scholarly journals Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions

2019 ◽  
Author(s):  
Stephen Hayes ◽  
Jennifer Mahony ◽  
Renaud Vincentelli ◽  
Laurie Ramond ◽  
Arjen Nauta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Bioinformatic Analysis ◽  
Infection Process ◽  
Ecological Niches ◽  
Carbohydrate Binding ◽  
Binding Assays ◽  
Protein Fusion ◽  
Carbohydrate Binding Modules ◽  
Structural Assembly ◽  
Green Fluorescent

Abstract: With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

scholarly journals Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 631 ◽  
Author(s):  
Stephen Hayes ◽  
Jennifer Mahony ◽  
Renaud Vincentelli ◽  
Laurie Ramond ◽  
Arjen Nauta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Bioinformatic Analysis ◽  
Infection Process ◽  
Ecological Niches ◽  
Carbohydrate Binding ◽  
Binding Assays ◽  
Protein Fusion ◽  
Carbohydrate Binding Modules ◽  
Structural Assembly ◽  
Green Fluorescent

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

scholarly journals The most abundant cyst wall proteins ofAcanthamoeba castellaniiare cellulose-binding lectins from three gene families that localize to distinct structures in cyst walls

2018 ◽  
Author(s):  
Pamela Magistrado-Coxen ◽  
Yousuf Aqeel ◽  
Angelo Lopez ◽  
John R. Haserick ◽  
Breeanna R. Urbanowicz ◽  
...  
Keyword(s):  
Cyst Wall ◽  
Fluorescent Protein ◽  
Acanthamoeba Castellanii ◽  
Gene Families ◽  
Carbohydrate Binding ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Carbohydrate Binding Modules ◽  
Green Fluorescent ◽  
Cellulose Binding

AbstractAcanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for glycopolymers to show that purified cyst walls ofA. castellaniiretain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight Cys residues each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for glycopolymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and the glycopolymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and their epitopes are mostly inaccessible in the ectocyst layer and ostioles. In summary, the most abundantA. castellaniiCWPs are three sets of lectins, which have conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique carbohydrate-binding modules (8-Cys of Jonah).Author summaryFifty years ago, the cyst wall ofAcanthamoeba castellaniiwas shown to contain cellulose and have an ectocyst layer, an endocyst layer, and conical ostioles that attach them. The goals here were to identify abundant cyst wall proteins (CWPs) and begin to determine how the wall is assembled. We used wheat germ agglutinin to show cyst walls also contain chitin fibrils. When trophozoites are starved of nutrients, they become immotile and make CWPs and glycopolymers in dozens of small vesicles. The primordial cyst wall is composed of a single, thin layer containing cellulose, chitin, and an abundant CWP we called Jonah. The primordial wall also has small, flat ostioles that contain another abundant CWP we called Luke. Jonah (the best candidate for diagnostic antibodies) is accessible in the ectocyst layer of mature cyst walls, while Luke and a third abundant CWP we termed Leo are present but mostly inaccessible in the endocyst layer and ostioles. WhileA. castellaniicyst walls contain cellulose (like plants) and chitin (like fungi), the glycopolymers are made in vesicles rather than at the plasma membrane, and the CWPs (Luke, Leo, and Jonah lectins) are unique to the protist.

scholarly journals Genetic Analysis of the Cell Division Protein FtsI (PBP3): Amino Acid Substitutions That Impair Septal Localization of FtsI and Recruitment of FtsN

2004 ◽  
Vol 186 (2) ◽  
pp. 490-502 ◽  
Author(s):  
Mark C. Wissel ◽  
David S. Weiss
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Transmembrane Helix ◽  
Binding Assays ◽  
Protein Fusion ◽  
Mutant Proteins ◽  
Domain Of Unknown Function ◽  
Green Fluorescent ◽  
Septal Localization ◽  
A Site

