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Michio Kurosu - One of the best experts on this subject based on the ideXlab platform.
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fluorescence based assay for polyprenyl phosphate glcnac 1 phosphate transferase weca and identification of novel antimycobacterial weca inhibitors
Analytical Biochemistry, 2016Co-Authors: Katsuhiko Mitachi, Shajila Siricilla, Dong Yang, Ying Kong, Karolina Skorupinskatudek, Ewa Swiezewska, Scott G Franzblau, Michio KurosuAbstract:Polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored Glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating states, making this enzyme a target for development of novel drugs. Here, we report a fluorescence-based method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C6-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C6-FITC (1) can be converted to the corresponding decaprenyl-P-P-Glucosamine-C6-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C6-FITC is readily extracted with n-butanol and can be quantified by ultraviolet–visible (UV–vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA inhibitors.
Imen Nouioui - One of the best experts on this subject based on the ideXlab platform.
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streptacidiphilus bronchialis sp nov a ciprofloxacin resistant bacterium from a human clinical specimen reclassification of streptomyces griseoplanus as streptacidiphilus griseoplanus comb nov and emended description of the genus streptacidiphilus
International Journal of Systematic and Evolutionary Microbiology, 2019Co-Authors: Imen Nouioui, Hanspeter Klenk, Jose M Igual, Christopher A Gulvik, Brent A Lasker, John R McquistonAbstract:The taxonomic position of strain 15-057AT, an acidophilic actinobacterium isolated from the bronchial lavage of an 80-year-old male, was determined using a polyphasic approach incorporating morphological, phenotypic, chemotaxonomic and genomic analyses. Pairwise 16S rRNA gene sequence similarities calculated using the GGDC web server between strain 15-057AT and its closest phylogenetic neighbours, Streptomyces griseoplanus NBRC 12779T and Streptacidiphilus oryzae TH49T, were 99.7 and 97.6 %, respectively. The G+C content of isolate 15-057AT was determined to be 72.6 mol%. DNA–DNA relatedness and average nucleotide identity between isolate 15-057AT and Streptomyces griseoplanus DSM 40009T were 29.2±2.5 % and 85.97 %, respectively. Chemotaxonomic features of isolate 15-057AT were consistent with its assignment within the genus Streptacidiphilus : the whole-cell hydrolysate contained ll-diaminopimelic acid as the diagnostic diamino acid and glucose, mannose and ribose as cell-wall sugars; the major menaquinone was MK9(H8); the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, Glycophospholipid, aminoGlycophospholipid and an unknown lipid; the major fatty acids were anteiso-C15 : 0 and iso-C16 : 0. Phenotypic and morphological traits distinguished isolate 15-057AT from its closest phylogenetic neighbours. The results of our taxonomic analyses showed that strain 15-057AT represents a novel species within the evolutionary radiation of the genus Streptacidiphilus , for which the name Streptacidiphilus bronchialis sp. nov. is proposed. The type strain is 15-057AT (=DSM 106435T=ATCC BAA-2934T).
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frankia irregularis sp nov an actinobacterium unable to nodulate its original host casuarina equisetifolia but effectively nodulates members of the actinorhizal rhamnales
International Journal of Systematic and Evolutionary Microbiology, 2018Co-Authors: Imen Nouioui, Faten Ghodhbanegtari, Hanspeter Klenk, Manfred Rhode, Vartul Sangal, Maher GtariAbstract:A red pigmented actinobacterium designated G2T, forming extremely branched vegetative hyphae, vesicles and mutilocular sporangia, was isolated from Casuarina equisetifolia nodules. The strain failed to nodulate its original host plant but effectively nodulated members of actinorhizal Rhamnales. The taxonomic position of G2T was determined using a polyphasic approach. The peptidoglycan of the strain contained meso-diaminopimelic acid as diagnostic diamino acid, galactose, glucose, mannose, rhamnose, ribose and xylose. The polar lipid pattern consisted of phosphatidylinositol (PI), diphosphatidylglycerol (DPG), Glycophospholipids (GPL1–2), phosphatidylglycerol (PG), aminophospholipid (APL) and unknown lipids (L). The predominant menaquinones were MK-9 (H4) and MK-9 (H6) while the major fatty acids were iso-C16 : 0, C17 : 1ω8c and C15 : 0. The size of the genome of G2T was 9.5 Mb and digital DNA G+C content was 70.9 %. The 16S rRNA gene showed 97.4–99.5 % sequence identity with the type strains of species of the genus Frankia . Digital DNA –DNA hybridisation (dDDH) values between G2T and its nearest phylogenetic neighbours Frankia elaeagni and Frankia discariae were below the threshold of 70 %. On the basis of these results, strain G2T (=DSM 45899T=CECT 9038T) is proposed to represent the type strain of a novel species Frankia irregularis sp. nov.
