scholarly journals Characterization of an ADP-Ribosyltransferase Toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

2002 ◽  
Vol 184 (7) ◽  
pp. 1851-1858 ◽  
Author(s):  
Martin Braun ◽  
Katja Stuber ◽  
Yvonne Schlatter ◽  
Thomas Wahli ◽  
Peter Kuhnert ◽  
...  
Keyword(s):  
Culture Medium ◽  
Morphological Changes ◽  
Polyclonal Antibodies ◽  
Sequence Similarity ◽  
Aeromonas Salmonicida ◽  
Wild Type ◽  
Significant Sequence Similarity ◽  
Fish Cells ◽  
Adp Ribosyltransferase

ABSTRACT An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.

scholarly journals Cloning and Characterization of theFlavobacterium johnsoniae Gliding Motility GenesgldD and gldE

2001 ◽  
Vol 183 (14) ◽  
pp. 4167-4175 ◽  
Author(s):  
David W. Hunnicutt ◽  
Mark J. McBride
Keyword(s):  
Membrane Protein ◽  
Cytoplasmic Membrane ◽  
Sequence Similarity ◽  
Gliding Motility ◽  
Wild Type ◽  
Bacteriophage Infection ◽  
Flavobacterium Johnsoniae ◽  
Latex Spheres ◽  
Serovar Typhimurium

ABSTRACT Cells of Flavobacterium johnsoniae move over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Cells of F. johnsoniaepropel latex spheres along their surfaces, which is thought to be a manifestation of the motility machinery. Three of the genes that are required for F. johnsoniae gliding motility,gldA, gldB, and ftsX, have recently been described. Tn4351 mutagenesis was used to identify another gene, gldD, that is needed for gliding. Tn4351-induced gldD mutants formed nonspreading colonies, and cells failed to glide. They also lacked the ability to propel latex spheres and were resistant to bacteriophages that infect wild-type cells. Introduction of wild-type gldD into the mutants restored motility, ability to propel latex spheres, and sensitivity to bacteriophage infection. gldD codes for a cytoplasmic membrane protein that does not exhibit strong sequence similarity to proteins of known function. gldE, which lies immediately upstream ofgldD, encodes another cytoplasmic membrane protein that may be involved in gliding motility. Overexpression ofgldE partially suppressed the motility defects of agldB point mutant, suggesting that GldB and GldE may interact. GldE exhibits sequence similarity to Borrelia burgdorferi TlyC and Salmonella enterica serovar Typhimurium CorC.

scholarly journals Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron

1990 ◽  
Vol 272 (3) ◽  
pp. 805-811 ◽  
Author(s):  
K Ramotar ◽  
B Boyd ◽  
G Tyrrell ◽  
J Gariepy ◽  
C Lingwood ◽  
...  
Keyword(s):  
Culture Medium ◽  
Polymyxin B ◽  
Exchange Chromatography ◽  
Structural Studies ◽  
Wild Type ◽  
Binding Studies ◽  
B Subunit ◽  
Tac Promoter ◽  
Glycolipid Receptor

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.

scholarly journals Trypanosome Telomeres Are Protected by a Homologue of Mammalian TRF2

2005 ◽  
Vol 25 (12) ◽  
pp. 5011-5021 ◽  
Author(s):  
Bibo Li ◽  
Amin Espinal ◽  
George A. M. Cross
Keyword(s):  
Dna Binding ◽  
Leishmania Major ◽  
Sequence Similarity ◽  
Significant Sequence Similarity ◽  
Telomere Protein ◽  
Binding Factor ◽  
Telomere Biology ◽  
Weak Similarity ◽  
Binding Component

ABSTRACT Putative TTAGGG repeat-binding factor (TRF) homologues in the genomes of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major were identified. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains but only weak similarity in their N-terminal domains. T. brucei TRF (tbTRF) is essential and was shown to bind to duplex TTAGGG repeats. The RNA interference-mediated knockdown of tbTRF arrested bloodstream cells at G2/M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knockdown diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and this also supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNA-binding protein and characterization of its function provide a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.

scholarly journals Inheritance of seedlessness and the molecular characterization of the INO gene in Annonaceae

2023 ◽  
Vol 83 ◽  
Author(s):  
B. R. R. M. Nassau ◽  
P. S. C. Mascarenhas ◽  
A. G. Guimarães ◽  
F. M. Feitosa ◽  
H. M. Ferreira ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Type A ◽  
Molecular Techniques ◽  
Wild Type ◽  
Annona Squamosa ◽  
High Sequence Similarity ◽  
Breeding Programs ◽  
Complete Dominance ◽  
Young Leaves

