scholarly journals HMW1 Is Required for Cytadhesin P1 Trafficking to the Attachment Organelle in Mycoplasma pneumoniae

1998 ◽  
Vol 180 (5) ◽  
pp. 1270-1276 ◽  
Author(s):  
Tae-Wook Hahn ◽  
Melisa J. Willby ◽  
Duncan C. Krause
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Genetic Manipulation ◽  
Full Length ◽  
Wild Type ◽  
Functional Importance ◽  
Normal Morphology ◽  
Terminal Domain ◽  
Terminal Structure

ABSTRACT Mycoplasma pneumoniae proteins HMW1-HMW3 collectively are essential for cytadherence, but the function or requirement for each has not been defined. Cytadherence mutant M6 lacks HMW1 because of a frameshift in hmw1 and produces a truncated adherence-associated protein P30 because of a deletion at the 3′ end ofp30. Genetic manipulation of this mutant was used to evaluate the role of HMW1 in cytadherence. Mutant M6 was transformed with a recombinant transposon containing a wild-type p30allele. Transformants synthesized both truncated and full-length P30, from the resident and recombinant alleles, respectively. However, these transformants remained hemadsorption negative, suggesting that HMW1 is required for cytadherence. Wild-type M. pneumoniaecells are generally elongated, tapering to form the attachment organelle at one end of the cell. The cytadhesin protein P1 is normally densely clustered on the mycoplasma surface at this differentiated terminal structure. However, both mutant M6 and M6 transformed with recombinant p30 had a striking ovoid morphology with no tapering at the tip structure, making the attachment organelle indistinguishable. Furthermore, protein P1 was randomly distributed on the mycoplasma surface rather than clustered at a polar location. In contrast, mutant M6 transformed with a recombinant transposon expressing the wild-type hmw1 allele exhibited a near-normal morphology and localized P1 to the attachment organelle. Significantly, M6 transformed with an hmw1 gene truncated slightly at the 3′ end failed to restore proper morphology or P1 localization to the attachment organelle, suggesting a functional importance to the C-terminal domain of HMW1.

scholarly journals Oligomerization and hemolytic properties of the C-terminal domain of pyolysin, a cholesterol-dependent cytolysin

2013 ◽  
Vol 91 (2) ◽  
pp. 59-66 ◽  
Author(s):  
Lisa Pokrajac ◽  
J. Robin Harris ◽  
Naghmeh Sarraf ◽  
Michael Palmer
Keyword(s):  
Full Length ◽  
Membrane Insertion ◽  
Wild Type ◽  
Terminal Domain ◽  
Large Size ◽  
Monomer Structure ◽  
Streptolysin O ◽  
Kinetics Of ◽  
Domain 4

Pyolysin (PLO) belongs to the homologous family of the cholesterol-dependent cytolysins (CDCs), which bind to cell membranes containing cholesterol to form oligomeric pores of large size. The CDC monomer structure consists of 4 domains. Among these, the C-terminal domain 4 has been implicated in membrane binding of the monomer, while the subsequent processes of oligomerization and membrane insertion have primarily been assigned to other domains of the molecule. Recombinantly expressed or proteolytic fragments that span domain 4 of the CDCs streptolysin O and perfringolysin O bind to membranes but fail to oligomerize, and they inhibit the activity of the respective wild-type toxins. We report here that the isolated domain 4 of pyolysin (PLO-D4) not only binds to membranes but also forms oligomers with itself, as well as hybrid oligomers with the full-length toxin. As expected, the pure PLO-D4 oligomers are devoid of pore-forming activity. Surprisingly, however, within hybrid oligomers, PLO-D4 not only fails to inhibit, but even amplifies the hemolytic activity of the full-length toxin, to an extent similar to that of doubling the amount of the full-length toxin alone. We propose that this amplification may be related to the kinetics of the oligomerization reaction. Overall, our findings indicate a greater role of domain 4 in the oligomerization of CDCs than previously demonstrated.

