Alteromonas Myovirus V22 Represents a New Genus of Marine Bacteriophages Requiring a Tail Fiber Chaperone for Host Recognition

ABSTRACT Marine phages play a variety of critical roles in regulating the microbial composition of our oceans. Despite constituting the majority of genetic diversity within these environments, there are relatively few isolates with complete genome sequences or in-depth analyses of their host interaction mechanisms, such as characterization of their receptor binding proteins (RBPs). Here, we present the 92,760-bp genome of the Alteromonas-targeting phage V22. Genomic and morphological analyses identify V22 as a myovirus; however, due to a lack of sequence similarity to any other known myoviruses, we propose that V22 be classified as the type phage of a new Myoalterovirus genus within the Myoviridae family. V22 shows gene homology and synteny with two different subfamilies of phages infecting enterobacteria, specifically within the structural region of its genome. To improve our understanding of the V22 adsorption process, we identified putative RBPs (gp23, gp24, and gp26) and tested their ability to decorate the V22 propagation strain, Alteromonas mediterranea PT11, as recombinant green fluorescent protein (GFP)-tagged constructs. Only GFP-gp26 was capable of bacterial recognition and identified as the V22 RBP. Interestingly, production of functional GFP-gp26 required coexpression with the downstream protein gp27. GFP-gp26 could be expressed alone but was incapable of host recognition. By combining size-exclusion chromatography with fluorescence microscopy, we reveal how gp27 is not a component of the final RBP complex but instead is identified as a new type of phage-encoded intermolecular chaperone that is essential for maturation of the gp26 RBP. IMPORTANCE Host recognition by phage-encoded receptor binding proteins (RBPs) constitutes the first step in all phage infections and the most critical determinant of host specificity. By characterizing new types of RBPs and identifying their essential chaperones, we hope to expand the repertoire of known phage-host recognition machineries. Due to their genetic plasticity, studying RBPs and their associated chaperones can shed new light onto viral evolution affecting phage-host interactions, which is essential for fields such as phage therapy or biotechnology. In addition, since marine phages constitute one of the most important reservoirs of noncharacterized genetic diversity on the planet, their genomic and functional characterization may be of paramount importance for the discovery of novel genes with potential applications.

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The Presence of Two Receptor-Binding Proteins Contributes to the Wide Host Range of Staphylococcal Twort-Like Phages

Applied and Environmental Microbiology ◽

10.1128/aem.01385-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5763-5774 ◽

Cited By ~ 32

Author(s):

Ippei Takeuchi ◽

Keita Osada ◽

Aa Haeruman Azam ◽

Hiroaki Asakawa ◽

Kazuhiko Miyanaga ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Host Range ◽