Abstract The inheritance of the seedless fruit characteristic of Annona squamosa has not yet been explained. Molecular techniques may aid breeding programs, mainly in the assisted selection of the target gene. The INO gene may be related to seed development in these fruits. The objective of the present paper was to investigate the inheritance of seedlessness in the 'Brazilian seedless' sugar apple and INO gene conservation in Annona squamosa and Annona cherimola x Annona squamosa genotypes by assessing their homology with the INO database genes. The F1 generation was obtained by crossing the mutant 'Brazilian seedless' (male genitor) (P1) with the wild-type A. squamosa with seeds (M1 and M2, female genitors). The INO gene was studied in mutant and wild-type A. squamosa (P1, M1, M2 and M3) and in the Gefner atemoya (A. cherimola x A. squamosa) (M4) cultivar. The DNA was extracted from young leaves, and four sets of specific primers flanking the INO gene were amplified. The seedless characteristic was identified as stenospermatic in the fruits of parental P1, suggesting monogenic inheritance with complete dominance. High sequence similarity of the INO gene amplifications in the sugar apple accessions (M1, M2, M3) and the atemoya cultivar Gefner (M4) reinforces the hypothesis of their conservation.

scholarly journals The ADP-Ribosylating Toxin, AexT, from Aeromonas salmonicida subsp. salmonicida Is Translocated via a Type III Secretion Pathway

2003 ◽  
Vol 185 (22) ◽  
pp. 6583-6591 ◽  
Author(s):  
Sarah E. Burr ◽  
Katja Stuber ◽  
Joachim Frey
Keyword(s):  
Type Iii Secretion ◽  
Aeromonas Salmonicida ◽  
Open Reading Frames ◽  
Type Iii ◽  
Secretion Pathway ◽  
Cultured Fish ◽  
Genes Encoding ◽  
Fish Cells ◽  
Adp Ribosyltransferase ◽  
Reading Frames

ABSTRACT AexT is an extracellular ADP ribosyltransferase produced by the fish pathogen Aeromonas salmonicida subsp. salmonicida. The protein is secreted by the bacterium via a recently identified type III secretion system. In this study, we have identified a further 12 open reading frames that possess high homology to genes encoding both structural and regulatory components of the Yersinia type III secretion apparatus. Using marker replacement mutagenesis of aopB, the A. salmonicida subsp. salmonicida homologue of yopB in Yersinia, we demonstrate that the bacterium translocates the AexT toxin directly into the cytosol of cultured fish cells via this type III secretion pathway. An acrV mutant of A. salmonicida subsp. salmonicida displays a calcium-blind phenotype, expressing and secreting significant amounts of AexT even in the presence of CaCl2 concentrations as high as 10 mM. This acrV mutant is also unable to translocate AexT into the cytosol of fish cells, indicating AcrV is involved in the translocation process. Inactivation of either the aopB or acrV gene in A. salmonicida subsp. salmonicida (resulting in an inability to translocate AexT) is accompanied by a loss of cytotoxicity that can be restored by trans complementation. Finally, we present data indicating that preincubation of the wild-type bacteria with antibodies directed against recombinant AcrV-His protein provides fish cells protection against the toxic effects of the bacterium.

scholarly journals Mutagenesis by Insertion of Tn4001 into the Genome of Spiroplasma citri: Characterization of Mutants Affected in Plant Pathogenicity and Transmission to the Plant by the Leafhopper Vector Circulifer haematoceps

1997 ◽  
Vol 10 (4) ◽  
pp. 454-461 ◽  
Author(s):  
X. Foissac ◽  
J. L. Danet ◽  
C. Saillard ◽  
P. Gaurivaud ◽  
F. Laigret ◽  
...  
Keyword(s):  
Type Strain ◽  
Culture Medium ◽  
Wild Type Strain ◽  
Single Copy ◽  
Wild Type ◽  
Spiroplasma Citri ◽  
Leafhopper Vector ◽  
Circulifer Haematoceps ◽  
Plant Pathogenicity

Two hundred and fifty-seven transposon Tn4001 mutants of Spiroplasma citri strain GII3 were used for transmission assays by the leafhopper vector Circulifer haematoceps into periwinkle (Catharanthus roseus) plants. Multiplication of the mutants in the two hosts, the leafhopper and the plant, as well as the symptom expression in the plant were studied. Two mutants, GMT 470 and GMT 553, caused no symptoms on plants. Tn4001 is inserted as a single copy in the genome of these mutants. Mutant GMT 470 did not multiply, or multiplied only poorly, in the leaf-hopper and was not transmitted by the insect to the plant, nor to culture medium through Parafilm membrane. The growth rate of GMT 470 in SP4 medium was twice as slow as that of wild-type strain GII3. Mutant GMT 553 multiplied in the leafhopper as well as the wild-type spiro-plasma, and was transmitted by the leafhoppers into the plants, where it reached the same titers as the wild-type strain but in approximately twice as much time. The plants containing high titers of mutant GMT 553 remained symptomless for several weeks. However, symptoms began to develop at a time when revertants that had lost the transposon were detected.

