components of the outer wall peptidoglycan (PG) and the outer membrane of Gram-negative bacteria and, in addition, when supplied exogenously their catabolism contributes valuable supplies of energy, carbon and nitrogen to the cell. The enzymes for both the synthesis and degradation of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are highly conserved but during evolution have become subject to different regulatory regimes. Escherichia coli grows more rapidly using GlcNAc as a carbon source than with GlcN.

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NagC and Mlc, paralogous members of the ROK family of proteins with almost identical helix-turn-helix DNA binding motifs, specifically regulate genes for transport and utilization of N-acetylglucosamine and glucose. We previously showed that two amino acids in a linker region outside the canonical helix-turn-helix motif are responsible for Mlc site specificity. In this work we identify four amino acids in the linker, which are required for recognition of NagC targets.

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In Bacillus subtilis separate sets of genes are implicated in the transport and metabolism of the amino sugars, glucosamine and N-acetylglucosamine. The genes for use of N-acetylglucosamine (nagAB and nagP) are found in most firmicutes and are controlled by a GntR family repressor NagR (YvoA). The genes for use of glucosamine (gamAP) are repressed by another GntR family repressor GamR (YbgA).

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Escherichia coli and Salmonella can use chitin-derived oligosaccharides as carbon and nitrogen sources. Chitosugars traverse the outer membrane through a dedicated chitoporin, ChiP, and are transported across the cytoplasmic membrane by the chitobiose transporter (ChbBCA). Previous work revealed that synthesis of the chitoporin, ChiP, requires transcription of the chbBCARFG operon.

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Article Synopsis
  • B. subtilis grows faster using glucosamine compared to N-acetylglucosamine, indicating that it has adapted to utilize glucosamine more efficiently due to the presence of the gamAP operon, which is specifically induced by glucosamine.
  • The research involved creating multiple deletion mutations in B. subtilis to study the roles of nag and gam operons in metabolizing these sugars, revealing that gamA is the primary gene responsible for N-acetylglucosamine catabolism.
  • The repression of the nagAB genes by a regulator called YvoA and the unique presence of the ybgA-gamAP operon in B. subtilis, which likely originated from lateral gene transfer, together support
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Transcriptional regulation is at the heart of biological functions such as adaptation to a changing environment or to new carbon sources. One of the mechanisms which has been found to modulate transcription, either positively (activation) or negatively (repression), involves the formation of DNA loops. A DNA loop occurs when a protein or a complex of proteins simultaneously binds to two different sites on DNA with looping out of the intervening DNA.

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Protein-DNA recognition is fundamental to transcriptional regulation. Transcription factors must be capable of locating their specific sites situated throughout the genome and distinguishing them from related sites. Mlc and NagC control uptake and use of the sugars, glucose and N-acetylglucosamine.

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The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.

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We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (O(SA1) and O(SA2)), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only.

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The ptsG gene, encoding the major glucose uptake system in Escherichia coli, is expressed from 2 promoters, a minor promoter p2 and a major downstream promoter p1. Transcription from both promoters is repressed by Mlc, and expression of p1 is activated by the cAMP/catabolite activator protein complex. Expression from p1 is also regulated post-transcriptionally in response to sugar stress via an sRNA, SgrS, which results in translational inhibition and mRNA degradation.

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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.

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A set of enzymes dedicated to recycling of the amino sugar components of peptidoglycan has previously been identified in Escherichia coli. The complete pathway includes the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase, of the catabolic pathway for use of N-acetylglucosamine (GlcNAc). Mutations in nagA result in accumulation of millimolar concentrations of GlcNAc6P, presumably by preventing peptidoglycan recycling.

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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.

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Mlc and NagC are two homologous transcription factors which bind to similar DNA targets but for which the inducing signals and mechanisms of activation are very different. Displacing Mlc from its DNA binding sites necessitates its sequestration to the inner membrane via an interaction with PtsG (EIICB(Glc)), while NagC is displaced from its DNA targets by interacting with GlcNAc6P. We have isolated mutations in both proteins which prevent the inactivation of the repressors by growth on glucose or GlcNAc.

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Expression of the FimB recombinase, and hence the OFF-to-ON switching of type 1 fimbriation in Escherichia coli, is inhibited by sialic acid (Neu(5)Ac) and by GlcNAc. NanR (Neu(5)Ac-responsive) and NagC (GlcNAc-6P-responsive) activate fimB expression by binding to operators (O(NR) and O(NC1) respectively) located more than 600 bp upstream of the fimB promoter within the large (1.4 kb) nanC-fimB intergenic region.

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Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase.

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The Escherichia coli yjhA (renamed nanC) gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from fimB, a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in E. coli.

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