scholarly journals The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

2009 ◽  
Vol 76 (4) ◽  
pp. 1267-1273 ◽  
Author(s):  
Camilla Oppegård ◽  
Linda Emanuelsen ◽  
Lisbeth Thorbek ◽  
Gunnar Fimland ◽  
Jon Nissen-Meyer
Keyword(s):  
Structural Model ◽  
Transmembrane Helix ◽  
Cellular Component ◽  
Expression Vectors ◽  
Wild Type ◽  
Terminal Part ◽  
Gene Encoding ◽  
Immunity Protein ◽  
Hybrid Peptides ◽  
Helix Structure

ABSTRACT Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (lagC) or the gene encoding the enterocin 1071 immunity protein (entI) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the lagC gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 519d-519 ◽  
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Height ◽  
Dry Weight ◽  
Nicotiana Alata ◽  
Wild Type ◽  
Gene Encoding ◽  
Delayed Senescence ◽  
Gene Construct ◽  
Shoot Dry Weight ◽  
Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals A BAFF-R mutation associated with non-Hodgkin lymphoma alters TRAF recruitment and reveals new insights into BAFF-R signaling

2010 ◽  
Vol 207 (12) ◽  
pp. 2569-2579 ◽  
Author(s):  
Joanne M. Hildebrand ◽  
Zhenghua Luo ◽  
Michelle K. Manske ◽  
Tammy Price-Troska ◽  
Steven C. Ziesmer ◽  
...  
Keyword(s):  
B Cell ◽  
Hodgkin Lymphoma ◽  
Novel Mutation ◽  
B Cell Development ◽  
Signaling Cascades ◽  
Binding Motif ◽  
Wild Type ◽  
Immunoglobulin Production ◽  
Gene Encoding ◽  
Non Hodgkin Lymphoma

The cytokine B cell activating factor (BAFF) and its receptor, BAFF receptor (BAFF-R), modulate signaling cascades critical for B cell development and survival. We identified a novel mutation in TNFRSF13C, the gene encoding human BAFF-R, that is present in both tumor and germline tissue from a subset of patients with non-Hodgkin lymphoma. This mutation encodes a His159Tyr substitution in the cytoplasmic tail of BAFF-R adjacent to the TRAF3 binding motif. Signaling through this mutant BAFF-R results in increased NF-κB1 and NF-κB2 activity and increased immunoglobulin production compared with the wild-type (WT) BAFF-R. This correlates with increased TRAF2, TRAF3, and TRAF6 recruitment to His159Tyr BAFF-R. In addition, we document a requirement for TRAF6 in WT BAFF-R signaling. Together, these data identify a novel lymphoma-associated mutation in human BAFF-R that results in NF-κB activation and reveals TRAF6 as a necessary component of normal BAFF-R signaling.

scholarly journals Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

2011 ◽  
Vol 25 (10) ◽  
pp. 1804-1818 ◽  
Author(s):  
K. Coopman ◽  
R. Wallis ◽  
G. Robb ◽  
A. J. H. Brown ◽  
G. F. Wilkinson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Agonist Affinity ◽  
Camp Generation ◽  
N Terminus ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

scholarly journals A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

2003 ◽  
Vol 14 (4) ◽  
pp. 1727-1743 ◽  
Author(s):  
Binggang Sun ◽  
Richard A. Firtel
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Pleckstrin Homology Domain ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

scholarly journals Enlarged Peroxisomes Are Present in Oleic Acid–grown Yarrowia lipolytica Overexpressing the PEX16 Gene Encoding an Intraperoxisomal Peripheral Membrane Peroxin

1997 ◽  
Vol 137 (6) ◽  
pp. 1265-1278 ◽  
Author(s):  
Gary A. Eitzen ◽  
Rachel K. Szilard ◽  
Richard A. Rachubinski
Keyword(s):  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Consensus Sequence ◽  
Wild Type ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
Peripheral Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

scholarly journals The Synechococcus Strain PCC 7942glnN Product (Glutamine Synthetase III) Helps Recovery from Prolonged Nitrogen Chlorosis

2000 ◽  
Vol 182 (19) ◽  
pp. 5615-5619 ◽  
Author(s):  
Jörg Sauer ◽  
Ulrike Dirmeier ◽  
Karl Forchhammer
Keyword(s):  
Glutamine Synthetase ◽  
Transcriptional Start Site ◽  
Binding Motif ◽  
Class Iii ◽  
Wild Type ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
Low Concentrations ◽  
Synechococcus Strain ◽  
Pcc 7942

ABSTRACT We report the cloning and sequencing of the glnN gene encoding a class III glutamine synthetase from the cyanobacteriumSynechococcus strain PCC 7942. Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif. Expression ofglnN is impaired in NtcA- and PII-deficient mutants. The only parameter which was negatively affected in theglnN mutant compared to the wild type was the recovery rate of prolonged nitrogen-starved cells with low concentrations of combined nitrogen.

scholarly journals Characterization of the Gene Encoding the Major Secreted Lysophospholipase A of Legionella pneumophila and Its Role in Detoxification of Lysophosphatidylcholine

2002 ◽  
Vol 70 (11) ◽  
pp. 6094-6106 ◽  
Author(s):  
Antje Flieger ◽  
Birgid Neumeister ◽  
Nicholas P. Cianciotto
Keyword(s):  
Fatty Acids ◽  
Legionella Pneumophila ◽  
Lipolytic Activity ◽  
Cholesterol Acyltransferase ◽  
Phospholipase A ◽  
Wild Type ◽  
Gene Encoding ◽  
Acyltransferase Activity ◽  
Lipolytic Enzymes ◽  
Cloned Gene

ABSTRACT We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An L. pneumophila plaA mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L. pneumophila. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA. Overexpression of plaA completely protected L. pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L. pneumophila. In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L. pneumophila.

scholarly journals IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

2009 ◽  
Vol 20 (13) ◽  
pp. 3055-3063 ◽  
Author(s):  
Raqual Bower ◽  
Kristyn VanderWaal ◽  
Eileen O'Toole ◽  
Laura Fox ◽  
Catherine Perrone ◽  
...  
Keyword(s):  
Double Mutant ◽  
Essential Role ◽  
Null Allele ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Mutant Strains ◽  
Dynein Arms ◽  
Image Averaging

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

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