scholarly journals Phage S144, a New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii

2020 ◽  
Vol 21 (15) ◽  
pp. 5196 ◽  
Author(s):  
Michela Gambino ◽  
Anders Nørgaard Sørensen ◽  
Stephen Ahern ◽  
Georgios Smyrlis ◽  
Yilmaz Emre Gencay ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
Trna Genes ◽  
Tail Fiber ◽  
Cronobacter Sakazakii ◽  
Salmonella Spp ◽  
Nucleotide Biosynthesis ◽  
C Terminus ◽  
Transmission Electron ◽  
Phage Receptor ◽  
Tail Fibers

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.

scholarly journals Molecular Analysis of Arthrobacter Myovirus vB_ArtM-ArV1: We Blame It on the Tail

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Laura Kaliniene ◽  
Eugenijus Šimoliūnas ◽  
Lidija Truncaitė ◽  
Aurelija Zajančkauskaitė ◽  
Juozas Nainys ◽  
...  
Keyword(s):  
Gc Content ◽  
Open Reading Frames ◽  
Trna Genes ◽  
Tail Fiber ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Transmission Electron ◽  
The Family ◽  
Contractile Tail ◽  
Phage Propagation

ABSTRACTThis is the first report on a myophage that infectsArthrobacter. A novel virus, vB_ArtM-ArV1 (ArV1), was isolated from soil usingArthrobactersp. strain 68b for phage propagation. Transmission electron microscopy showed its resemblance to members of the familyMyoviridae: ArV1 has an isometric head (∼74 nm in diameter) and a contractile, nonflexible tail (∼192 nm). Phylogenetic and comparative sequence analyses, however, revealed that ArV1 has more genes in common with phages from the familySiphoviridaethan it does with any myovirus characterized to date. The genome of ArV1 is a linear, circularly permuted, double-stranded DNA molecule (71,200 bp) with a GC content of 61.6%. The genome includes 101 open reading frames (ORFs) yet contains no tRNA genes. More than 50% of ArV1 genes encode unique proteins that either have no reliable identity to database entries or have homologues only inArthrobacterphages, both sipho- and myoviruses. Using bioinformatics approaches, 13 ArV1 structural genes were identified, including those coding for head, tail, tail fiber, and baseplate proteins. A further 6 ArV1 ORFs were annotated as encoding putative structural proteins based on the results of proteomic analysis. Phylogenetic analysis based on the alignment of four conserved virion proteins revealed thatArthrobactermyophages form a discrete clade that seems to occupy a position somewhat intermediate between myo- and siphoviruses. Thus, the data presented here will help to advance our understanding of genetic diversity and evolution of phages that constitute the orderCaudovirales.IMPORTANCEBacteriophages, which likely originated in the early Precambrian Era, represent the most numerous population on the planet. Approximately 95% of known phages are tailed viruses that comprise three families:Podoviridae(with short tails),Siphoviridae(with long noncontractile tails), andMyoviridae(with contractile tails). Based on the current hypothesis, myophages, which may have evolved from siphophages, are thought to have first emerged among Gram-negative bacteria, whereas they emerged only later among Gram-positive bacteria. The results of the molecular characterization of myophage vB_ArtM-ArV1 presented here conform to the aforementioned hypothesis, since, at a glance, bacteriophage vB_ArtM-ArV1 appears to be a siphovirus that possesses a seemingly functional contractile tail. Our work demonstrates that such “chimeric” myophages are of cosmopolitan nature and are likely characteristic of the ecologically important soil bacterial genusArthrobacter.

scholarly journals Genetically modified human type II collagen for N- and C-terminal covalent tagging

2018 ◽  
Vol 96 (2) ◽  
pp. 204-211
Author(s):  
Andrew Wieczorek ◽  
Clara K. Chan ◽  
Suzana Kovacic ◽  
Cindy Li ◽  
Thomas Dierks ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Connective Tissue Diseases ◽  
Sulfhydryl Group ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Structural Protein ◽  
Connective Tissues ◽  
Human Type ◽  
C Terminus ◽  
Transmission Electron

