scholarly journals Molecular characterization of the Agrobacterium tumefaciens DNA transfer protein VirB6

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3483-3492 ◽  
Author(s):  
Paul K. Judd ◽  
David Mahli ◽  
Anath Das
Keyword(s):  
Amino Acids ◽  
Agrobacterium Tumefaciens ◽  
Transmembrane Domain ◽  
Ti Plasmid ◽  
Dna Transfer ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Cell Pole ◽  
A Cell

The VirB proteins of Agrobacterium tumefaciens assemble a T-pilus and a type IV secretion (T4S) apparatus for the transfer of DNA and proteins to plant cells. VirB6 is essential for DNA transfer and is a polytopic integral membrane protein with at least four membrane-spanning domains. VirB6 is postulated to function in T-pilus biogenesis and to be a component of the T4S apparatus. To identify amino acids required for VirB6 function, random mutations were introduced into virB6, and mutants that failed to complement a deletion in virB6 in tumour formation assays were isolated. Twenty-one non-functional mutants were identified, eleven of which had a point mutation that led to a substitution in a single amino acid. Characterization of the mutants indicated that the N-terminal large periplasmic domain and the transmembrane domain TM3 are required for VirB6 function. TM3 has an unusual sequence feature in that it is rich in bulky hydrophobic amino acids. This feature is found conserved in the VirB6 family of proteins. Studies on the effect of VirB6 on other VirB proteins showed that the octopine Ti-plasmid VirB6, unlike its nopaline Ti-plasmid counterpart, does not affect accumulation of VirB3 and VirB5, but has a strong negative effect on the accumulation of the VirB7-VirB7 dimer. Using indirect immunofluorescence microscopy the authors recently demonstrated that VirB6 localizes to a cell pole in a VirB-dependent manner. Mutations identified in the present study did not affect polar localization of the protein or the formation of the VirB7-VirB7 dimer. A VirB6-GFP fusion that contained the entire VirB6 ORF did not localize to a cell pole in either the presence or the absence of the other VirB proteins. IMF studies using dual labelling demonstrated that VirB6 colocalizes with VirB3 and VirB9, and not with VirB4, VirB5 and VirB11. These results support the conclusion that VirB6 is a structural component of the T4S apparatus.

scholarly journals Correction: characterization of the virB operon from Agrobacterium tumefaciens Ti plasmid.

1990 ◽  
Vol 265 (8) ◽  
pp. 4768
Author(s):  
J E Ward ◽  
D E Akiyoshi ◽  
D Regier ◽  
A Datta ◽  
M P Gordon ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Ti Plasmid ◽  
Virb Operon

scholarly journals The Presence and Characterization of a virF Gene on Agrobacterium vitis Ti Plasmids

1998 ◽  
Vol 11 (5) ◽  
pp. 429-433 ◽  
Author(s):  
B. Schrammeijer ◽  
J. Hemelaar ◽  
P. J. J. Hooykaas
Keyword(s):  
Amino Acids ◽  
Agrobacterium Tumefaciens ◽  
Molecular Mass ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Tumor Formation ◽  
Nicotiana Glauca ◽  
Agrobacterium Vitis ◽  
Ti Plasmids

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plas-mids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.

Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

scholarly journals Complex Response of the CpxAR Two-Component System to β-Lactams on Antibiotic Resistance and Envelope Homeostasis in Enterobacteriaceae

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Muriel Masi ◽  
Elizabeth Pinet ◽  
Jean-Marie Pagès
Keyword(s):  
Efflux Pump ◽  
Multidrug Resistant ◽  
Diaminopimelic Acid ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Content Type ◽  
Promising Therapeutic Target ◽  
Abnormal Accumulation ◽  
A Cell ◽  
Mutational Activation

