Identification and Characterization of trans-Isopentenyl Diphosphate Synthases Involved in Herbivory-Induced Volatile Terpene Formation in Populus trichocarpa

In response to insect herbivory, poplar releases a blend of volatiles that plays important roles in plant defense. Although the volatile bouquet is highly complex and comprises several classes of compounds, it is dominated by mono- and sesquiterpenes. The most common precursors for mono- and sesquiterpenes, geranyl diphosphate (GPP) and (E,E)-farnesyl diphosphate (FPP), respectively, are in general produced by homodimeric or heterodimeric trans-isopentenyl diphosphate synthases (trans-IDSs) that belong to the family of prenyltransferases. To understand the molecular basis of herbivory-induced terpene formation in poplar, we investigated the trans-IDS gene family in the western balsam poplar Populus trichocarpa. Sequence comparisons suggested that this species possesses a single FPP synthase gene (PtFPPS1) and four genes encoding two large subunits (PtGPPS1.LSU and PtGPPS2.LSU) and two small subunits (PtGPPS.SSU1 and PtGPPS.SSU2) of GPP synthases. Transcript accumulation of PtGPPS1.LSU and PtGPPS.SSU1 was significantly upregulated upon leaf herbivory, while the expression of PtFPPS1, PtGPPS2.LSU, and PtGPPS.SSU2 was not influenced by the herbivore treatment. Heterologous expression and biochemical characterization of recombinant PtFPPS1, PtGPPS1.LSU, and PtGPPS2.LSU confirmed their respective IDS activities. Recombinant PtGPPS.SSU1 and PtGPPS.SSU2, however, had no enzymatic activity on their own, but PtGPPS.SSU1 enhanced the GPP synthase activities of PtGPPS1.LSU and PtGPPS2.LSU in vitro. Altogether, our data suggest that PtGPPS1.LSU and PtGPPS2.LSU in combination with PtGPPS.SSU1 may provide the substrate for herbivory-induced monoterpene formation in P. trichocarpa. The sole FPP synthase PtFPPS1 likely produces FPP for both primary and specialized metabolism in this plant species.

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Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

Applied and Environmental Microbiology ◽

10.1128/aem.69.9.5423-5432.2003 ◽

2003 ◽

Vol 69 (9) ◽

pp. 5423-5432 ◽

Cited By ~ 23

Author(s):

Armelle Cochu ◽

Christian Vadeboncoeur ◽

Sylvain Moineau ◽

Michel Frenette

Keyword(s):

Biochemical Characterization ◽

Sugar Transport ◽

Streptococcus Thermophilus ◽

Phosphate Group ◽

Phosphotransferase System ◽

Whole Cells ◽

Genes Encoding ◽

Regulatory Functions

ABSTRACT In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABMan as well as membrane fragments containing IICMan and IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIABMan.

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Genome-Wide Identification and Functional Characterization of the Trans-Isopentenyl Diphosphate Synthases Gene Family in Cinnamomum camphora

Frontiers in Plant Science ◽

10.3389/fpls.2021.708697 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zerui Yang ◽

Chunzhu Xie ◽

Ting Zhan ◽

Linhuan Li ◽

Shanshan Liu ◽

...

Keyword(s):

Essential Oil ◽

Gene Family ◽

Functional Characterization ◽

Cinnamomum Camphora ◽

Isopentenyl Diphosphate ◽

Geranyl Diphosphate ◽

Wide Range ◽

Camphor Tree

Trans-isopentenyl diphosphate synthases (TIDSs) genes are known to be important determinants for terpene diversity and the accumulation of terpenoids. The essential oil of Cinnamomum camphora, which is rich in monoterpenes, sesquiterpenes, and other aromatic compounds, has a wide range of pharmacological activities and has therefore attracted considerable interest. However, the TIDS gene family, and its relationship to the camphor tree (C. camphora L. Presl.), has not yet been characterized. In this study, we identified 10 TIDS genes in the genome of the C. camphora borneol chemotype that were unevenly distributed on chromosomes. Synteny analysis revealed that the TIDS gene family in this species likely expanded through segmental duplication events. Furthermore, cis-element analyses demonstrated that C. camphora TIDS (CcTIDS) genes can respond to multiple abiotic stresses. Finally, functional characterization of eight putative short-chain TIDS proteins revealed that CcTIDS3 and CcTIDS9 exhibit farnesyl diphosphate synthase (FPPS) activity, while CcTIDS1 and CcTIDS2 encode geranylgeranyl diphosphate synthases (GGPPS). Although, CcTIDS8 and CcTIDS10 were found to be catalytically inactive alone, they were able to bind to each other to form a heterodimeric functional geranyl diphosphate synthase (GPPS) in vitro, and this interaction was confirmed using a yeast two-hybrid assay. Furthermore, transcriptome analysis revealed that the CcTIDS3, CcTIDS8, CcTIDS9, and CcTIDS10 genes were found to be more active in C. camphora roots as compared to stems and leaves, which were verified by quantitative real-time PCR (qRT-PCR). These novel results provide a foundation for further exploration of the role of the TIDS gene family in camphor trees, and also provide a potential mechanism by which the production of camphor tree essential oil could be increased for pharmacological purposes through metabolic engineering.

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Human- and Plant-Pathogenic Pseudomonas Species Produce Bacteriocins Exhibiting Colicin M-Like Hydrolase Activity towards Peptidoglycan Precursors

Journal of Bacteriology ◽

10.1128/jb.01824-08 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3657-3664 ◽

Cited By ~ 52

Author(s):

Hélène Barreteau ◽

Ahmed Bouhss ◽

Martine Fourgeaud ◽

Jean-Luc Mainardi ◽

Thierry Touzé ◽

...

