scholarly journals Cultivation of a lytic double-stranded RNA bacteriophage infecting Microvirgula aerodenitrificans reveals a mutualistic parasitic lifestyle

2021 ◽  
Author(s):  
Xiaoyao Cai ◽  
Fengjuan Tian ◽  
Li Teng ◽  
Hongmei Liu ◽  
Yigang Tong ◽  
...  
Keyword(s):  
Double Layer ◽  
Sequence Similarity ◽  
Double Stranded Rna ◽  
Bacteria Growth ◽  
A Genome ◽  
Domains Of Life ◽  
Parasitic Lifestyle ◽  
Dsrna Viruses ◽  
Fungal Viruses ◽  
Classical Protocol

Bacteriophages are considered the most abundant entities on earth. However, there are merely seven sequenced double-stranded (ds)RNA phages compared with thousands of dsDNA phages. Interestingly, dsRNA viruses are quite common in fungi and usually have a lifestyle of commensalism or mutualism. Thus, the classical protocol of using double-layer agar plates to characterize phage plaques might be significantly biased in the isolation of dsRNA phages beyond strictly lytic lifestyles. Thus, we applied the protocol of isolating fungal viruses to identify RNA phages in bacteria and successfully isolated a novel dsRNA phage, phiNY, from Microvirgula aerodenitrificans . phiNY has a genome of three dsRNA segments, and its genome sequence has no nucleotide sequence similarity with any other phage. Although phiNY encodes a lytic protein of glycoside hydrolase and phage particles are consistently released during bacterial growth, phiNY replication did not block bacteria growth, nor did it form any plaque on agar plates. More strikingly, the phiNY-infected strain grew faster than the phiNY-negative strain, indicating a mutualistic parasitic lifestyle. Thus, this study not only reveals a new mutualistic parasitic dsRNA phage but also implies that other virus isolation methods would be valuable to identify phages with other lifestyles. Importance Viruses with dsRNA genomes are quite diverse and infect organisms in all three domains of life. Though dsRNA viruses infecting humans, plants and fungi are quite common, dsRNA viruses infecting bacteria, known as bacteriophages, are quite understudied and only seven dsRNA phages have been sequenced so far. One possible explanation for the rare isolation of dsRNA phages might be the protocols of double-layer agar plates assay. Phages beyond strictly lytic lifestyles might not form plaques. Thus, we applied the protocol of isolating fungal viruses to identify RNA phages inside bacteria and successfully isolated a novel dsRNA phage phiNY with a mutualistic parasitic lifestyle. This study implies dsRNA phages beyond strictly lytic lifestyle might be common in nature and deserves more investigations.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates

2021 ◽  
Vol 102 (7) ◽  
Author(s):  
Caitlin A. O'Brien ◽  
Jessica J. Harrison ◽  
Agathe M. G. Colmant ◽  
Renee J. Traves ◽  
Devina Paramitha ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Untranslated Regions ◽  
Fixation Method ◽  
Enveloped Viruses ◽  
Double Stranded Rna ◽  
Vertebrate Cell ◽  
Infected Cells ◽  
Replicative Intermediates ◽  
Dsrna Viruses ◽  
Arboviral Disease

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

Nonomuraea montanisoli sp. nov., isolated from mountain forest soil

2021 ◽  
Author(s):  
Suchart Chanama ◽  
Chanwit Suriyachadkun ◽  
Manee Chanama
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Related Species ◽  
Sequence Similarity ◽  
Diaminopimelic Acid ◽  
Mountain Forest ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
A Genome

A novel actinomycete, strain SMC 257T, was isolated from a soil sample collected from mountain forest, Nan Province, Thailand. Strain SMC 257T formed tightly closed spiral spore chains on aerial mycelia. A polyphasic approach was used for the taxonomic study of this strain. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SMC 257T belonged to the genus Nonomuraea , and the closest phylogenetically related species were Nonomuraea roseoviolacea subsp. carminata JCM 9946T (98.9 % 16S rRNA gene sequence similarity), Nonomuraea rhodomycinica TBRC 6557T (98.4 %), and Nonomuraea roseoviolacea subsp. roseoviolacea JCM 3145T (98.3 %). Genome sequencing revealed a genome size of 9.76 Mbp and a G+C content of 72.3 mol%. The genome average nucleotide identity (ANI) and the digital DNA–DNA hybridization (dDDH) values that distinguished this novel strain from its closest related species were species boundary of 95–96 % and 70 %, respectively. The cell wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars were glucose, ribose, madurose and mannose. The major menaquinone was MK-9(H4). The polar lipid profile consisted of phosphatidylethanolamine, hydroxyphosphatidylethanolamine, lysophosphatidylethanolamine, diphosphatidylglycerol, N-phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. The predominant cellular fatty acids were C17 : 0 10-methyl and iso-C16 : 0. Based on comparative analysis of phenotypic, chemotaxonomic and genotypic data, strain SMC 257T is considered to represent a novel species of the genus Nonomuraea , for which the name Nonomuraea montanisoli is proposed. The type strain is SMC 257T (=TBRC 13065T=NBRC 114772T).