ABSTRACT The Cpx stress response is widespread among Enterobacteriaceae. We previously reported a mutation in cpxA in a multidrug-resistant strain of Klebsiella aerogenes isolated from a patient treated with imipenem. This mutation yields a single-amino-acid substitution (Y144N) located in the periplasmic sensor domain of CpxA. In this work, we sought to characterize this mutation in Escherichia coli by using genetic and biochemical approaches. Here, we show that cpxAY144N is an activated allele that confers resistance to β-lactams and aminoglycosides in a CpxR-dependent manner, by regulating the expression of the OmpF porin and the AcrD efflux pump, respectively. We also demonstrate the effect of the intimate interconnection between the Cpx system and peptidoglycan integrity on the expression of an exogenous AmpC β-lactamase by using imipenem as a cell wall-active antibiotic or by inactivating penicillin-binding proteins. Moreover, our data indicate that the Y144N substitution abrogates the interaction between CpxA and CpxP and increases phosphotransfer activity on CpxR. Because the addition of a strong AmpC inducer such as imipenem is known to cause abnormal accumulation of muropeptides (disaccharide-pentapeptide and N-acetylglucosamyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamy-meso-diaminopimelic-acid-d-alanyl-d-alanine) in the periplasmic space, we propose these molecules activate the Cpx system by displacing CpxP from the sensor domain of CpxA. Altogether, these data could explain why large perturbations to peptidoglycans caused by imipenem lead to mutational activation of the Cpx system and bacterial adaptation through multidrug resistance. These results also validate the Cpx system, in particular, the interaction between CpxA and CpxP, as a promising therapeutic target.

scholarly journals The Epigenetic Repertoire of Daphnia magna Includes Modified Histones

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Nicole F. Robichaud ◽  
Jeanette Sassine ◽  
Margaret J. Beaton ◽  
Vett K. Lloyd
Keyword(s):  
Life Cycle ◽  
Histone H3 ◽  
Dependent Manner ◽  
Nurse Cells ◽  
Important Species ◽  
Clonal Population ◽  
A Cell ◽  
Cycle Dependent

Daphnids are fresh water microcrustaceans, many of which follow a cyclically parthenogenetic life cycle. Daphnia species have been well studied in the context of ecology, toxicology, and evolution, but their epigenetics remain largely unexamined even though sex determination, the production of sexual females and males, and distinct adult morphological phenotypes, are determined epigenetically. Here, we report on the characterization of histone modifications in Daphnia. We show that a number of histone H3 and H4 modifications are present in Daphnia embryos and histone H3 dimethylated at lysine 4 (H3K4me2) is present nonuniformly in the nucleus in a cell cycle-dependent manner. In addition, this histone modification, while present in blastula and gastrula cells as well as the somatic cells of adults, is absent or reduced in oocytes and nurse cells. Thus, the epigenetic repertoire of Daphnia includes modified histones and as these epigenetic forces act on a genetically homogeneous clonal population Daphnia offers an exceptional tool to investigate the mechanism and role of epigenetics in the life cycle and development of an ecologically important species.

scholarly journals Determinants of Dengue Virus NS4A Protein Oligomerization

2015 ◽  
Vol 89 (12) ◽  
pp. 6171-6183 ◽  
Author(s):  
Chia Min Lee ◽  
Xuping Xie ◽  
Jing Zou ◽  
Shi-Hua Li ◽  
Michelle Yue Qi Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dengue Virus ◽  
Viral Replication ◽  
Transmembrane Domain ◽  
Antiviral Drug ◽  
Protein Oligomerization ◽  
Dependent Manner ◽  
Wild Type ◽  
Replication Complex ◽  
Host Membrane

ABSTRACTFlavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1.transcomplementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication.IMPORTANCEWe report that DENV NS4A forms oligomers. Such NS4A oligomerization is mediated mainly through amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]). The biological importance of NS4A oligomerization is demonstrated by results showing that mutations of flavivirus conserved residues (E50A and G67A located within TMD1) reduced the oligomerization and stability of the NS4A protein, leading to attenuated viral replication. A systematic mutagenesis analysis demonstrated that flavivirus conserved NS4A residues are important for DENV replication. A successfultranscomplementation of replication-lethal NS4A mutant virus requires wild-type NS4A in the context of the viral replication complex. The wild-type NS4A protein alone is not sufficient to rescue the replication defect of NS4A mutants. Intriguingly, distinct NS4A mutants yielded different complementation outcomes in the replicon-containing cells. Overall, the study has enhanced our understanding of flavivirus NS4A at the molecular level. The results also suggest that inhibitor blocking of NS4A oligomerization could be explored for antiviral drug discovery.