Keyword(s):

Biochemical Characterization ◽

Hydrolase Activity ◽

New Class ◽

Low Concentrations ◽

Pseudomonas Species ◽

Genes Encoding ◽

Colicin M ◽

Undecaprenyl Phosphate

ABSTRACT Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae, and P. fluorescens. These genes were detected only in a restricted number of strains. In P. aeruginosa, for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit Mg2+-dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.

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Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

Veterinary Pathology ◽

10.1177/030098588502200413 ◽

1985 ◽

Vol 22 (4) ◽

pp. 375-386 ◽

Cited By ~ 6

Author(s):

H. C. Wimberly ◽

D. O. Slauson ◽

N. R. Neilsen

Keyword(s):

Functional Activity ◽

Biochemical Characterization ◽

Platelet Activating Factor ◽

Biochemical Properties ◽

Naturally Occurring ◽

Rabbit Platelets ◽

Rapid Reaction ◽

Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

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Molecular and biochemical characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria

Biochemical Journal ◽

10.1042/bj20042042 ◽

2005 ◽

Vol 389 (1) ◽

pp. 117-125 ◽

Cited By ~ 86

Author(s):

Taisei KIKUCHI ◽

Hajime SHIBUYA ◽

John T. JONES

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Biochemical Characterization ◽

Glycosyl Hydrolase ◽

Functional Characterization ◽

Nematode Species ◽

Bursaphelenchus Xylophilus ◽

Pinewood Nematode ◽

Sequence Comparisons

We report the cloning and functional characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria. This is the first gene of this type from any nematode species. We show that a similar cDNA is also present in another closely related species B. mucronatus, but that similar sequences are not present in any other nematode studied to date. The B. xylophilus gene is expressed solely in the oesophageal gland cells of the nematode and the protein is present in the nematode's secretions. The deduced amino acid sequence of the gene is very similar to glycosyl hydrolase family 16 proteins. The recombinant protein, expressed in Escherichia coli, preferentially hydrolysed the β-1,3-glucan laminarin, and had very low levels of activity on β-1,3-1,4-glucan, lichenan and barley β-glucan. Laminarin was degraded in an endoglucanase mode by the enzyme. The optimal temperature and pH for activity of the recombinant enzyme were 65 °C and pH 4.9. The protein is probably important in allowing the nematodes to feed on fungi. Sequence comparisons suggest that the gene encoding the endo-β-1,3-glucanase was acquired by horizontal gene transfer from bacteria. B. xylophilus therefore contains genes that have been acquired by this process from both bacteria and fungi. These findings support the idea that multiple independent horizontal gene transfer events have helped in shaping the evolution of several different life strategies in nematodes.

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Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.427 ◽

1985 ◽

Vol 101 (2) ◽

pp. 427-440 ◽

Cited By ~ 40

Author(s):

E Bartnik ◽

M Osborn ◽

K Weber

Keyword(s):

Positive Reaction ◽

Intermediate Filaments ◽

Biochemical Characterization ◽

Helix Pomatia ◽

Molar Ratio ◽

Negative Stain ◽

Epithelial Morphology ◽

Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

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In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins

Methods in Molecular Biology - Yeast Cytokinesis ◽

10.1007/978-1-4939-3145-3_12 ◽

2016 ◽

pp. 151-179 ◽

Cited By ~ 11

Author(s):

Dennis Zimmermann ◽

Alisha N. Morganthaler ◽

David R. Kovar ◽

Cristian Suarez

Keyword(s):

Binding Proteins ◽

Biochemical Characterization ◽

Actin Binding ◽

Actin Binding Proteins

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Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

10.1101/303594 ◽

2018 ◽

Author(s):

Krithika Rajagopalan ◽

Jonathan Dworkin

Keyword(s):

Biochemical Characterization ◽

Regulatory Protein ◽

Biochemical Properties ◽

Bacterial Genomes ◽

Phosphatase Inhibitors ◽

E Coli ◽

Specificity And Sensitivity ◽

Genes Encoding ◽

Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

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Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis

Microbiology ◽

10.1099/mic.0.27534-0 ◽

2005 ◽

Vol 151 (5) ◽

pp. 1453-1464 ◽

Cited By ~ 103

Author(s):

M. Gabriela Bowden ◽

Wei Chen ◽

Jenny Singvall ◽

Yi Xu ◽

Sharon J. Peacock ◽

...

Keyword(s):

Cell Wall ◽

Staphylococcus Epidermidis ◽

Tertiary Structure ◽

Genomic Sequence ◽

Human Infection ◽

Pcr Analysis ◽

Genes Encoding ◽

Antibody Titres

Staphylococcus epidermidis is a ubiquitous human skin commensal that has emerged as a major cause of foreign-body infections. Eleven genes encoding putative cell-wall-anchored proteins were identified by computer analysis of the publicly available S. epidermidis unfinished genomic sequence. Four genes encode previously described proteins (Aap, Bhp, SdrF and SdrG), while the remaining seven have not been characterized. Analysis of primary sequences of the Staphylococcus epidermidis surface (Ses) proteins indicates that they have a structural organization similar to the previously described cell-wall-anchored proteins from S. aureus and other Gram-positive cocci. However, not all of the Ses proteins are direct homologues of the S. aureus proteins. Secondary and tertiary structure predictions suggest that most of the Ses proteins are composed of several contiguous subdomains, and that the majority of these predicted subdomains are folded into β-rich structures. PCR analysis indicates that certain genes may be found more frequently in disease isolates compared to strains isolated from healthy skin. Patients recovering from S. epidermidis infections had higher antibody titres against some Ses proteins, implying that these proteins are expressed during human infection. Western blot analyses of early-logarithmic and late-stationary in vitro cultures suggest that different regulatory mechanisms control the expression of the Ses proteins.

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