Receptor Binding ◽

Polyclonal Antibody ◽

Binding Proteins ◽

Host Recognition ◽

Resistant Bacteria ◽

Tail Fiber ◽

Content Type ◽

Wide Host Range

ABSTRACTThanks to their wide host range and virulence, staphylococcal bacteriophages (phages) belonging to the genusTwortlikevirus(staphylococcal Twort-like phages) are regarded as ideal candidates for clinical application forStaphylococcus aureusinfections due to the emergence of antibiotic-resistant bacteria of this species. To increase the usability of these phages, it is necessary to understand the mechanism underlying host recognition, especially the receptor-binding proteins (RBPs) that determine host range. In this study, we found that the staphylococcal Twort-like phage ΦSA012 possesses at least two RBPs. Genomic analysis of five mutant phages of ΦSA012 revealed point mutations inorf103, in a region unique to staphylococcal Twort-like phages. Phages harboring mutated ORF103 could not infectS. aureusstrains in which wall teichoic acids (WTAs) are glycosylated with α-N-acetylglucosamine (α-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection by ΦSA012 in the presence of α-GlcNAc, suggesting that ORF103 binds to α-GlcNAc. In contrast, a polyclonal antibody against ORF105, a short tail fiber component previously shown to be an RBP, inhibited phage infection irrespective of the presence of α-GlcNAc. Immunoelectron microscopy indicated that ORF103 is a tail fiber component localized at the bottom of the baseplate. From these results, we conclude that ORF103 binds α-GlcNAc in WTAs, whereas ORF105, the primary RBP, is likely to bind the WTA backbone. These findings provide insight into the infection mechanism of staphylococcal Twort-like phages.IMPORTANCEStaphylococcusphages belonging to the genusTwortlikevirus(called staphylococcal Twort-like phages) are considered promising agents for control ofStaphylococcus aureusdue to their wide host range and highly lytic capabilities. Although staphylococcal Twort-like phages have been studied widely for therapeutic purposes, the host recognition process of staphylococcal Twort-like phages remains unclear. This work provides new findings about the mechanisms of host recognition of the staphylococcal Twort-like phage ΦSA012. The details of the host recognition mechanism of ΦSA012 will allow us to analyze the mechanisms of infection and expand the utility of staphylococcal Twort-like phages for the control ofS. aureus.

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Structural and functional characterization of the receptor binding proteins of Escherichia coli O157 phages EP75 and EP335

Computational and Structural Biotechnology Journal ◽

10.1016/j.csbj.2021.06.001 ◽

2021 ◽

Author(s):

Sander Witte ◽

Léa V. Zinsli ◽

Rafael Gonzalez-Serrano ◽

Cassandra I. Matter ◽

Martin J. Loessner ◽

...

Keyword(s):

Escherichia Coli ◽

Receptor Binding ◽

Binding Proteins ◽

Functional Characterization ◽

Escherichia Coli O157

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Design, expression, purification and characterization of a YFP-tagged 2019-nCoV spike receptor-binding domain construct

10.1101/2020.09.29.318196 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tobias Bierig ◽

Gabriella Collu ◽

Alain Blanc ◽

Emiliya Poghosyan ◽

Roger. M. Benoit

Keyword(s):

Fusion Protein ◽

Receptor Binding ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Fluorescent Labeling ◽

Binding Domain ◽

Size Exclusion ◽

Antibody Detection ◽

Receptor Binding Domain ◽

Binding Studies

Abstract2019-nCoV is the causative agent of the serious, still ongoing, worldwide COVID-19 pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA IMAC purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.

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Articles selected by Faculty of 1000: phage receptor-binding proteins; genetic diversity in Toxoplasma; ribosome biogenesis and cell size; gene regulation by retrotransposons; manipulation of the cancer epigenome

Genome Biology ◽

10.1186/gb-2004-5-12-361 ◽

2004 ◽

Vol 5 (12) ◽

Author(s):

Keyword(s):

Genetic Diversity ◽

Gene Regulation ◽

Cell Size ◽

Receptor Binding ◽

Ribosome Biogenesis ◽

Binding Proteins ◽

Phage Receptor

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Detailed Genomic Analysis of the Wβ and γ Phages Infecting Bacillus anthracis: Implications for Evolution of Environmental Fitness and Antibiotic Resistance

Journal of Bacteriology ◽

10.1128/jb.188.8.3037-3051.2006 ◽

2006 ◽

Vol 188 (8) ◽

pp. 3037-3051 ◽

Cited By ~ 70

Author(s):

Raymond Schuch ◽

Vincent A. Fischetti

Keyword(s):