scholarly journals A Novel Bipartite Double-Stranded RNA Mycovirus from the White Root Rot Fungus Rosellinia necatrix: Molecular and Biological Characterization, Taxonomic Considerations, and Potential for Biological Control

2009 ◽  
Vol 83 (24) ◽  
pp. 12801-12812 ◽  
Author(s):  
Sotaro Chiba ◽  
Lakha Salaipeth ◽  
Yu-Hsin Lin ◽  
Atsuko Sasaki ◽  
Satoko Kanematsu ◽  
...  
Keyword(s):  
Biological Control ◽  
Root Rot ◽  
Sequence Similarity ◽  
Peptide Mass Fingerprinting ◽  
Dsrna Virus ◽  
Rosellinia Necatrix ◽  
Virus Family ◽  
White Root Rot ◽  
Double Stranded Rna ◽  
Peptide Mass

ABSTRACT White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5′ (24 nt) and 3′ (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5′ untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3′-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions ∼50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.

scholarly journals Hansschlegelia quercus sp. nov., a novel methylotrophic bacterium isolated from oak buds

2020 ◽  
Vol 70 (8) ◽  
pp. 4646-4652 ◽  
Author(s):  
Nadezhda V. Agafonova ◽  
Elena N. Kaparullina ◽  
Denis S. Grouzdev ◽  
Nina V. Doronina
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Sequence Similarity ◽  
Methylotrophic Bacterium ◽  
Rrna Gene ◽  
Methylotrophic Bacteria ◽  
Content Type ◽  
Link Type ◽  
A Genome

Novel aerobic, restricted facultatively methylotrophic bacteria were isolated from buds of English oak (Quercus robur L.; strain DubT) and northern red oak (Quercus rubra L.; strain KrD). The isolates were Gram-negative, asporogenous, motile short rods that multiplied by binary fisson. They utilized methanol, methylamine and a few polycarbon compounds as carbon and energy sources. Optimal growth occurred at 25 °C and pH 7.5. The dominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and phoshatidylglycerol. The major cellular fatty acids of cells were C18 : 1 ω7c, 11-methyl C18 : 1 ω7c and C16 : 0. The major ubiquinone was Q-10. Analysis of 16S rRNA gene sequences showed that the strains were closely related to the members of the genus Hansschlegelia : Hansschlegelia zhihuaiae S113T(97.5–98.0 %), Hansschlegelia plantiphila S1T (97.4–97.6 %) and Hansschlegelia beijingensis PG04T(97.0–97.2 %). The 16S rRNA gene sequence similarity between strains DubT and KrD was 99.7 %, and the DNA–DNA hybridization (DDH) result between the strains was 85 %. The ANI and the DDH values between strain DubT and H. zhihuaiae S113T were 80.1 and 21.5  %, respectively. Genome sequencing of the strain DubT revealed a genome size of 3.57 Mbp and a G+C content of 67.0 mol%. Based on the results of the phenotypic, chemotaxonomic and genotypic analyses, it is proposed that the isolates be assigned to the genus Hansschlegelia as Hansschlegelia quercus sp. nov. with the type strain DubT (=VKM B-3284T=CCUG 73648T=JCM 33463T).

scholarly journals Siblings or doppelgängers? Deciphering the evolution of structured cis-regulatory RNAs beyond homology

2020 ◽  
Vol 48 (5) ◽  
pp. 1941-1951
Author(s):  
Elizabeth C. Gray ◽  
Daniel M. Beringer ◽  
Michelle M. Meyer
Keyword(s):  
Functional Diversity ◽  
Sequence Similarity ◽  
Functional Validation ◽  
Rna Sequences ◽  
Full Spectrum ◽  
Regulatory Molecule ◽  
Mrna Transcripts ◽  
Domains Of Life ◽  
Regulatory Rnas