Collagen is the predominant structural protein in vertebrates, where it contributes to connective tissues and the ECM; it is also widely used in biomaterials and tissue engineering. Dysfunction of this protein and its processing can lead to a wide variety of developmental disorders and connective tissue diseases. Recombinantly engineering the protein is challenging due to post-translational modifications generally required for its stability and secretion from cells. Introducing end labels into the protein is problematic, because the N- and C-termini of the physiologically relevant tropocollagen lie internal to the initially flanking N- and C-propeptide sequences. Here, we introduce mutations into human type II procollagen in a manner that addresses these concerns and purify the recombinant protein from a stably transfected HT1080 human fibrosarcoma cell line. Our approach introduces chemically addressable groups into the N- and C-telopeptide termini of tropocollagen. Simultaneous overexpression of formylglycine generating enzyme (FGE) allows the endogenous production of an aldehyde tag in a defined, substituted sequence in the N terminus of the mutated collagen, whereas the C-terminus of each chain presents a sulfhydryl group from an introduced cysteine. These modifications are designed to enable specific covalent end-labelling of collagen. We find that the doubly mutated protein folds and is secreted from cells. Higher order assembly into well-ordered collagen fibrils is demonstrated through transmission electron microscopy. Chemical tagging of thiols is successful; however, background from endogenous aldehydes present in wild-type collagen has thus far obscured the desired specific N-terminal labelling. Strategies to overcome this challenge are proposed.

scholarly journals Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

2020 ◽  
Author(s):  
Ramakrishnan B. Kumar ◽  
Pasi Purhonen ◽  
Hans Hebert ◽  
Caroline Jegerschöld
Keyword(s):  
Arachidonic Acid ◽  
Complex Formation ◽  
Calcium Ions ◽  
Dependent Manner ◽  
Present Communication ◽  
Nuclear Membranes ◽  
His Tag ◽  
C Terminus ◽  
Transmission Electron

AbstractAmong the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectroscopy. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

scholarly journals Isolation and characterization of bacteriophages from India, with lytic activity againstMycobacterium tuberculosis

2018 ◽  
Vol 64 (7) ◽  
pp. 483-491 ◽  
Author(s):  
Urmi Bajpai ◽  
Abhishek Kumar Mehta ◽  
Kandasamy Eniyan ◽  
Avni Sinha ◽  
Ankita Ray ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Sequence Similarity ◽  
Gc Content ◽  
Polymerase Chain Reaction Analysis ◽  
Open Reading Frames ◽  
Genome Maintenance ◽  
Isolation And Characterization ◽  
Virion Release ◽  
Double Stranded Dna ◽  
Transmission Electron

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages — PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.

scholarly journals Specific Detection of Yersinia pestis Based on Receptor Binding Proteins of Phages

Pathogens ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 611 ◽  
Author(s):  
Friederike Born ◽  
Peter Braun ◽  
Holger C. Scholz ◽  
Gregor Grass
Keyword(s):  
Yersinia Pestis ◽  
Receptor Binding ◽  
Fluorescent Proteins ◽  
Diagnostic Methods ◽  
Tail Fiber ◽  
Yersinia Species ◽  
Recombinant Receptor ◽  
F1 Antigen ◽  
Phage Receptor ◽  
Bacterial Plaque

The highly pathogenic bacterium Yersinia pestis is the causative agent of plague, a notorious infectious zoonotic disease. When transmitted from person to person as pneumonic plague via droplets, Y. pestis is highly contagious and in most cases is fatal if left untreated. Thus, when plague is suspected, rapid diagnosis is crucial, as a serious course of the infection is only averted by early antibiotic therapy. The bacterium is easy to cultivate, accessible and has a high potential for nefarious use such as bioterrorism. Highly specific, rapid and easy-to-use confirmatory diagnostic methods are required to reliably identify the pathogen independently from PCR-based methods or F1 antigen-based immunological detection. Yersinia pestis specific phages such as L-413C and ΦA1122 are already used for detection of Y. pestis in bacterial plaque or biosensor assays. Here, we made use of the host specificities conferred by phage receptor binding (or tail fiber/spike) proteins (RBP) for developing a specific, fast and simple fluorescence-microscopy-based detection method for Y. pestis. Genes of putative RBP of phages L-413C (gpH) and ΦA1122 (gp17) were fused with those of fluorescent proteins and recombinant receptor-reporter fusion proteins were produced heterologously in Escherichia coli. When first tested on attenuated Y. pestis strain EV76, RBP-reporters bound to the bacterial cell surface. This assay could be completed within a few minutes using live or formaldehyde-inactivated cells. Specificity tests using cultures of closely related Yersinia species and several inactivated fully virulent Y. pestis strains exhibited high specificities of the RBP-reporters against Y. pestis. The L-413C RBP proved to be especially specific, as it only detected Y. pestis at all temperatures tested, whereas the RBP of ΦA1122 also bound to Y. pseudotuberculosis strains at 37 °C (but not at 28, 20 or 6 °C). Finally, the Y. pestis-specific capsule, produced when grown at 37 °C, significantly reduced binding of phage ΦA1122 RBP, whereas the capsule only slightly diminished binding of L-413C RBP.