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Frankia discariae sp. nov.: an infective and effective microsymbiont isolated from the root nodule of Discaria trinervis
Archives of Microbiology, 2017Co-Authors: Imen Nouioui, Manfred Rohde, Hanspeter Klenk, Maria Carmen Montero-calasanz, Faten Ghodhbane-gtari, Louis S. Tisa, Maher GtariAbstract:Strain BCU110501^T was the first isolate reported to fulfill Koch’s postulates by inducing effective nodules on its host plant of origin Discaria trinervis ( Rhalmnaceae ). Based on 16S rRNA gene sequence similarities, the strain was found to be most closely related to the type strain of Frankia elaeagni DSM 46783^T (98.6%) followed by F. alni DSM 45986^T (98.2%), F. casuarinae DSM 45818^T (97.8%) and F. inefficacies DSM 45817^T (97.8%). Digital DNA:DNA hybridizations (dDDH) between strain BCU110501^Tand the type strains of other Frankia species were clearly below the cutoff point of 70%. The G+C content of DNA is 72.36%. The cell wall of strain BCU110501^T contained meso -diaminopimelic acid and the cell sugars were galactose, glucose, mannose, xylose and ribose. Polar lipids were phosphatidylinositol (PI), diphosphatidylglycerol (DPG), Glycophospholipid (GPL_1−3), phosphatidylglycerol (PG) and an unknown lipid (L). The major fatty acids of strain BCU110501^T consisted of iso -C16:0, C17:1 w8c and C16:0. Major menaquinones were MK9 (H_4), MK9 (H_6) and MK9 (H_2). Based on these analyses, strain BCU110501^T (=DSM 46785^T=CECT 9042^T) should be classified as the type strain of a novel Frankia species, for which the name Frankia discariae sp. nov. is proposed.
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frankia coriariae sp nov an infective and effective microsymbiont isolated from coriaria japonica
International Journal of Systematic and Evolutionary Microbiology, 2017Co-Authors: Imen Nouioui, Faten Ghodhbanegtari, Manfred Rohde, Hanspeter Klenk, Maher GtariAbstract:The taxonomic description of a nitrogen-fixing actinobacterium, strain BMG5.1T, as a novel species within the genus Frankia was based on a polyphasic approach. The strain was isolated from the root nodules of Coriaria japonica, and it fulfilled Koch’s postulates by inducing effective nodules on Coriaria spp. and Datisca spp. Based on phenotypic and chemotaxonomic features, strain BMG5.1T is distinguishable from all other species of the genus Frankia . It is characterized by the presence of phosphatidylinositol, diphosphatidylglycerol, phosphatidylglycerol and Glycophospholipids in its polar lipids; galactose, glucose, mannose and a trace of ribose as cellular sugars; meso-diaminopimelic acid as cell-wall peptidoglycan; C18 : 1ω9c as major fatty acid (>30 %); and MK-9(H6) (44.7 %) as predominant isoprenolog (>30 %). The 16S rRNA gene sequence similarities and the digital DNA–DNA hybridization values between the described novel strain and strains of the other species of the genus Frankia correspond to a range of 97–98.4 % and 22.1–24 %, respectively. The DNA G+C content is 70.2 mol%. On the basis of these results, strain BMG5.1T (=CECT 9032T=DSM 100624T) is proposed as the type strain of a novel species of the genus Frankia , named Frankia coriariae sp. nov.
