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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 25 September 2012, Vol.109(39), pp.15906-11
    Description: Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria.
    Keywords: Models, Biological ; Bacterial Proteins -- Metabolism ; Escherichia Coli -- Metabolism ; Phosphoenolpyruvate Sugar Phosphotransferase System -- Metabolism ; Protein Multimerization -- Physiology ; RNA-Binding Proteins -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    In: Molecular Microbiology, August 2012, Vol.85(4), pp.597-601
    Description: Transcriptional regulators are controlled through various, mostly well‐understood, principles. In the study of Richet ., published in this issue of , fluorescence microscopy was used to uncover an unorthodox mechanism that relies on the dynamic shuttling of a gene regulator between the membrane and the chromosome. When not occupied with transport, the maltose‐specific ABC transporter sequesters and thereby inactivates its cognate transcriptional regulator MalT. Upon maltose transport, MalT is released from the membrane and activates the maltose utilization and transport genes. This mechanism prevents induction of MalT by endogenously produced maltotriose, which is the inducer. Thus, the maltose uptake system is a trigger transporter with a bi‐functional role in transport and regulation.
    Keywords: Transcription (Genetics) -- Genetic Aspects ; Genetic Research -- Genetic Aspects ; Genes -- Genetic Aspects ; Television Broadcasting Industry ; Disaccharides;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2012, Vol.109(39), pp.15906-15911
    Description: Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria. ; p. 15906-15911.
    Keywords: Models ; Transporters ; Phosphotransferases (Kinases) ; Gram-Negative Bacteria ; Cytoplasm ; Escherichia Coli ; Transcription (Genetics) ; Phosphorylation ; Histidine
    ISSN: 0027-8424
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  • 4
    Language: English
    In: Nucleic acids research, 29 January 2016, Vol.44(2), pp.824-37
    Description: In E. coli, small RNA GlmZ activates the glmS mRNA by base-pairing in an Hfq dependent manner. When not required, GlmZ is bound by adaptor protein RapZ and recruited to RNase E, which cleaves GlmZ in its base-pairing sequence. Small RNA GlmY counteracts cleavage of GlmZ by sequestration of RapZ. Although both sRNAs are highly homologous, only GlmZ specifically binds Hfq and undergoes cleavage by RNase E. We used domain swapping to identify the responsible modules. Two elements, the 3' terminal oligo(U) stretch and the base-pairing region enable GlmZ to interact with Hfq. Accordingly, Hfq inhibits cleavage of GlmZ, directing it to base-pairing. Intriguingly, the central stem loop of GlmZ is decisive for cleavage, whereas the sequence comprising the actual cleavage site is dispensable. Assisted by RapZ, RNase E cleaves any RNA fused to the 3' end of this module. These results suggest a novel mode for RNase E recognition, in which one of the required handholds in the substrate is replaced by an RNA binding protein. This device can generate RNAs of interest in their 5' monophosphorylated form on demand. As these species are rapidly degraded, this tool allows to regulate gene expression post-transcriptionally by modulation of RapZ levels.
    Keywords: Aptamers, Nucleotide -- Metabolism ; Endoribonucleases -- Metabolism ; Escherichia Coli Proteins -- Metabolism ; Host Factor 1 Protein -- Metabolism ; RNA, Bacterial -- Metabolism
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 5
    In: Molecular Microbiology, October 2012, Vol.86(1), pp.96-110
    Description: Many possess the paralogous PTS, in addition to the sugar transport phosphotransferase system (PTS). In the PTS phosphoryl‐groups are transferred from phosphoenolpyruvate to protein EIIA via the phosphotransferases EI and NPr. The PTS has been implicated in regulation of diverse physiological processes. In , the PTS plays a role in potassium homeostasis. In particular, EIIA binds to and stimulates activity of a two‐component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high‐affinity K transporter KdpFABC. Here, we show that the phosphate () regulon is likewise modulated by PTS. The regulon, which comprises more than 30 genes, is activated by the two‐component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA are unable to fully activate the genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, expression is increased above the wild‐type level in mutants deficient for EIIA phosphorylation suggesting that non‐phosphorylated EIIA modulates . Protein interaction analyses reveal binding of EIIA to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in .
    Keywords: Gene Expression Regulation, Bacterial ; Protein Interaction Mapping ; Bacterial Proteins -- Metabolism ; Escherichia Coli -- Genetics ; Escherichia Coli Proteins -- Metabolism ; Phosphoenolpyruvate Sugar Phosphotransferase System -- Metabolism;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 6
    Language: English
    In: The Journal of biological chemistry, 20 April 2018, Vol.293(16), pp.5781-5792
    Description: Utilization of energy-rich carbon sources such as glucose is fundamental to the evolutionary success of bacteria. Glucose can be catabolized via glycolysis for feeding the intermediary metabolism. The methylglyoxal synthase MgsA produces methylglyoxal from the glycolytic intermediate dihydroxyacetone phosphate. Methylglyoxal is toxic, requiring stringent regulation of MgsA activity. In the Gram-positive bacterium , an interaction with the phosphoprotein Crh controls MgsA activity. In the absence of preferred carbon sources, Crh is present in the nonphosphorylated state and binds to and thereby inhibits MgsA. To better understand the mechanism of regulation of MgsA, here we performed biochemical and structural analyses of MgsA and of its interaction with Crh. Our results indicated that MgsA forms a hexamer ( a trimer of dimers) in the crystal structure, whereas it seems to exist in an equilibrium between a dimer and hexamer in solution. In the hexamer, two alternative dimers could be distinguished, but only one appeared to prevail in solution. Further analysis strongly suggested that the hexamer is the biologically active form. cross-linking studies revealed that Crh interacts with the N-terminal helices of MgsA and that the Crh-MgsA binding inactivates MgsA by distorting and thereby blocking its active site. In summary, our results indicate that dimeric and hexameric MgsA species exist in an equilibrium in solution, that the hexameric species is the active form, and that binding to Crh deforms and blocks the active site in MgsA.
