scholarly journals The phosphohistidine phosphatase SixA dephosphorylates the phosphocarrier NPr

2020 ◽  
pp. jbc.RA120.015121
Author(s):  
Jane E. Schulte ◽  
Manuela Roggiani ◽  
Hui Shi ◽  
Jun Zhu ◽  
Mark Goulian
Keyword(s):  
Protein Function ◽  
Growth Defect ◽  
Phosphoryl Transfer ◽  
Post Translational Modification ◽  
Phosphotransferase System ◽  
E Coli ◽  
System A ◽  
Mouse Intestine ◽  
Histidine Phosphorylation

Histidine phosphorylation is a post-translational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr, and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are out-competed by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The wide-spread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

scholarly journals SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

Open Biology ◽  
2013 ◽  
Vol 3 (10) ◽  
pp. 120173 ◽  
Author(s):  
Ingrid Kassner ◽  
Anneli Andersson ◽  
Monika Fey ◽  
Martin Tomas ◽  
Elisa Ferrando-May ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Interactions ◽  
Protein Function ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Protein Methyltransferase

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo . ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo . Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

scholarly journals Lysine methylation is an endogenous post-translational modification of tau protein in human brain and a modulator of aggregation propensity

2014 ◽  
Vol 462 (1) ◽  
pp. 77-88 ◽  
Author(s):  
Kristen E. Funk ◽  
Stefani N. Thomas ◽  
Kelsey N. Schafer ◽  
Grace L. Cooper ◽  
Zhongping Liao ◽  
...  
Keyword(s):  
Human Brain ◽  
Tau Protein ◽  
Protein Function ◽  
Lysine Methylation ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Aggregation Propensity ◽  
Normal Human ◽  
Tau Aggregation

Diverse post-translational modifications regulate tau protein function and misfolding. In the present study we identified lysine methylation as a tau post-translational modification in normal human brain, and found it depressed tau aggregation propensity when modelled in vitro.

scholarly journals Acetate Metabolism in a pta Mutant ofEscherichia coli W3110: Importance of Maintaining Acetyl Coenzyme A Flux for Growth and Survival

1999 ◽  
Vol 181 (21) ◽  
pp. 6656-6663 ◽  
Author(s):  
Dong-Eun Chang ◽  
Sooan Shin ◽  
Joon-Shick Rhee ◽  
Jae-Gu Pan
Keyword(s):  
Amino Acids ◽  
Coenzyme A ◽  
Physiological Role ◽  
Normal Growth ◽  
Growth Defect ◽  
Acetate Metabolism ◽  
Phosphotransferase System ◽  
Acetyl Coenzyme A ◽  
E Coli ◽  
Acetyl Coenzyme

ABSTRACT In order to study the physiological role of acetate metabolism inEscherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate,d-lactate, and l-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by thepta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The ptamutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-β-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

scholarly journals The Streptomycin-Treated Mouse Intestine SelectsEscherichia coli envZMissense Mutants That Interact with Dense and Diverse Intestinal Microbiota

2012 ◽  
Vol 80 (5) ◽  
pp. 1716-1727 ◽  
Author(s):  
Mary P. Leatham-Jensen ◽  
Jakob Frimodt-Møller ◽  
Jimmy Adediran ◽  
Matthew E. Mokszycki ◽  
Megan E. Banner ◽  
...  
Keyword(s):  
Treated Mouse ◽  
Bacterial Biofilms ◽  
Deletion Mutants ◽  
Content Type ◽  
E Coli ◽  
Minority Member ◽  
Mouse Intestine ◽  
Colicin V ◽  
Colonizing Ability

ABSTRACTPreviously, we reported that the streptomycin-treated mouse intestine selected nonmotileEscherichia coliMG1655flhDCdeletion mutants ofE. coliMG1655 with improved colonizing ability that grow 15% fasterin vitroin mouse cecal mucus and 15 to 30% faster on sugars present in mucus (M. P. Leatham et al., Infect. Immun. 73:8039–8049, 2005). Here, we report that the 10 to 20% remaining motileE. coliMG1655 areenvZmissense mutants that are also better colonizers of the mouse intestine thanE. coliMG1655. One of theflhDCmutants,E. coliMG1655 ΔflhD, and one of theenvZmissense mutants,E. coliMG1655 mot-1, were studied further.E. coliMG1655 mot-1 is more resistant to bile salts and colicin V thanE. coliMG1655 ΔflhDand grows ca. 15% slowerin vitroin mouse cecal mucus and on several sugars present in mucus compared toE. coliMG1655 ΔflhDbut grows 30% faster on galactose. Moreover,E. coliMG1655 mot-1 andE. coliMG1655 ΔflhDappear to colonize equally well in one intestinal niche, butE. coliMG1655 mot-1 appears to use galactose to colonize a second, smaller intestinal niche either not colonized or colonized poorly byE. coliMG1655 ΔflhD. Evidence is also presented thatE. coliMG1655 is a minority member of mixed bacterial biofilms in the mucus layer of the streptomycin-treated mouse intestine. We offer a hypothesis, which we call the “Restaurant” hypothesis, that explains how nutrient acquisition in different biofilms comprised of different anaerobes can account for our results.

scholarly journals Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

2006 ◽  
Vol 75 (4) ◽  
pp. 1661-1666 ◽  
Author(s):  
Theresa Deland Ho ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Genetic Manipulation ◽  
Enterohemorrhagic Escherichia Coli ◽  
Deletion Strain ◽  
Growth Defect ◽  
Intestinal Colonization ◽  
Wild Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

scholarly journals TetR-Based Gene Regulation Systems for Francisella tularensis

2012 ◽  
Vol 78 (19) ◽  
pp. 6883-6889 ◽  
Author(s):  
Eric D. LoVullo ◽  
Cheryl N. Miller ◽  
Martin S. Pavelka ◽  
Thomas H. Kawula
Keyword(s):  
Gene Expression ◽  
Francisella Tularensis ◽  
Protein Function ◽  
Expression System ◽  
Promoter Sequence ◽  
Growth Defect ◽  
Regulate Gene Expression ◽  
Content Type ◽  
System A ◽  
Tet Repressor

ABSTRACTThere are a number of genetic tools available for studyingFrancisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems forF. tularensisbased on the Tet repressor, TetR. For the inducible system, atetoperator sequence was cloned into a modifiedF. tularensis groESLpromoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon,luxCDABE, was cloned under the hybridFrancisellatetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association withtetO.Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml−1. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists ofFTRpregulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with theluxreporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploringF. tularensisprotein function.

Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Expression System ◽  
High Yield ◽  
Gene Synthesis ◽  
Post Translational Modification ◽  
Iptg Induction ◽  
E Coli ◽  
Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

scholarly journals Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evgeniya Schastnaya ◽  
Zrinka Raguz Nakic ◽  
Christoph H. Gruber ◽  
Peter Francis Doubleday ◽  
Aarti Krishnan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pentose Phosphate ◽  
Acetate Metabolism ◽  
Post Translational Modification ◽  
E Coli ◽  
In Vivo Experiments ◽  
Functional Relevance ◽  
Regulation Of Enzyme Activity

AbstractProtein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.

scholarly journals Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

2020 ◽  
Author(s):  
Fabienne Benz ◽  
Jana S. Huisman ◽  
Erik Bakkeren ◽  
Joana A. Herter ◽  
Tanja Stadler ◽  
...  
Keyword(s):  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Transfer Genes ◽  
Serovar Typhimurium ◽  
Mouse Intestine ◽  
Specific Factors ◽  
Natural Strains

Abstract Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.

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