scholarly journals Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

2020 ◽  
Author(s):  
Aniefon Ibuot ◽  
Rachel E. Webster ◽  
Lorraine E. Williams ◽  
Jon K. Pittman
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Metal Binding ◽  
Metal Tolerance ◽  
Alginate Beads ◽  
Full Length ◽  
Wild Type ◽  
Microalgal Biomass ◽  
Terminal Domain ◽  
Metal Biosorption ◽  
Transgenic Cells

AbstractThe use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalisation of both metals compared to wild type. AtHMA4 was expressed either as the full-length protein or just the C-terminal tail, which is known to have metal binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the full-length protein or C-terminal domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilisation in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilisation maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild type cells. This demonstrates that expressing AtHMA4 full-length or C-terminus has great potential as a strategy for bioremediation using microalgal biomass.

Genetic Manipulation of Key Photosynthetic Enzymes in the C4 Plant Flaveria bidentis

1997 ◽  
Vol 24 (4) ◽  
pp. 477 ◽  
Author(s):  
Robert T. Furbank ◽  
Julie A. Chitty ◽  
Colin L.D. Jenkins ◽  
William C. Taylor ◽  
Stephen J. Trevanion ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Malate Dehydrogenase ◽  
Genetic Manipulation ◽  
Photosynthetic Performance ◽  
Covalent Modification ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Enzymes ◽  
Flaveria Bidentis

The NADP-malic enzyme type C4 dicot Flaveria bidentis (L.) Kuntze was transformed with antisense and cosense gene constructs that resulted in specific decreases in single photosynthetic enzymes. The enzymes targeted were ribulose-1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39] (Rubisco), pyruvate, Pi dikinase [EC 2.7.9.1] (PPDK) and NADP malate dehydrogenase [EC 1.1.1.82] (NADP-MDH). These enzymes were chosen as targets because they have low activity compared to photosynthetic rates (Rubisco), are subject to complex covalent regulation (NADP-MDH), or both (PPDK). T1 progeny of a number of lines of these transformants were examined for the effects of these gene constructs on enzyme levels and photosynthetic performance. Rubisco antisense transformants expressing between 15 and 100% of wild-type enzyme activity were obtained. Pyruvate, Pi dikinase antisense lines were obtained with 40–100% wild-type levels. NADP malate dehydrogenase sense constructs caused a co-suppression of enzyme activity with some lines containing less than 2% of wild- type activity. Under saturating illumination, the control coefficients for these enzymes were determined to be up to 0.7 for Rubisco, 0.2–0.3 for PPDK and effectively zero for NADP-MDH. The implications of these observations for the regulation of photosynthetic flux and metabolism in C4 plants and the role of regulation by covalent modification are discussed.

scholarly journals Identification and Complementation of a Mutation Associated with Loss of Mycoplasma pneumoniae Virulence-Specific Proteins B and C

2005 ◽  
Vol 187 (2) ◽  
pp. 747-751 ◽  
Author(s):  
Robert H. Waldo ◽  
Jarrat L. Jordan ◽  
Duncan C. Krause
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Wild Type Allele ◽  
Wild Type ◽  
Type Allele ◽  
Specific Proteins

ABSTRACT A mutation in gene MPN142 (orf6) was identified in the Mycoplasma pneumoniae cytadherence mutant III-4. MPN142 encodes virulence-specific proteins P90 and P40 (proteins B and C, respectively). Analysis of MPN142 in a cytadhering revertant and complementation using a recombinant wild-type allele confirmed the role of this mutation in the cytadherence defect.

scholarly journals The N-Terminal Domain of Enterococcal Surface Protein, Esp, Is Sufficient for Esp-Mediated Biofilm Enhancement in Enterococcus faecalis

2005 ◽  
Vol 187 (17) ◽  
pp. 6213-6222 ◽  
Author(s):  
Preeti M. Tendolkar ◽  
Arto S. Baghdayan ◽  
Nathan Shankar
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Tandem Repeats ◽  
Full Length ◽  
Specific Factor ◽  
Dependent Manner ◽  
Wild Type ◽  
Specific Domain ◽  
Terminal Domain ◽  
Tract Infections