    Keywords: Bacillus ; Crh ; Hpr ; Bacteria ; Bacterial Genetics ; Bacterial Metabolism ; Catabolite Regulation ; Crystal Structure ; Glucose Catabolism ; Glycolysis ; Metabolic Regulation ; Methylglyoxal Synthase ; Methylglyoxal Toxicity ; Phosphoprotein ; Prokaryotic Signal-Transduction ; Protein Cross-Linking ; Protein Structure ; Protein-Protein Interaction ; Protein Interaction Maps ; Bacillus Subtilis -- Metabolism ; Bacterial Proteins -- Metabolism ; Carbon-Oxygen Lyases -- Metabolism ; Phosphoproteins -- Metabolism
    E-ISSN: 1083-351X
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  • 7
    Language: English
    In: Journal of bacteriology, May 2013, Vol.195(10), pp.2146-54
    Description: Bacillus subtilis transports β-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the β-glucoside permease BglP is induced in the presence of the substrate salicin, and this induction requires the binding of the antiterminator protein LicT to a specific RNA target in the 5' region of the bglP mRNA to prevent the formation of a transcription terminator. LicT is composed of an N-terminal RNA-binding domain and two consecutive PTS regulation domains, PRD1 and PRD2. In the absence of salicin, LicT is phosphorylated on PRD1 by BglP and thereby inactivated. In the presence of the inducer, the phosphate group from PRD1 is transferred back to BglP and consequently to the incoming substrate, resulting in the activation of LicT. In this study, we have investigated the intracellular localization of LicT. While the protein was evenly distributed in the cell in the absence of the inducer, we observed a subpolar localization of LicT if salicin was present in the medium. Upon addition or removal of the inducer, LicT rapidly relocalized in the cells. This dynamic relocalization did not depend on the binding of LicT to its RNA target sites, since the localization pattern was not affected by deletion of all LicT binding sites. In contrast, experiments with mutants affected in the PTS components as well as mutations of the LicT phosphorylation sites revealed that phosphorylation of LicT by the PTS components plays a major role in the control of the subcellular localization of this RNA-binding transcription factor.
    Keywords: Bacillus Subtilis -- Metabolism ; Bacterial Proteins -- Metabolism ; Transcription Factors -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 8
    Language: English
    In: Nucleic acids research, March 2011, Vol.39(4), pp.1294-309
    Description: Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.
    Keywords: Gene Expression Regulation, Bacterial ; Transcription, Genetic ; Enterobacteriaceae -- Genetics ; RNA, Bacterial -- Genetics ; RNA, Small Untranslated -- Genetics
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 9
    Language: English
    In: The Journal of biological chemistry, 21 November 2003, Vol.278(47), pp.46219-29
    Description: Activity of antiterminator protein BglG regulating the beta-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner. It requires HPr phosphorylation to be active, whereas phosphorylation by the beta-glucoside-specific transport protein EIIBgl inhibits its activity. BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines. For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site. In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2. In this work, a screen for mutant BglG proteins that escape repression by EIIBgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins. Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EIIBgl and that His-160 contributes to negative regulation. His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr. Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208. However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG. These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer. The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EIIBgl-catalyzed phosphorylation at His-101.
    Keywords: Escherichia Coli Proteins -- Metabolism ; RNA-Binding Proteins -- Metabolism
    ISSN: 0021-9258
    E-ISSN: 1083351X
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  • 10
    Language: English
    In: Nucleic acids research, 13 October 2017, Vol.45(18), pp.10845-10860
    Description: In phylogenetically diverse bacteria, the conserved protein RapZ plays a central role in RNA-mediated regulation of amino-sugar metabolism. RapZ contributes to the control of glucosamine phosphate biogenesis by selectively presenting the regulatory small RNA GlmZ to the essential ribonuclease RNase E for inactivation. Here, we report the crystal structures of full length Escherichia coli RapZ at 3.40 Å and 3.25 Å, and its isolated C-terminal domain at 1.17 Å resolution. The structural data confirm that the N-terminal domain of RapZ possesses a kinase fold, whereas the C-terminal domain bears closest homology to a subdomain of 6-phosphofructokinase, an important enzyme in the glycolytic pathway. RapZ self-associates into a domain swapped dimer of dimers, and in vivo data support the importance of quaternary structure in RNA-mediated regulation of target gene expression. Based on biochemical, structural and genetic data, we suggest a mechanism for binding and presentation by RapZ of GlmZ and the closely related decoy sRNA, GlmY. We discuss a scenario for the molecular evolution of RapZ through re-purpose of enzyme components from central metabolism.
    Keywords: Escherichia Coli Proteins -- Chemistry ; RNA-Binding Proteins -- Chemistry
    ISSN: 03051048
    E-ISSN: 1362-4962
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