Structured cis-regulatory RNAs have evolved across all domains of life, highlighting the utility and plasticity of RNA as a regulatory molecule. Homologous RNA sequences and structures often have similar functions, but homology may also be deceiving. The challenges that derive from trying to assign function to structure and vice versa are not trivial. Bacterial riboswitches, viral and eukaryotic IRESes, CITEs, and 3′ UTR elements employ an array of mechanisms to exert their effects. Bioinformatic searches coupled with biochemical and functional validation have elucidated some shared and many unique ways cis-regulators are employed in mRNA transcripts. As cis-regulatory RNAs are resolved in greater detail, it is increasingly apparent that shared homology can mask the full spectrum of mRNA cis-regulator functional diversity. Furthermore, similar functions may be obscured by lack of obvious sequence similarity. Thus looking beyond homology is crucial for furthering our understanding of RNA-based regulation.

scholarly journals Colibacter massiliensis gen. nov. sp. nov., a novel Gram-stain-positive anaerobic diplococcal bacterium, isolated from the human left colon

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hussein Anani ◽  
Rita Abou Abdallah ◽  
May Khoder ◽  
Anthony Fontanini ◽  
Morgane Mailhe ◽  
...  
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Left Colon ◽  
Deficiency Anemia ◽  
Rrna Gene ◽  
Gram Stain ◽  
16S Rrna Sequence ◽  
A Genome ◽  
The Family ◽  
Bacterium Strain

AbstractThe gut microbiota is considered to play a key role in human health. As a consequence, deciphering its microbial diversity is mandatory. A polyphasic taxonogenomic strategy based on the combination of phenotypic and genomic analyses was used to characterize a new bacterium, strain Marseille-P2911. This strain was isolated from a left colon sample of a 60-year old man who underwent a colonoscopy for an etiological investigation of iron-deficiency anemia in Marseille, France. On the basis of 16S rRNA sequence comparison, the closest phylogenetic neighbor was Anaeroglobus geminatus (94.59% 16S rRNA gene sequence similarity) within the family Veillonellaceae. Cells were anaerobic, Gram-stain-positive, non-spore-forming, catalase/oxidase negative cocci grouped in pairs. The bacterium was able to grow at 37 °C after 2 days of incubation. Strain Marseille-P2911 exhibited a genome size of 1,715,864-bp with a 50.2% G + C content, and digital DNA-DNA hybridization (dDDH) and OrthoANI values with A. geminatus of only 19.1 ± 4.5% and 74.42%, respectively. The latter value being lower than the threshold for genus delineation (80.5%), we propose the creation of the new genus Colibacter gen. nov., with strain Marseille-P2911T (=DSM 103304 = CSUR P2911) being the type strain of the new species Colibacter massiliensis gen. nov., sp. nov.

scholarly journals Probing the Transcription Mechanisms of Reovirus Cores with Molecules That Alter RNA Duplex Stability

2009 ◽  
Vol 83 (11) ◽  
pp. 5659-5670 ◽  
Author(s):  
Alexander A. Demidenko ◽  
Max L. Nibert
Keyword(s):  
Small Molecules ◽  
Dimethyl Sulfoxide ◽  
Active Fraction ◽  
Core Particle ◽  
Double Stranded Rna ◽  
Promoter Escape ◽  
Duplex Stability ◽  
Rna Duplexes ◽  
Dsrna Viruses ◽  
Transcript Initiation

ABSTRACT The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps.

scholarly journals Inferring Genome Trees by Using a Filter To Eliminate Phylogenetically Discordant Sequences and a Distance Matrix Based on Mean Normalized BLASTP Scores

2002 ◽  
Vol 184 (8) ◽  
pp. 2072-2080 ◽  
Author(s):  
G. D. Paul Clarke ◽  
Robert G. Beiko ◽  
Mark A. Ragan ◽  
Robert L. Charlebois
Keyword(s):  
Sequence Similarity ◽  
Distance Matrix ◽  
Bootstrap Support ◽  
Systematic Bias ◽  
Reading Frame ◽  
Topological Features ◽  
A Genome ◽  
Pairwise Sequence Similarity ◽  
Similarity Relationships ◽  
Genome Tree

