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The New Phytologist Jul 2014Two-component signalling (TCS) systems play important roles in cytokinin and ethylene signalling in Arabidopsis thaliana. Although the involvement of histidine kinases...
Two-component signalling (TCS) systems play important roles in cytokinin and ethylene signalling in Arabidopsis thaliana. Although the involvement of histidine kinases (AHKs) in drought stress responses has been described, their role and that of histidine phosphotransferases (AHPs) in guard cell signalling remain to be fully elucidated. Here, we investigated the roles of TCS genes, the histidine phosphotransferase AHP2 and the histidine kinases AHK2 and AHK3, previously reported to play roles in cytokinin and abscisic acid (ABA) signalling. We show that AHP2 is present in the nucleus and the cytoplasm, and is involved in light-induced opening. We also present evidence that there is some redistribution of AHP2 from the nucleus to the cytoplasm on addition of ABA. In addition, we provide data to support a role for the cytokinin receptors AHK2 and AHK3 in light-induced stomatal opening and, by inference, in controlling the stomatal sensitivity to ABA. Our results provide new insights into the operation of TCS in plants, cross-talk in stomatal signalling and, in particular, the process of light-induced stomatal opening.
Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Cell Nucleus; Cytoplasm; Gene Expression Regulation, Plant; Histidine Kinase; Light; Phosphotransferases; Plant Cells; Plant Leaves; Plant Stomata; Plants, Genetically Modified; Protein Kinases; Signal Transduction
PubMed: 24758561
DOI: 10.1111/nph.12813 -
Molecules (Basel, Switzerland) Oct 2020is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. lacks complete...
is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. lacks complete respiratory chain and relies on carbohydrate fermentation for energy generation. One of the essential components for this includes the mannose phosphotransferase system (Man-PTS), which plays a central role in glucose transport and exhibits a broad specificity for a range of hexoses. Importantly, Man-PTS is involved in the global regulation of gene expression for virulence determinants. We herein report the three-dimensional structure of the EIIA domain of mannose phosphotransferase system (SpEIIA-Man). Our structure shows a dimeric arrangement of EIIA and reveals a detailed molecular description of the active site. Since PTS transporters are exclusively present in microbes and sugar transporters have already been suggested as valid targets for antistreptococcal antibiotics, our work sets foundation for the future development of antimicrobial strategies against .
Topics: Bacterial Proteins; Crystallography, X-Ray; Mannose; Phosphotransferases; Streptococcus pneumoniae; Substrate Specificity
PubMed: 33053673
DOI: 10.3390/molecules25204633 -
PloS One 2015Upon encystment induction, Azotobacter vinelandii produces the phenolic lipids alkylresorcinols (ARs) that are structural components of the cysts. The enzymes...
Upon encystment induction, Azotobacter vinelandii produces the phenolic lipids alkylresorcinols (ARs) that are structural components of the cysts. The enzymes responsible for the ARs synthesis are encoded in the arsABCD operon, whose expression is activated by ArpR. The transcription of arpR is initiated from an RpoS dependent promoter. The nitrogen-related phosphotransferase system (PTS(Ntr)) is a global regulatory system present in Gram negative bacteria. It comprises the EI(Ntr), NPr and EIIA(Ntr) proteins encoded by ptsP, ptsO and ptsN genes respectively. These proteins participate in a phosphoryl-group transfer from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. In A. vinelandii, the non-phosphorylated form of EIIA(Ntr) was previously shown to repress the synthesis of poly-ß-hydroxybutyrate. In this work, we show that PTS(Ntr) also regulates the synthesis of ARs. In a strain that carries unphosphorylated EIIA(Ntr), the expression of arpR was reduced, while synthesis of ARs and transcription of arsA were almost abrogated. The expression of arpR from an RpoS-independent promoter in this strain restored the ARs synthesis. Taken together these results indicate that unphosphorylated EIIA(Ntr) negatively affects activation of arpR transcription by RpoS.
Topics: Azotobacter vinelandii; Bacterial Proteins; Gene Expression Regulation, Bacterial; Mutation; Phosphorylation; Phosphotransferases; Resorcinols; Transcriptional Activation
PubMed: 25642700
DOI: 10.1371/journal.pone.0117184 -
Proceedings of the National Academy of... Sep 1983The DNA sequence of an aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae ATCC 10745 (a neomycin producer) was determined. The gene was localized by...
The DNA sequence of an aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae ATCC 10745 (a neomycin producer) was determined. The gene was localized by in vitro subcloning and insertional inactivation. Molecular weight, amino acid composition, and amino-terminal analysis of the purified aph gene product confirmed the accuracy and position of the aph gene sequence. Pairwise comparisons of S. fradiae aph with the aph genes encoded by bacterial transposons Tn5 and Tn903 showed significant nucleotide and amino acid homologies which indicated a common evolutionary origin for these antibiotic-resistance genes.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Codon; DNA Restriction Enzymes; Drug Resistance, Microbial; Genes; Genes, Bacterial; Kanamycin Kinase; Neomycin; Phosphotransferases; Plasmids; Streptomyces
PubMed: 6310563
DOI: 10.1073/pnas.80.17.5190 -
Cell Research Aug 2019
Topics: Anti-Bacterial Agents; Bacillus subtilis; Bacteriocins; Bacteriophage lambda; Biological Transport, Active; Cryoelectron Microscopy; Drug Discovery; Escherichia coli; Food Preservatives; Mannose; Phosphotransferases
PubMed: 31209249
DOI: 10.1038/s41422-019-0194-z -
European Journal of Biochemistry Oct 1977The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose...