ABSTRACT FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of several proteins that localize to the septal ring. FtsI comprises a small cytoplasmic domain, a transmembrane helix, a noncatalytic domain of unknown function, and a catalytic (transpeptidase) domain. The last two domains reside in the periplasm. We used PCR to randomly mutagenize ftsI, ligated the products into a green fluorescent protein fusion vector, and screened ∼7,500 transformants for gfp-ftsI alleles that failed to complement an ftsI null mutant. Western blotting and penicillin-binding assays were then used to weed out proteins that were unstable, failed to insert into the cytoplasmic membrane, or were defective in catalysis. The remaining candidates were tested for septal localization and ability to recruit another division protein, FtsN, to the septal ring. Mutant proteins severely defective in localization to the septal ring all had lesions in one of three amino acids—R23, L39, or Q46—that are in or near the transmembrane helix and implicate this region of FtsI in septal localization. Mutant FtsI proteins defective in recruitment of FtsN all had lesions in one of eight residues in the noncatalytic domain. The most interesting of these mutants had lesions at G57, S61, L62, or R210. Although separated by ∼150 residues in the primary sequence, these amino acids are close together in the folded protein and might constitute a site of FtsI-FtsN interaction.

scholarly journals Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules

Materials ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3175
Author(s):  
Mariana Barbosa ◽  
Hélvio Simões ◽  
Duarte Miguel F. Prazeres
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Protein A ◽  
Chemical Properties ◽  
Main Idea ◽  
Bioactive Molecules ◽  
Scaffolding Protein ◽  
Carbohydrate Binding ◽  
Biological Interactions ◽  
Carbohydrate Binding Modules

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

A novel linker histone-like protein is associated with cytoplasmic filaments inCaenorhabditis elegans

2002 ◽  
Vol 115 (14) ◽  
pp. 2881-2891
Author(s):  
Monika A. Jedrusik ◽  
Stefan Vogt ◽  
Peter Claus ◽  
Ekkehard Schulze
Keyword(s):  
Rna Interference ◽  
Fluorescent Protein ◽  
Muscle Growth ◽  
Egg Laying ◽  
Globular Domain ◽  
Protein Fusion ◽  
Linker Histones ◽  
C Elegans ◽  
Fusion Protein Expression ◽  
Green Fluorescent

The histone H1 complement of Caenorhabditis elegans contains a single unusual protein, H1.X. Although H1.X possesses the globular domain and the canonical three-domain structure of linker histones, the amino acid composition of H1.X is distinctly different from conventional linker histones in both terminal domains. We have characterized H1.X in C. elegans by antibody labeling, green fluorescent protein fusion protein expression and RNA interference. Unlike normal linker histones, H1.X is a cytoplasmic as well as a nuclear protein and is not associated with chromosomes. H1.X is most prominently expressed in the marginal cells of the pharynx and is associated with a peculiar cytoplasmic cytoskeletal structure therein, the tonofilaments. Additionally H1.X::GFP is expressed in the cytoplasm of body and vulva muscle cells, neurons, excretory cells and in the nucleoli of embryonic blastomeres and adult gut cells. RNA interference with H1.X results in uncoordinated and egg laying defective animals, as well as in a longitudinally enlarged pharynx. These phenotypes indicate a cytoplasmic role of H1.X in muscle growth and muscle function.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

scholarly journals Developmental-Stage-Specific Assembly of ParB Complexes in Streptomyces coelicolor Hyphae

2005 ◽  
Vol 187 (10) ◽  
pp. 3572-3580 ◽  
Author(s):  
Dagmara Jakimowicz ◽  
Bertolt Gust ◽  
Jolanta Zakrzewska-Czerwinska ◽  
Keith F. Chater
Keyword(s):  
Green Fluorescent Protein ◽  
Developmental Stage ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Chromosome Partitioning ◽  
Aerial Hyphae ◽  
Green Fluorescent

ABSTRACT In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

Sign in / Sign up

Export Citation Format

Share Document