ABSTRACT Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.

scholarly journals Male courtship song drives escape responses that are suppressed for successful mating

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eliane Arez ◽  
Cecilia Mezzera ◽  
Ricardo M. Neto-Silva ◽  
Márcia M. Aranha ◽  
Sophie Dias ◽  
...  
Keyword(s):  
Escape Response ◽  
Walking Speed ◽  
Genetic Manipulation ◽  
Courtship Song ◽  
Male Courtship ◽  
Wild Type ◽  
Crucial Component ◽  
Escape Responses ◽  
Sexually Mature

AbstractPersuasion is a crucial component of the courtship ritual needed to overcome contact aversion. In fruit flies, it is well established that the male courtship song prompts receptivity in female flies, in part by causing sexually mature females to slow down and pause, allowing copulation. Whether the above receptivity behaviours require the suppression of contact avoidance or escape remains unknown. Here we show, through genetic manipulation of neurons we identified as required for female receptivity, that male song induces avoidance/escape responses that are suppressed in wild type flies. First, we show that silencing 70A09 neurons leads to an increase in escape, as females increase their walking speed during courtship together with an increase in jumping and a reduction in pausing. The increase in escape response is specific to courtship, as escape to a looming threat is not intensified. Activation of 70A09 neurons leads to pausing, confirming the role of these neurons in escape modulation. Finally, we show that the escape displays by the female result from the presence of a courting male and more specifically from the song produced by a courting male. Our results suggest that courtship song has a dual role, promoting both escape and pause in females and that escape is suppressed by the activity of 70A09 neurons, allowing mating to occur.

scholarly journals Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Baisakhi Banerjee ◽  
Rajat Banerjee
Keyword(s):  
Analytical Ultracentrifugation ◽  
Coiled Coil ◽  
Tryptophan Fluorescence ◽  
Trna Synthetase ◽  
Full Length ◽  
Spectroscopic Techniques ◽  
Wild Type ◽  
Native Form ◽  
E Coli ◽  
Terminal Domain

E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700) forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D) also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS.

SK3 Trafficking in Hippocampal Cells: The Role of Different Molecular Domains

2006 ◽  
Vol 26 (6) ◽  
pp. 399-412 ◽  
Author(s):  
Ilaria Decimo ◽  
Renza Roncarati ◽  
Silvia Grasso ◽  
Marcel Clemens ◽  
Christian Chiamulera ◽  
...  
Keyword(s):  
Cell Body ◽  
Fluorescent Protein ◽  
Full Length ◽  
Differential Distribution ◽  
Calmodulin Binding ◽  
Quantitative Expression ◽  
Terminal Domain ◽  
Control Fusion ◽  
Green Fluorescent

The regulative steps that control trafficking of ion channels are fundamental determinants of their qualitative and quantitative expression on the cell membrane. In this work the trafficking of the small conductance calcium-activated potassium channel, SK3 was studied in neurons in order to identify relevant molecular domains involved in this process. Hippocampal cell cultures were transfected with fusion proteins of green fluorescent protein (GFP) and different SK3 subunit truncations. The differential distribution of the mutants was analyzed by confocal microscopy and compared to the localization of the control fusion protein with full length SK3. The transport of chimeric proteins was quantified from fluorescence images by developing a morphometric analytical method. We found that the full length SK3 was distributed in cell body, axon and dendrites, whereas the deleted forms GFPΔ578–736 (deletion of the entire C-terminal domain), GFPΔCaMBD (deletion of the calmodulin-binding site) and GFPΔN (deletion of the N-terminal domain) were not transported into cell processes but accumulated in the cell body. The GFPΔ640–736 (deletion of the distal C-terminal domain) showed a distribution similar to control. The quantification and statistical analysis confirmed the differences in distribution across the three groups. In conclusion, the current work provides evidence for a fundamental role of the N-terminal domain and the calmodulin binding domain in SK3 trafficking in neurons.