The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose 1-phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsI1), and a transport activity (fruA). Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1-phosphate, identified in vivo and in vitro or in fructose 6-phosphate identified by indirect lines of evidence. These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems. The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression. The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene. When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6-phosphate as well as fructose 1-phosphate. A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose.
Topics: Bacillus subtilis; Biological Transport, Active; Fructose; Fructosephosphates; Genes; Mutation; Phosphoenolpyruvate; Phosphotransferases
PubMed: 200418
DOI: 10.1111/j.1432-1033.1977.tb11817.x -
Journal of the American Chemical Society Sep 2010The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr...
Solution structure of the 128 kDa enzyme I dimer from Escherichia coli and its 146 kDa complex with HPr using residual dipolar couplings and small- and wide-angle X-ray scattering.
The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr (∼146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS data that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C(2) symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large (∼70-90°) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.
Topics: Bacterial Proteins; Crystallography, X-Ray; Escherichia coli; Models, Molecular; Molecular Weight; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Phosphotransferases; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Scattering, Small Angle; Solutions; X-Ray Diffraction
PubMed: 20731394
DOI: 10.1021/ja105485b -
Proceedings of the National Academy of... May 1980Facts relating to the mechanism of phosphoryl transfer by acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) are reviewed. They point to the existence of at...
Facts relating to the mechanism of phosphoryl transfer by acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) are reviewed. They point to the existence of at least one experimentally established phosphoenzyme (E-P) intermediate on the reaction pathway. Sterically, the phosphoryl transfer occurs with a net inversion of the configuration of the phosphorus atom. These facts are best in accord with a triple-displacement mode of action for acetate kinase, with two E-P intermediates and three steric inversions on phosphorus. It follows that a second E-P for acetate kinase must exist.
Topics: Acetate Kinase; Adenosine Diphosphate; Adenosine Triphosphate; Binding Sites; Catalysis; Escherichia coli; Mercury; Phosphates; Phosphotransferases; Stereoisomerism
PubMed: 6248856
DOI: 10.1073/pnas.77.5.2626 -
Antimicrobial Agents and Chemotherapy Sep 1981An Escherichia coli strain with a plasmidic amikacin resistance has been selected for which the evidence strongly indicates that resistance is mediated by aminoglycoside...
An Escherichia coli strain with a plasmidic amikacin resistance has been selected for which the evidence strongly indicates that resistance is mediated by aminoglycoside phosphotransferase [APH(3')-II]: (i) this resistance was coupled with resistance against kanamycin and neomycin; (ii) partially purified APH(3')-II[APH(3") free] modified amikacin by phosphorylation; (iii) the product of the APH(3')-II mediated reaction (i.e., 3'-O-phosphoryl-amikacin) lost its antibacterial activity; and (iv) the amikacin-modifying APH(3')-II activity increased 5- to 10-fold after adaptation of the cells to higher concentrations of amikacin. The substrate spectrum of this enzyme showed a low activity against amikacin as compared with neomycin. It is argued that the enzyme level rather than its substrate spectrum is important for enzyme-mediated resistance. The increase in enzyme levels was found to be correlated with an increase in copy number of a 110-Megadalton plasmid (pBN66) which coded for the APH(3')-II and the APH(3") activity. The increase in copy number was irreversible, and therefore this phenomenon is ascribed to a mutation of a gene which affects the copy number. In transconjugants, the original low copy number was present, and therefore the mutation must be located on the chromosome and not on the plasmid.
Topics: Amikacin; Bacterial Proteins; Cell-Free System; Conjugation, Genetic; Drug Resistance, Microbial; Escherichia coli; Kanamycin; Kanamycin Kinase; Microbial Sensitivity Tests; Phosphorylation; Phosphotransferases; Plasmids
PubMed: 6272630
DOI: 10.1128/AAC.20.3.344 -
Nature Communications Jun 2014The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyses the transfer of a substituted phosphate group from a CDP-linked donor to an...
The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyses the transfer of a substituted phosphate group from a CDP-linked donor to an alcohol acceptor. This is an essential reaction for phospholipid biosynthesis across all kingdoms of life, and it is catalysed solely by CDP-APs. Here we report the 2.0 Å resolution crystal structure of a representative CDP-AP from Archaeoglobus fulgidus. The enzyme (AF2299) is a homodimer, with each protomer consisting of six transmembrane helices and an N-terminal cytosolic domain. A polar cavity within the membrane accommodates the active site, lined with the residues from an absolutely conserved CDP-AP signature motif (D(1)xxD(2)G(1)xxAR...G(2)xxxD(3)xxxD(4)). Structures in the apo, CMP-bound, CDP-bound and CDP-glycerol-bound states define functional roles for each of these eight conserved residues and allow us to propose a sequential, base-catalysed mechanism universal for CDP-APs, in which the fourth aspartate (D4) acts as the catalytic base.
Topics: Alcohols; Amino Acid Motifs; Amino Acid Sequence; Archaeal Proteins; Archaeoglobus fulgidus; Binding Sites; Biocatalysis; Catalytic Domain; Models, Molecular; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor); Protein Structure, Tertiary; Sequence Alignment
PubMed: 24923293
DOI: 10.1038/ncomms5068