scholarly journals l-Rhamnose Transport Is Sugar Kinase (RhaK) Dependent in Rhizobium leguminosarum bv. trifolii

2007 ◽  
Vol 189 (23) ◽  
pp. 8437-8446 ◽  
Author(s):  
Jason S. Richardson ◽  
Ivan J. Oresnik
Keyword(s):  
Abc Transporter ◽  
Rhizobium Leguminosarum ◽  
Nodule Occupancy ◽  
Vitro Assay ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Pentose Sugar ◽  
Sugar Kinase

ABSTRACT Strains of Rhizobium leguminosarum which are unable to catabolize l-rhamnose, a methyl-pentose sugar, are compromised in the ability to compete for nodule occupancy versus wild-type strains. Previous characterization of the 11-kb region necessary for the utilization of rhamnose identified a locus carrying catabolic genes and genes encoding the components of an ABC transporter. Genetic evidence suggested that the putative kinase RhaK carried out the first step in the catabolism of rhamnose. Characterization of this kinase led to the observation that strains carrying rhamnose kinase mutations were unable to transport rhamnose into the cell. The absence of a functional rhamnose kinase did not stop the transcription and translation of the ABC transporter components. By developing an in vitro assay for RhaK activity, we have been able to show that (i) RhaK activity is consistent with RhaK phosphorylating rhamnose and (ii) biochemical activity of RhaK is necessary for rhamnose transport.

scholarly journals A Genetic Locus Necessary for Rhamnose Uptake and Catabolism in Rhizobium leguminosarum bv. trifolii

2004 ◽  
Vol 186 (24) ◽  
pp. 8433-8442 ◽  
Author(s):  
Jason S. Richardson ◽  
Michael F. Hynes ◽  
Ivan J. Oresnik
Keyword(s):  
Abc Transporter ◽  
Rhizobium Leguminosarum ◽  
Genetic Locus ◽  
Negative Regulator ◽  
Nodule Occupancy ◽  
Structural Genes ◽  
Dot Blot ◽  
Genes Encoding ◽  
Sugar Kinase ◽  
Dot Blot Analysis

ABSTRACT Rhizobium leguminosarum bv. trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy. In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp. Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized. The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose. The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence. RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions. Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus. Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways.

scholarly journals Characterization of an L-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

2019 ◽  
Author(s):  
Tyler Stack ◽  
Katelyn Morrison ◽  
Thomas Dettmer ◽  
Brendan Wille ◽  
Chan Kim ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Sequence Similarity ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Benzilic Acid ◽  
Amidohydrolase Superfamily ◽  
Sequence Similarity Networks

<p>L-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. <i>Ralstonia eutropha </i>H16 (<i>Cupriavidus necator </i>ATCC 17699) uses L-ascorbate as sole carbon source but lacks the genes encoding the known catabolic pathways. RNAseq identified eight candidate catabolic genes. Sequence similarity networks and genome neighborhood networks guided predictions for function of the encoded proteins; the predictions were confirmed by <i>in vitro</i> assays and <i>in vivo</i> growth phenotypes of gene deletion mutants. L-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b<sub>561</sub> and gluconolactonase families, respectively, to form 2,3-diketo-L-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-L-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-L-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD<sup>+</sup> catalytically. The product, L-lyxonate, is catabolized to alpha-ketoglutarate by a previously characterized pathway.</p>

scholarly journals The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

2015 ◽  
Vol 197 (24) ◽  
pp. 3812-3821 ◽  
Author(s):  
Damien M. R. Rivers ◽  
Ivan J. Oresnik
Keyword(s):  
Abc Transporter ◽  
Rhizobium Leguminosarum ◽  
Kinase Activity ◽  
Bioinformatic Analysis ◽  
Nodule Occupancy ◽  
Alanine Scanning Mutagenesis ◽  
Content Type ◽  
Abc Protein ◽  
N Terminus ◽  
Sugar Kinase

ABSTRACTRhamnose catabolism inRhizobium leguminosarumwas found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded byrhaSTPQand on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis ofrhaKshowed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the allelesrhaK72andrhaK73were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined byrhaSTPQ. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either therhaK72orrhaK73allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly.IMPORTANCEABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are thecarbohydrateuptaketransporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport.

scholarly journals Characterization of an L-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

2019 ◽  
Author(s):  
Tyler Stack ◽  
Katelyn Morrison ◽  
Thomas Dettmer ◽  
Brendan Wille ◽  
Chan Kim ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Sequence Similarity ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Benzilic Acid ◽  
Amidohydrolase Superfamily ◽  
Sequence Similarity Networks

<p>L-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. <i>Ralstonia eutropha </i>H16 (<i>Cupriavidus necator </i>ATCC 17699) uses L-ascorbate as sole carbon source but lacks the genes encoding the known catabolic pathways. RNAseq identified eight candidate catabolic genes. Sequence similarity networks and genome neighborhood networks guided predictions for function of the encoded proteins; the predictions were confirmed by <i>in vitro</i> assays and <i>in vivo</i> growth phenotypes of gene deletion mutants. L-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b<sub>561</sub> and gluconolactonase families, respectively, to form 2,3-diketo-L-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-L-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-L-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD<sup>+</sup> catalytically. The product, L-lyxonate, is catabolized to alpha-ketoglutarate by a previously characterized pathway.</p>

In Vitro Assay for Single-cell Characterization of Impaired Deformability in Red Blood Cells under Recurrent Episodes of Hypoxia

Lab on a Chip ◽  
2021 ◽  
Author(s):  
YUHAO QIANG ◽  
Jia Liu ◽  
Ming Dao ◽  
E Du
Keyword(s):  
Shear Stress ◽  
Red Blood Cells ◽  
Single Cell ◽  
Blood Cells ◽  
Blood Circulation ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Cyclic Shear

Red blood cells (RBCs) are subjected to recurrent changes in shear stress and oxygen tension during blood circulation. The cyclic shear stress has been identified as an important factor that...

scholarly journals In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens

2009 ◽  
Vol 191 (7) ◽  
pp. 2033-2041 ◽  
Author(s):  
Meriyem Aktas ◽  
Franz Narberhaus
Keyword(s):  
Agrobacterium Tumefaciens ◽  
In Vitro Assay ◽  
Enzyme Properties ◽  
Vitro Assay ◽  
Plant Infection ◽  
In Vitro Characterization ◽  
End Products ◽  
And Control

ABSTRACT Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

scholarly journals In Vitro Import of a Nuclearly Encoded tRNA into Mitochondria of Solanum tuberosum

2003 ◽  
Vol 23 (11) ◽  
pp. 4000-4012 ◽  
Author(s):  
Ludovic Delage ◽  
André Dietrich ◽  
Anne Cosset ◽  
Laurence Maréchal-Drouard
Keyword(s):  
Solanum Tuberosum ◽  
Higher Plants ◽  
Plant Mitochondria ◽  
Protein Translation ◽  
Trna Genes ◽  
Vitro Assay ◽  
Trna Import

ABSTRACT Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNAAla, tRNAPhe, and tRNAMet-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNAAla than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

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

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1453-1464 ◽  
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.

scholarly journals A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

2005 ◽  
Vol 187 (10) ◽  
pp. 3374-3383 ◽  
Author(s):  
Christopher Stead ◽  
An Tran ◽  
Donald Ferguson ◽  
Sara McGrath ◽  
Robert Cotter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Lipid A ◽  
In Vitro Assay ◽  
Spectrometric Analysis ◽  
Vitro Assay ◽  
The Core ◽  
H Pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

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