Bacillus Anthracis ◽

Fluorescent Protein ◽

Point Mutations ◽

Genomic Analysis ◽

Open Reading Frames ◽

Host Recognition ◽

Pcr Analysis ◽

Tail Fiber ◽

Green Fluorescent ◽

Laboratory Tool

ABSTRACT Phage-mediated lysis has been an essential laboratory tool for rapidly identifying Bacillus anthracis for more than 40 years, relying on the γ phage derivative of a Bacillus cereus prophage called W. The complete genomic sequences of the temperate W phage, referred to as Wβ, and its lytic variant γ were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that γ evolved through mutations at key loci controlling host recognition, lysogenic growth, and possibly host phenotypic modification. Included are a cluster of point mutations at the gp14 tail fiber locus of γ, encoding a protein that, when fused to green fluorescent protein, binds specifically to B. anthracis. A large 2,003-bp deletion was also identified at the γ lysogeny module, explaining its shift from a temperate to a lytic lifestyle. Finally, evidence of recombination was observed at a dicistronic Wβ locus, encoding putative bacterial cell surface-modifying proteins, replaced in γ with a locus, likely obtained from a B. anthracis prophage, encoding demonstrable fosfomycin resistance. Reverse transcriptase PCR analysis confirmed strong induction at the dicistronic Wβ locus and at four other phage loci in B. anthracis and/or B. cereus lysogens. In all, this study represents the first genomic and functional description of two historically important phages and is part of a broader investigation into contributions of phage to the B. anthracis life cycle. Initial findings suggest that lysogeny of B. anthracis promotes ecological adaptation, rather than virulence, as with other gram-positive pathogens.

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Conserved and Diverse Traits of Adhesion Devices from Siphoviridae Recognizing Proteinaceous or Saccharidic Receptors

Viruses ◽

10.3390/v12050512 ◽

2020 ◽

Vol 12 (5) ◽

pp. 512 ◽

Cited By ~ 5

Author(s):

Adeline Goulet ◽

Silvia Spinelli ◽

Jennifer Mahony ◽

Christian Cambillau

Keyword(s):

Receptor Binding ◽

Binding Proteins ◽

Phage Therapy ◽

Activation Mechanism ◽

Host Recognition ◽

Tape Measure Protein ◽

Tape Measure ◽

Receptor Recognition ◽

Contractile Tail ◽

Tail Protein

Bacteriophages can play beneficial roles in phage therapy and destruction of food pathogens. Conversely, they play negative roles as they infect bacteria involved in fermentation, resulting in serious industrial losses. Siphoviridae phages possess a long non-contractile tail and use a mechanism of infection whose first step is host recognition and binding. They have evolved adhesion devices at their tails’ distal end, tuned to recognize specific proteinaceous or saccharidic receptors on the host’s surface that span a large spectrum of shapes. In this review, we aimed to identify common patterns beyond this apparent diversity. To this end, we analyzed siphophage tail tips or baseplates, evaluating their known structures, where available, and uncovering patterns with bioinformatics tools when they were not. It was thereby identified that a triad formed by three proteins in complex, i.e., the tape measure protein (TMP), the distal tail protein (Dit), and the tail-associated lysozyme (Tal), is conserved in all phages. This common scaffold may harbor various functional extensions internally while it also serves as a platform for plug-in ancillary or receptor-binding proteins (RBPs). Finally, a group of siphophage baseplates involved in saccharidic receptor recognition exhibits an activation mechanism reminiscent of that observed in Myoviridae.

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Mex67p of Schizosaccharomyces pombeInteracts with Rae1p in Mediating mRNA Export

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8767-8782.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8767-8782 ◽

Cited By ~ 52

Author(s):

Jin Ho Yoon ◽

Dona C. Love ◽

Anjan Guhathakurta ◽

John A. Hanover ◽

Ravi Dhar

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Fluorescent Protein ◽

Synthetic Lethality ◽

Sequence Similarity ◽

Mrna Export ◽

Nuclear Periphery ◽

Nuclear Pore ◽

Multicopy Suppressor ◽

Accessory Factor

ABSTRACT We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 tsmutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast toscMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with therae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of therae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

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T4-like Bacteriophages Isolated from Pig Stools Infect Yersinia pseudotuberculosis and Yersinia pestis Using LPS and OmpF as Receptors