Study on biocharacteristics and puruvate production of moderately halophilic bacteria isolated from dunalliella tertiolecta’s culture medium

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Hoang Thi Lan Anh ◽  
Luu Thi Tam ◽  
Dang Diem Hong
Keyword(s):  
Fatty Acids ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Culture Medium ◽  
Sequence Similarity ◽  
Halophilic Bacteria ◽  
Rrna Gene ◽  
Wild Type ◽  
Moderately Halophilic ◽  
Moderately Halophilic Bacteria

A moderately halophilic bacteria designed strain D34 was isolated from the culture medium of green microalga Dunaliella tertiolecta. The isolate was Gram-negative, aerobic, rod-shaped, approximately 0.45–0.60 mm wide and 1.25–5.10 mm long, occuring singly, non-motile, and flagellum- less. Colonies on solid media are cream, circular, and smooth. This strain was able to produce exopolysaccharide, poly hydroxybutyrate, oxidase and catalase positive. Growth occurred in a temparature range of 20–40°C, a salts concentration of 0.1–25% (w/v), and pH range 6–12. The major fatty acids were C16:0 (35.59%), C16:1w-7 (20.54%), C18: 1w-7 (30.14%), and C12:0 (10.03% of total fatty acids). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain D34 belonged to the genus Halomonas. The highest levels of 16S rRNA gene sequence similarity were found between the strain D34 and H. aquamarina (sequence similarity 98.6 %).Pyruvate, a central intermediate in metabolism processes in all organisms, is widely used for the synthesis of various chemicals and polymers as well as ingredient or additive in food, cosmetics, and pharmaceuticals. In this study, pyruvate production by strain D34 following changes in culture medium, glucose and nitrate concentrations and culture temperature were also studied. In 84 hours of batch- cultivation, pyruvate production by wild-type Halomonas sp. D34 reached 37.24 g/L at 37°C with 20% glucose and 30 g/L sodium nitrate adding to SOT medium. These data provided evidences for pyruvate production using novel wild-type strains. 

scholarly journals Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils.

1996 ◽  
Vol 183 (6) ◽  
pp. 2437-2448 ◽  
Author(s):  
P D Ponath ◽  
S Qin ◽  
T W Post ◽  
J Wang ◽  
L Wu ◽  
...  
Keyword(s):  
Chemokine Receptors ◽  
Cell Lymphoma ◽  
Sequence Similarity ◽  
B Cell Lymphoma ◽  
Binding Studies ◽  
Significant Sequence Similarity ◽  
High Affinity ◽  
Chemotactic Protein ◽  
And Migration

The chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxin binding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues.

scholarly journals Mutagenesis and Functional Characterization of theglnB, glnA, and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

2000 ◽  
Vol 182 (4) ◽  
pp. 983-992 ◽  
Author(s):  
Yaoping Zhang ◽  
Edward L. Pohlmann ◽  
Paul W. Ludden ◽  
Gary P. Roberts
Keyword(s):  
Nitrogen Fixation ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Functional Characterization ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation ofnif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase–dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization ofglnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for PII) were constructed. While PII-Y51F showed a lower nitrogenase activity than that of wild type, a PIIdeletion mutant showed very little nif expression. This effect of PII on nif expression is apparently the result of a requirement of PII for NifA activation, whose activity is regulated by NH4 + in R. rubrum. The modification of glutamine synthetase (GS) in theseglnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of PII might exist inR. rubrum and regulate the modification of GS. PII also appears to be involved in the regulation of DRAT activity, since an altered response to NH4 + was found in a mutant expressing PII-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH4 + and darkness treatments.

Isolation and Characterization of Magbane, a Magnesium-Lethal Mutant of Paramecium

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1061-1069
Author(s):  
Jocelyn A Hammond ◽  
Robin R Preston
Keyword(s):  
Culture Medium ◽  
Single Gene ◽  
Wild Type ◽  
Lethal Mutant ◽  
Isolation And Characterization ◽  
Highly Sensitive ◽  
K Current ◽  
Mutant Phenotypes ◽  
Genetic Mutants

Abstract Discerning the mechanisms responsible for membrane excitation and ionic control in Paramecium has been facilitated by the availability of genetic mutants that are defective in these pathways. Such mutants typically are selected on the basis of behavioral anomalies or resistance to ions. There have been few attempts to isolate ion-sensitive strains, despite the insights that might be gained from studies of their phenotypes. Here, we report isolation of “magbane,” an ion-sensitive strain that is susceptible to Mg2+. Whereas the wild type tolerated the addition of ≥20 mm MgCl2 to the culture medium before growth was slowed and ultimately suppressed (at >40 mm), mgx mutation slowed growth at 10 mm. Genetic analysis indicated that the phenotype resulted from a recessive single-gene mutation that had not been described previously. We additionally noted that a mutant that was well described previously (restless) is also highly sensitive to Mg2+. This mutant is characterized by an inability to control membrane potential when extracellular K+ concentrations are lowered, due to inappropriate regulation of a Ca2+-dependent K+ current. However, comparing the mgx and rst mutant phenotypes suggested that two independent mechanisms might be responsible for their Mg2+ lethality. The possibility that mgx mutation may adversely affect a transporter that is required for maintaining low intracellular Mg2+ is considered.

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