scholarly journals Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter

1988 ◽  
Vol 8 (3) ◽  
pp. 1327-1335
Author(s):  
J V Cox ◽  
E Lazarides
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Transporter Protein ◽  
Isolation And Characterization ◽  
Anion Transporter ◽  
Primary Structures ◽  
Membrane Spanning

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The approximately 90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as well as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid cells in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments.

scholarly journals Amyloid like Aggregates Formed by the Self-Assembly of Proline and Hydroxyproline

2021 ◽  
Author(s):  
Bharti Koshti ◽  
Ramesh, Singh ◽  
Vivekshinh Kshtriya ◽  
Shanka Walia ◽  
Dhiraj Bhatia ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Self Assembly ◽  
Metabolic Disorders ◽  
Research Work ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Force Microscopy ◽  
Aggregation Behaviour ◽  
Significant Research

<p>Single amino acid based self-assembled structures have gained a lot of interest recently owing to their pathological significance in metabolite disorders. There is plethora of significant research work which illustrate amyloid like characteristics of assemblies formed by aggregation of single amino acids like Phenylalanine, Tyrosine, Tryptophan, Cysteine and Methionine and its implications in pathophysiology of single amino acid metabolic disorders like phenylketonuria, tyrosinemia, hypertryptophanemia, cystinuria and hypermethioninemia respectively. Hence, studying aggregation behaviour of single amino acids is very crucial to assess the underlying molecular mechanism behind metabolic disorders. In this manuscript we report for the very first time the aggregation properties of non-aromatic single amino acids Hydroxy-proline and Proline. The morphologies of these were studied extensively by Optical microscopy (OM), ThT binding fluorescence microscopy, Scanning Electron Microscopy (SEM) and Atomic force microscopy (AFM). It can be assessed that these amino acids form globular structures at lower concentrations and gradually changes to tape like structures on increasing the concentration as assessed by AFM. ThT and CR binding assay reveal the aggregates do have amyloid like characteristics. Further MTT assays on SHSY5Y neural cell lines reveal cytotoxicity and the aggregates caused significant cell death in dose dependent manner. These results have important implications in understanding the pathophysiology of single amino acid disorders like Hyperprolinemia and Hydroxyprolinemia in association with amyloid diseases. The symptoms of these diseases are also accompanied by extensive neurological problems like intellectual disability, seizures and psychiatric problems which further evince amyloid like etiology for these rare in-born errors of metabolism.</p>

Anionic amino acid uptake by microvillous membrane vesicles from human placenta

1989 ◽  
Vol 257 (5) ◽  
pp. C1005-C1011 ◽  
Author(s):  
A. J. Moe ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamate Uptake ◽  
Membrane Vesicles ◽  
Amino Acid Uptake ◽  
Maternal Blood ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Concentration Dependent Manner

The transport mechanisms for anionic amino acids in trophoblast microvillous (maternal facing) membrane were investigated by characterization of L-[3H]aspartate and L-[3H]glutamate uptake in membrane vesicles. Uptake of the anionic amino acids was by a single high-affinity Na+-dependent K+-stimulated cotransporter that is pH sensitive and electrogenic. A second Na+-dependent transporter could not be discriminated, and there was no observable Na+-independent uptake. An outwardly directed K+ gradient (100 mM KCl inside) resulted in a 5- to 10-fold stimulation in glutamate uptake in the presence of Na+. Intravesicular KCl had no effect on transporter affinity but increased transporter velocity in a concentration-dependent manner. Inhibition of Na+-K+-dependent uptake of L-aspartate and L-glutamate (20 mM, 30 s) by 2 mM unlabeled amino acids demonstrated stereoselectivity for L-glutamate but not for L-aspartate. The neutral amino acids (L-alanine, L-threonine, L-serine, L-cysteine, L-phenylalanine) were not effective inhibitors. These data are consistent with an anionic amino acid transporter in the microvillous membrane of the trophoblast, which has characteristics qualitatively similar to the X-AG system found in other epithelia. This system may mediate the concentrative placental uptake of anionic amino acids from maternal blood in utero.

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