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Frankia inefficax sp. nov., an actinobacterial endophyte inducing ineffective, non nitrogen-fixing, root nodules on its actinorhizal host plants
Antonie van Leeuwenhoek, 2017Co-Authors: Imen Nouioui, Manfred Rohde, Maher Gtari, Maria Carmen Montero-calasanz, Faten Ghodhbane-gtari, Louis S. Tisa, Hanspeter KlenkAbstract:Strain EuI1c^T is the first actinobacterial endophyte isolated from Elaeagnus umbellata that was shown to be infective on members of Elaeagnaceae and Morella but lacking the ability to form effective root nodules on its hosts. The strain can be easily distinguished from strains of other Frankia species based on its inability to produce vesicles, the specialized thick-walled structures where nitrogen fixation occurs. Chemotaxonomically, strain EuI1c^T contains phosphatidylinositol, diphosphatidylglycerol, two Glycophospholipids and phosphatidylglycerol as phospholipids. The whole cell sugars were composed of glucose, galactose, mannose, ribose, rhamnose and fucose as diagnostic sugars of the species. Major fatty acids were iso -C_16:0, C_17:1 ω 8c and C_15:0 and C_17:0 and the predominant menaquinones were MK-9(H_6), MK-9(H_8) and MK-9(H_4). Analysis of the 16S rRNA gene sequence of strain EuI1c^T showed 97, 97.4 and 97.9% identity with Frankia elaeagni DSM 46783^T, Frankia casuarinae DSM 45818^T and Frankia alni DSM 45986^T, respectively. Digital DNA:DNA hybridizations with type strains of the three Frankia species with validly/effectively published names are significantly below 70%. These results warrant distinction of EuI1c^T (= DSM 45817^T = CECT 9037^T) as the type strain of a novel species designated Frankia inefficax sp. nov.
Katsuhiko Mitachi - One of the best experts on this subject based on the ideXlab platform.
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fluorescence based assay for polyprenyl phosphate glcnac 1 phosphate transferase weca and identification of novel antimycobacterial weca inhibitors
Analytical Biochemistry, 2016Co-Authors: Katsuhiko Mitachi, Shajila Siricilla, Dong Yang, Ying Kong, Karolina Skorupinskatudek, Ewa Swiezewska, Scott G Franzblau, Michio KurosuAbstract:Polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored Glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating states, making this enzyme a target for development of novel drugs. Here, we report a fluorescence-based method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C6-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C6-FITC (1) can be converted to the corresponding decaprenyl-P-P-Glucosamine-C6-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C6-FITC is readily extracted with n-butanol and can be quantified by ultraviolet–visible (UV–vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA inhibitors.
Ingrid W Caras - One of the best experts on this subject based on the ideXlab platform.
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An internally positioned signal can direct attachment of a Glycophospholipid membrane anchor.
The Journal of cell biology, 1991Co-Authors: Ingrid W CarasAbstract:All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.
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Glycophospholipid membrane anchor attachment molecular analysis of the cleavage attachment site
Journal of Biological Chemistry, 1991Co-Authors: Paul Moran, Helga Raab, William J Kohr, Ingrid W CarasAbstract:The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.
John R Mcquiston - One of the best experts on this subject based on the ideXlab platform.
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streptacidiphilus bronchialis sp nov a ciprofloxacin resistant bacterium from a human clinical specimen reclassification of streptomyces griseoplanus as streptacidiphilus griseoplanus comb nov and emended description of the genus streptacidiphilus
International Journal of Systematic and Evolutionary Microbiology, 2019Co-Authors: Imen Nouioui, Hanspeter Klenk, Jose M Igual, Christopher A Gulvik, Brent A Lasker, John R McquistonAbstract:The taxonomic position of strain 15-057AT, an acidophilic actinobacterium isolated from the bronchial lavage of an 80-year-old male, was determined using a polyphasic approach incorporating morphological, phenotypic, chemotaxonomic and genomic analyses. Pairwise 16S rRNA gene sequence similarities calculated using the GGDC web server between strain 15-057AT and its closest phylogenetic neighbours, Streptomyces griseoplanus NBRC 12779T and Streptacidiphilus oryzae TH49T, were 99.7 and 97.6 %, respectively. The G+C content of isolate 15-057AT was determined to be 72.6 mol%. DNA–DNA relatedness and average nucleotide identity between isolate 15-057AT and Streptomyces griseoplanus DSM 40009T were 29.2±2.5 % and 85.97 %, respectively. Chemotaxonomic features of isolate 15-057AT were consistent with its assignment within the genus Streptacidiphilus : the whole-cell hydrolysate contained ll-diaminopimelic acid as the diagnostic diamino acid and glucose, mannose and ribose as cell-wall sugars; the major menaquinone was MK9(H8); the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, Glycophospholipid, aminoGlycophospholipid and an unknown lipid; the major fatty acids were anteiso-C15 : 0 and iso-C16 : 0. Phenotypic and morphological traits distinguished isolate 15-057AT from its closest phylogenetic neighbours. The results of our taxonomic analyses showed that strain 15-057AT represents a novel species within the evolutionary radiation of the genus Streptacidiphilus , for which the name Streptacidiphilus bronchialis sp. nov. is proposed. The type strain is 15-057AT (=DSM 106435T=ATCC BAA-2934T).