scholarly journals Sinorhizobium meliloti Phage ΦM9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid

2015 ◽  
Vol 89 (21) ◽  
pp. 10945-10958 ◽  
Author(s):  
Matthew C. Johnson ◽  
Kelsey B. Tatum ◽  
Jason S. Lynn ◽  
Tess E. Brewer ◽  
Stephen Lu ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Open Reading Frames ◽  
Host Cells ◽  
Close Relative ◽  
Tail Fiber ◽  
Nitrogen Fixing ◽  
Related Factors ◽  
Content Type ◽  
Fiber Assembly ◽  
Host Infection

ABSTRACTRelatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of theSinorhizobium meliloti-infecting T4 superfamily phage ΦM9. This phage and its close relativeRhizobiumphage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-likeS. melilotiphages of the ΦM12 group. Structurally, ΦM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ΦM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ΦM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ΦM9 andS. melilotiphage ΦM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide.IMPORTANCEDespite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail through which the DNA is delivered to host cells. This phylogenetic and structural study ofS. meliloti-infecting T4 superfamily phage ΦM9 provides new insight into the diversity of this family. The comparison of structure-related genes in both ΦM9 andS. meliloti-infecting T4 superfamily phage ΦM12, which comes from a completely different lineage of these phages, allows the identification of host infection-related factors.

scholarly journals Salmonellagenomic island 3 is an integrative and conjugative element and contributes to copper and arsenic resistance ofSalmonella enterica

2019 ◽  
Author(s):  
Nobuo Arai ◽  
Tsuyoshi Sekizuka ◽  
Yukino Tamamura ◽  
Masahiro Kusumoto ◽  
Atsushi Hinenoya ◽  
...  
Keyword(s):  
Mobile Genetic Element ◽  
Open Reading Frames ◽  
Trna Genes ◽  
Sequencing Analysis ◽  
Arsenic Resistance ◽  
Filter Mating ◽  
Serovar Typhimurium ◽  
Resistance Island ◽  
Reading Frames ◽  
Integrative And Conjugative Element

ABSTRACTSalmonellagenomic island 3 (SGI3) was first described as a chromosomal island inSalmonella4,[5],12:i:-, a monophasic variant ofSalmonella entericasubsp.entericaserovar Typhimurium. The SGI3 DNA sequence detected fromSalmonella4,[5],12:i:-isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic resistance operon in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter mating experiments using theS. entericaserovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genespheVorpheR. The length of the target site was 52 or 55 bp, and a 55-bpattIsequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4compared with the recipient strains under anaerobic conditions. Resistance was defined by thecusgene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4compared with recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic resistance ofS. enterica.

scholarly journals Cloning, Expression, Purification, and Oligomeric Characterization of the AopB-C-terminus Domain in T3SS Major Translocator Protein of Aeromonas hydrophila

2021 ◽  
Vol 37 (3) ◽  
Author(s):  
Nguyen Van Sang ◽  
Nguyen Thi Uyen
Keyword(s):  
Aeromonas Hydrophila ◽  
Structural Information ◽  
Secretion System ◽  
Host Cells ◽  
Translocator Protein ◽  
Dimensional Structure ◽  
Soluble Form ◽  
Salmonella Spp ◽  
Host Cell Membrane ◽  
C Terminus