Viruses ◽

10.3390/v13020296 ◽

2021 ◽

Vol 13 (2) ◽

pp. 296

Author(s):

Mabruka Salem ◽

Maria I. Pajunen ◽

Jin Woo Jun ◽

Mikael Skurnik

Keyword(s):

Yersinia Pseudotuberculosis ◽

Host Recognition ◽

Tail Fiber ◽

Long Tail ◽

Phage T4 ◽

One Step ◽

In Trans ◽

Step Growth ◽

Resistant Strains ◽

Salmonella Phage

The Yersinia bacteriophages fPS-2, fPS-65, and fPS-90, isolated from pig stools, have long contractile tails and elongated heads, and they belong to genus Tequatroviruses in the order Caudovirales. The phages exhibited relatively wide host ranges among Yersinia pseudotuberculosis and related species. One-step growth curve experiments revealed that the phages have latent periods of 50–80 min with burst sizes of 44–65 virions per infected cell. The phage genomes consist of circularly permuted dsDNA of 169,060, 167,058, and 167,132 bp in size, respectively, with a G + C content 35.3%. The number of predicted genes range from 267 to 271. The phage genomes are 84–92% identical to each other and ca 85% identical to phage T4. The phage receptors were identified by whole genome sequencing of spontaneous phage-resistant mutants. The phage-resistant strains had mutations in the ompF, galU, hldD, or hldE genes. OmpF is a porin, and the other genes encode lipopolysaccharide (LPS) biosynthetic enzymes. The ompF, galU, and hldE mutants were successfully complemented in trans with respective wild-type genes. The host recognition was assigned to long tail fiber tip protein Gp38, analogous to that of T-even phages such as Salmonella phage S16, specifically to the distal β-helices connecting loops.

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Assessment of Genetic Diversity and Symbiotic Efficiency of Selected Rhizobia Strains Nodulating Lentil (Lens culinaris Medik.)

Plants ◽

10.3390/plants10010015 ◽

2020 ◽

Vol 10 (1) ◽

pp. 15

Author(s):

Badreddine Sijilmassi ◽

Abdelkarim Filali-Maltouf ◽

Hassan Boulahyaoui ◽

Aymane Kricha ◽

Kenza Boubekri ◽

...

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

16S Rrna ◽

Rhizobium Leguminosarum ◽

Sequence Similarity ◽

P Value ◽

Rrna Gene ◽

Research Station ◽

Symbiotic Efficiency ◽

Rhizobium Strains

A total of 14 Rhizobium strains were isolated from lentil accessions grown at the ICARDA experimental research station at Marchouch in Morocco and used for molecular characterization and symbiotic efficiency assessment. Individual phylogenetic analysis using the 16S rRNA gene, house-keeping genes rpoB, recA, and gyrB, and symbiotic genes nodD and nodA along with Multilocus Sequence Analysis (MLSA) of the concatenated genes (16S rRNA-rpoB-recA-gyrB) was carried out for the identification and clustering of the isolates. The symbiotic efficiency of the strains was assessed on three Moroccan lentil cultivars (Bakria, Chakkouf, and Zaria) based on the number of nodules, plant height, plant dry weight, and total nitrogen content in leaves. The results showed that the individual phylogenetic analysis clustered all the strains into Rhizobium laguerreae and Rhizobium leguminosarum with sequence similarity ranging from 94 to 100%, except one strain which clustered with Mesorhizobium huakuii with sequence similarity of 100%. The MLSA of the concatenated genes and the related percentages of similarity clustered these strains into two groups of Rhizobium species, with one strain as a new genospecies when applying the threshold of 96%. For symbiotic efficiency, the Bakria variety showed the best association with 10 strains compared to its non-inoculated control (p-value ≤ 0.05), followed by Chakkouf and Zaria. The present study concluded that the genetic diversity and the symbiotic efficiency of Rhizobium strains appeared to be mainly under the control of the lentil genotypes.

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