ABSTRACT Darwin's paradigm holds that the diversity of present-day organisms has arisen via a process of genetic descent with modification, as on a bifurcating tree. Evidence is accumulating that genes are sometimes transferred not along lineages but rather across lineages. To the extent that this is so, Darwin's paradigm can apply only imperfectly to genomes, potentially complicating or perhaps undermining attempts to reconstruct historical relationships among genomes (i.e., a genome tree). Whether most genes in a genome have arisen via treelike (vertical) descent or by lateral transfer across lineages can be tested if enough complete genome sequences are used. We define a phylogenetically discordant sequence (PDS) as an open reading frame (ORF) that exhibits patterns of similarity relationships statistically distinguishable from those of most other ORFs in the same genome. PDSs represent between 6.0 and 16.8% (mean, 10.8%) of the analyzable ORFs in the genomes of 28 bacteria, eight archaea, and one eukaryote (Saccharomyces cerevisiae). In this study we developed and assessed a distance-based approach, based on mean pairwise sequence similarity, for generating genome trees. Exclusion of PDSs improved bootstrap support for basal nodes but altered few topological features, indicating that there is little systematic bias among PDSs. Many but not all features of the genome tree from which PDSs were excluded are consistent with the 16S rRNA tree.

scholarly journals Dual Role of a Viral Polymerase in Viral Genome Replication and Particle Self-Assembly

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Xiaoyu Sun ◽  
Serban L. Ilca ◽  
Juha T. Huiskonen ◽  
Minna M. Poranen
Keyword(s):  
Self Assembly ◽  
Viral Genome ◽  
Rna Viruses ◽  
The Self ◽  
Genome Replication ◽  
Double Stranded Rna ◽  
Polymerase Complex ◽  
Viral Genome Replication ◽  
Dsrna Viruses

ABSTRACTDouble-stranded RNA (dsRNA) viruses package several RNA-dependent RNA polymerases (RdRp) together with their dsRNA genome into an icosahedral protein capsid known as the polymerase complex. This structure is highly conserved among dsRNA viruses but is not found in any other virus group. RdRp subunits typically interact directly with the main capsid proteins, close to the 5-fold symmetric axes, and perform viral genome replication and transcription within the icosahedral protein shell. In this study, we utilizedPseudomonasphage Φ6, a well-established virus self-assembly model, to probe the potential roles of the RdRp in dsRNA virus assembly. We demonstrated that Φ6 RdRp accelerates the polymerase complex self-assembly process and contributes to its conformational stability and integrity. We highlight the role of specific amino acid residues on the surface of the RdRp in its incorporation during the self-assembly reaction. Substitutions of these residues reduce RdRp incorporation into the polymerase complex during the self-assembly reaction. Furthermore, we determined that the overall transcription efficiency of the Φ6 polymerase complex increased when the number of RdRp subunits exceeded the number of genome segments. These results suggest a mechanism for RdRp recruitment in the polymerase complex and highlight its novel role in virion assembly, in addition to the canonical RNA transcription and replication functions.IMPORTANCEDouble-stranded RNA viruses infect a wide spectrum of hosts, including animals, plants, fungi, and bacteria. Yet genome replication mechanisms of these viruses are conserved. During the infection cycle, a proteinaceous capsid, the polymerase complex, is formed. An essential component of this capsid is the viral RNA polymerase that replicates and transcribes the enclosed viral genome. The polymerase complex structure is well characterized for many double-stranded RNA viruses. However, much less is known about the hierarchical molecular interactions that take place in building up such complexes. Using the bacteriophage Φ6 self-assembly system, we obtained novel insights into the processes that mediate polymerase subunit incorporation into the polymerase complex for generation of functional structures. The results presented pave the way for the exploitation and engineering of viral self-assembly processes for biomedical and synthetic biology applications. An understanding of viral assembly processes at the molecular level may also facilitate the development of antivirals that target viral capsid assembly.

scholarly journals Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Maxwell J. Scott ◽  
Joshua B. Benoit ◽  
Rebecca J. Davis ◽  
Samuel T. Bailey ◽  
Virag Varga ◽  
...  
Keyword(s):  
Genetic Control ◽  
New World ◽  
Strain Analysis ◽  
Gene Drive ◽  
Screwworm Fly ◽  
Control Programs ◽  
Host Seeking ◽  
A Genome ◽  
Long Read ◽  
Parasitic Lifestyle

AbstractThe New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

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