scholarly journals Tuning microscale, retractable, membrane-breaking protein needles

2015 ◽  
Author(s):  
Jessica K Polka ◽  
Pamela A Silver
Keyword(s):  
Lipid Bilayers ◽  
Protein Structures ◽  
Open Reading Frames ◽  
Paramecium Tetraurelia ◽  
E Coli ◽  
Body State ◽  
High Throughput Assay ◽  
Reading Frames

The refractile (R) bodies found in Caedibacter taeniospiralis, a bacterial endosymbiont of Paramecium tetraurelia, are large, polymeric protein structures that can switch between two conformations. At cytoplasmic pH, they resemble coiled ribbons of protein 500nm in diameter. At low pH, they extend to form hollow needles up to 20 microns long. They can be expressed heterologously from an operon containing four short open reading frames and can function in vitro in diverse buffer conditions. In this study, R bodies purified from Escherichia coli were found to be capable of undergoing many consecutive extension-contraction cycles. Furthermore, the solubility of R bodies, which can easily be interpreted by eye, was found to correlate with their extension state. This macroscopic phenotype was used to develop a quantitative, high-throughput assay for R body state, enabling a visual screen of R body mutants defective in extension. The role of specific amino acids in extension was determined, and this information was used to construct rationally-designed mutants tailored to extend at higher pH. Furthermore, R bodies were able to rupture E. coli spheroplasts to release soluble proteins across lipid bilayers. Taken together, these results show that R bodies act as tunable, pH-actuated pistons suitable for a variety of membrane-breaking applications.

scholarly journals Phage composition of a fermented milk and colostrum product assessed by microbiome array; putative role of open reading frames in reference to cell signaling and neurological development

2019 ◽  
Author(s):  
Stefania Pacini ◽  
Marco Ruggiero
Keyword(s):  
Dna Sequences ◽  
Bovine Milk ◽  
Fermented Milk ◽  
Open Reading Frames ◽  
Neurological Development ◽  
Putative Role ◽  
Biological Entities ◽  
Reading Frames

AbstractBacteriophages (phages), Earth’s most numerous biological entities, are natural constituents of alimentary matrices; in this study we describe the characterization of phage populations in a product obtained by fermentation of bovine milk and colostrum. Such characterizations were achieved using a microarray consisting of a chip covered in short DNA sequences that are specific to certain target organisms for a total of approximately 12,000 species. The only viruses evidenced by the array belonged to Siphoviridae, the largest phage family that targets bacteria and archea. The array yielded 27 iterations corresponding to a unique target. We discuss the putative role of some open reading frames of these phages in conferring health-supporting properties with particular reference to cells signaling and neurological development. We also describe the in vitro interaction of this fermented product with alpha-N-acetylgalactosaminidase, an enzyme whose activity in serum is elevated in neurodevelopmental disorders.

scholarly journals Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome

mSystems ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Kaneyoshi Yamamoto ◽  
Yuki Yamanaka ◽  
Tomohiro Shimada ◽  
Paramita Sarkar ◽  
Myu Yoshida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Small Subunit ◽  
Open Reading Frames ◽  
Pcr Assay ◽  
E Coli ◽  
K 12 ◽  
Chip Chip ◽  
Reading Frames

The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (β′, of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature.

In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide

2020 ◽  
Vol 167 (5) ◽  
pp. 451-462
Author(s):  
Taisho Abe ◽  
Riku Nagai ◽  
Shunta Shimazaki ◽  
Shunta Kondo ◽  
Satoshi Nishimura ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
Translation System ◽  
Ribosome Stalling ◽  
Internal Position ◽  
Polyproline Motif ◽  
Reading Frames

Abstract We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

scholarly journals ORF157 from the Archaeal Virus Acidianus Filamentous Virus 1 Defines a New Class of Nuclease

2010 ◽  
Vol 84 (10) ◽  
pp. 5025-5031 ◽  
Author(s):  
Adeline Goulet ◽  
Mery Pina ◽  
Peter Redder ◽  
David Prangishvili ◽  
Laura Vera ◽  
...  
Keyword(s):  
Hot Springs ◽  
Protein Structures ◽  
Three Dimensional ◽  
Nuclease Activity ◽  
Open Reading Frames ◽  
Double Stranded Dna ◽  
Substitution Mutations ◽  
Reading Frames