scholarly journals The Role of GRASP55 in Golgi Fragmentation and Entry of Cells into Mitosis

2008 ◽  
Vol 19 (6) ◽  
pp. 2579-2587 ◽  
Author(s):  
Juan Manuel Duran ◽  
Matt Kinseth ◽  
Carine Bossard ◽  
David W. Rose ◽  
Roman Polishchuk ◽  
...  
Keyword(s):  
Peptide Mapping ◽  
Full Length ◽  
Wild Type ◽  
Experimental Conditions ◽  
Golgi Membranes ◽  
Golgi Fragmentation ◽  
Per Se ◽  
Golgi Organization

GRASP55 is a Golgi-associated protein, but its function at the Golgi remains unclear. Addition of full-length GRASP55, GRASP55-specific peptides, or an anti-GRASP55 antibody inhibited Golgi fragmentation by mitotic extracts in vitro, and entry of cells into mitosis. Phospho-peptide mapping of full-length GRASP55 revealed that threonine 225 and 249 were mitotically phosphorylated. Wild-type peptides containing T225 and T249 inhibited Golgi fragmentation and entry of cells into mitosis. Mutant peptides containing T225E and T249E, in contrast, did not affect Golgi fragmentation and entry into mitosis. These findings reveal a role of GRASP55 in events leading to Golgi fragmentation and the subsequent entry of cell into mitosis. Surprisingly, however, under our experimental conditions, >85% knockdown of GRASP55 did not affect the overall organization of Golgi organization in terms of cisternal stacking and lateral connections between stacks. Based on our findings we suggest that phosphorylation of GRASP55 at T225/T249 releases a bound component, which is phosphorylated and necessary for Golgi fragmentation. Thus, GRASP55 has no role in the organization of Golgi membranes per se, but it controls their fragmentation by regulating the release of a partner, which requires a G2-specific phosphorylation at T225/T249.

Comparison of full-length and conserved segments of wheat dehydrin DHN-5 overexpressed in Arabidopsis thaliana showed different responses to abiotic and biotic stress

2016 ◽  
Vol 43 (11) ◽  
pp. 1048 ◽  
Author(s):  
Marwa Drira ◽  
Moez Hanin ◽  
Khaled Masmoudi ◽  
Faiçal Brini
Keyword(s):  
Arabidopsis Thaliana ◽  
Fungal Infections ◽  
Alternaria Solani ◽  
Triticum Aestivum L ◽  
Full Length ◽  
Plant Tolerance ◽  
Wild Type ◽  
Biotic And Abiotic Stresses ◽  
Related Stress

Dehydrins (DHNs) are among the most common proteins accumulated in plants under water-related stress. They typically contain at least three conserved sequences designated as the Y-, S- and K-segments. The present work aims to highlight the role of the K-segments in plant tolerance to biotic and abiotic stresses. For this purpose, transgenic Arabidopsis thaliana (L.) Heyhn. lines expressing distinct wheat (Triticum aestivum L.) DHN-5 truncated constructs with or without the K-segments were generated. Our results showed that unlike the derivative lacking a K-segment, constructs containing only one or two K-segments enhanced the tolerance of A. thaliana to diverse stresses and were similar to the full-length wheat DHN-5. Moreover, compared with the wild-type and the YS form, the transgenic plants overexpressing wheat DHN-5 with K-segments maintained higher superoxide dismutase, catalase and peroxide dismutase enzymatic activity, and accumulated lower levels of H2O2 and malondialdehyde. In addition, we demonstrated that lines like A. thaliana overexpressing wheat DHN-5 showed increased resistance to fungal infections caused by Botrytis cinerea and Alternaria solani. Finally, the overexpression of the different forms of wheat DHN-5 led to the regulation of the expression of several genes involved in the jasmonic acid signalling pathway.

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