Type three secretion system (T3SS) is found exclusively in gram-negative pathogens such as Yersinia spp., Escherichia coli, Salmonella spp., Shigella spp., Pseudomonas spp., Vibrio parahaemolyticus, and Aeromonas hydrophila. The translocon pore of T3SS comprises major and minor translocator proteins that assemble to provide passage of effectors through the host cell membrane. Major translocator protein AopB from Aeromonas hydrophila plays an important role in translocon pore formation. Despite tremendous efforts, structural information regarding the C-terminus domain of major translocator AopB remains elusive. In this study, the DNA fragment encoding for the C-terminus domain of the AopB major translocator from Aeromonas hydrophila AH-1 was cloned in to pET-M expression vector and expressed in BL21 (DE3) host cells. The recombinant AopB-C-terminus domain was successfully purified using immobilized nickel affinity chromatography as a soluble form. Crosslinking analysis among AopB-C-terminus molecules in solution showed that this domain exists as a mixture of tetramer, trimer, dimer and monomer forms. The three-dimensional structure model of AopB-C-terminus oligomerization was built by SWISS-MODEL and PyMol. The oligomeric model of AopB-C-terminus can be used for structural studies of the AopB-C-terminus domain which can contribute to the elucidation of the structure of the type III secretion system.

scholarly journals Patterns of Sequence Conservation in the S-Layer Proteins and Related Sequences in Clostridium difficile

2002 ◽  
Vol 184 (14) ◽  
pp. 3886-3897 ◽  
Author(s):  
Emanuela Calabi ◽  
Neil Fairweather
Keyword(s):  
Clostridium Difficile ◽  
Open Reading Frames ◽  
C Terminus ◽  
Antibiotic Associated Diarrhea ◽  
Processing Site ◽  
Different Strains ◽  
Related Proteins ◽  
High Degree ◽  
Insight Into ◽  
Reading Frames

ABSTRACT Clostridium difficile is the etiological agent of antibiotic-associated diarrhea. Among the factors that may play a role in infection are S-layer proteins (SLPs). Previous work has shown these to consist mainly of two components, resulting from the cleavage of a precursor encoded by the slpA gene. The high-molecular-weight (MW) subunit is related both to amidases from B. subtilis and to at least another 28 gene products in C. difficile strain 630. To gain insight into the functions of the SLPs and related proteins, we have further investigated the pattern of variability both at the slpA locus and at six nearby paralogs. Sequencing of the slpA gene from an S-layer group II strain and a variant S-layer group strain confirms a high degree of divergence in the low-MW SLP, which may result from diversifying selection. A highly conserved motif, however, is found at the C terminus in all low-MW subunits and may be essential for SlpA precursor cleavage. In strain 167, a variant cleavage product is present, suggesting a secondary processing site. Southern blotting analysis shows slpA-like open reading frames (ORFs) 2 to 7 to be conserved in all nine strains tested, with one exception: ORF2, which encodes a 66-kDa polypeptide coextracted at low pH with the main SLPs in strain 630, may be partially deleted in strain 167. Polymorphism within the slpA-ORF7 cluster may be more pronounced in the region proximal to the slpA gene. Unexpectedly, a high-MW subunit probe cross hybridizes to sequences outside the slpA locus, which appear to vary in number in different strains.

scholarly journals SalmonellaGenomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance ofSalmonella enterica

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Nobuo Arai ◽  
Tsuyoshi Sekizuka ◽  
Yukino Tamamura ◽  
Masahiro Kusumoto ◽  
Atsushi Hinenoya ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
Trna Genes ◽  
Sequencing Analysis ◽  
Content Type ◽  
Arsenic Tolerance ◽  
Filter Mating ◽  
Serovar Typhimurium ◽  
Resistance Island ◽  
Reading Frames ◽  
Integrative And Conjugative Element

ABSTRACTSalmonellagenomic island 3 (SGI3) was first described as a chromosomal island inSalmonella4,[5],12:i:–, a monophasic variant ofSalmonella entericasubsp.entericaserovar Typhimurium. The SGI3 DNA sequence detected fromSalmonella4,[5],12:i:– isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using theS. entericaserovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genespheVorpheR. The length of the target site was 52 or 55 bp, and a 55-bpattIsequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4compared to the recipient strains under anaerobic conditions. Tolerance was defined by thecusgene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance ofS. enterica.

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