ABSTRACT Acidianus filamentous virus 1 (AFV1) (Lipothrixviridae) is an enveloped filamentous virus that was characterized from a crenarchaeal host. It infects Acidianus species that thrive in the acidic hot springs (>85°C and pH <3) of Yellowstone National Park, WY. The AFV1 20.8-kb, linear, double-stranded DNA genome encodes 40 putative open reading frames whose sequences generally show little similarity to other genes in the sequence databases. Because three-dimensional structures are more conserved than sequences and hence are more effective at revealing function, we set out to determine protein structures from putative AFV1 open reading frames (ORF). The crystal structure of ORF157 reveals an α+β protein with a novel fold that remotely resembles the nucleotidyltransferase topology. In vitro, AFV1-157 displays a nuclease activity on linear double-stranded DNA. Alanine substitution mutations demonstrated that E86 is essential to catalysis. AFV1-157 represents a novel class of nuclease, but its exact role in vivo remains to be determined.

scholarly journals Phage composition of a fermented milk and colostrum product assessed by microbiome array; putative role of open reading frames in reference to cell signaling and neurological development

2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Stefania Pacini ◽  
Marco Ruggiero S
Keyword(s):  
Dna Sequences ◽  
Bovine Milk ◽  
Fermented Milk ◽  
Open Reading Frames ◽  
Neurological Development ◽  
Putative Role ◽  
Biological Entities ◽  
Reading Frames

Bacteriophages (phages), Earth's most numerous biological entities, are natural constituents of alimentary matrices; in this study we describe the characterization of phage populations in a product obtained by fermentation of bovine milk and colostrum. Such characterizations were achieved using a microarray consisting of a chip covered in short DNA sequences that are specific to certain target organisms for a total of approximately 12,000 species. The only viruses evidenced by the array belonged to Siphoviridae, the largest phage family that targets bacteria and archea. The array yielded 27 iterations corresponding to a unique target. We discuss the putative role of some open reading frames of these phages in conferring health-supporting properties with particular reference to cells signaling and neurological development. We also describe the in vitro interaction of this fermented product with alpha-N-acetylgalactosaminidase, an enzyme whose activity in serum is elevated in neurodevelopmental disorders.

scholarly journals Functional Incorporation of Chimeric b Subunits into F1Fo ATP Synthase

2007 ◽  
Vol 189 (15) ◽  
pp. 5463-5471 ◽  
Author(s):  
Shane B. Claggett ◽  
Tammy Bohannon Grabar ◽  
Stanley D. Dunn ◽  
Brian D. Cain
Keyword(s):  
Atp Synthase ◽  
Photosynthetic Bacteria ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
E Coli ◽  
Peripheral Stalk ◽  
Catalytic Sites ◽  
Reading Frames ◽  
Better Than

ABSTRACT F1Fo ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F1 sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b′ peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b′ genes in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Δb). Although not all of the chimeric subunits were incorporated into F1Fo ATP synthase complexes, plasmids expressing either chimeric b E39-I86 or b′E39-I86 were capable of functionally complementing strain KM2 (Δb). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b′E39-D53 or b L54-I86 subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b′ subunit in vitro. Coexpression of the b E39-I86 and b′E39-I86 subunits in strain KM2 (Δb) yielded F1Fo complexes containing heterodimeric peripheral stalks composed of both subunits.

scholarly journals Identification of the Structural Gene for the TDP-Fuc4NAc:Lipid II Fuc4NAc Transferase Involved in Synthesis of Enterobacterial Common Antigen in Escherichia coliK-12

2001 ◽  
Vol 183 (22) ◽  
pp. 6509-6516 ◽  
Author(s):  
Arifur Rahman ◽  
Kathleen Barr ◽  
Paul D. Rick
Keyword(s):  
Gene Cluster ◽  
Structural Gene ◽  
Open Reading Frames ◽  
E Coli ◽  
Lipid Ii ◽  
Enterobacterial Common Antigen ◽  
Repeat Units ◽  
Cell Extracts ◽  
Reading Frames

ABSTRACT The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-d-galactose, ManNAcA is N-acetyl-d-mannosaminuronic acid, and GlcNAc is N-acetyl-d-glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the wec gene cluster located at ca. 85.4 min on the Escherichia coli chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the wec gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of thewec gene cluster in the region betweeno416 and wecG revealed that it contained three open reading frames: o74, o204, ando450. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames, o359 and o450. Mutants ofE. coli possessing null mutations in o359were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [3H]FucNAc from TDP-[3H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-typeo359 allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified o359 gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [3H]Fuc4NAc from TDP-[3H]Fuc4NAc to lipid II. Our data support the conclusion that o359 of the wec gene cluster of E. coli is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P &lt; 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P &lt; 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P &lt; 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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