scholarly journals Raman Microspectroscopy Goes Viral: Infection Dynamics in the Cosmopolitan Microalga, Emiliania huxleyi

2021 ◽  
Vol 12 ◽  
Author(s):  
Elena Yakubovskaya ◽  
Tatiana Zaliznyak ◽  
Joaquín Martínez Martínez ◽  
Gordon T. Taylor
Keyword(s):  
Nuclear Dna ◽  
Isotopic Signature ◽  
Emiliania Huxleyi ◽  
Raman Microspectroscopy ◽  
Stable Isotope Probing ◽  
Marine Phytoplankton ◽  
Host Cells ◽  
Genome Replication ◽  
Label Free ◽  
Material Transfer

Emiliania huxleyi is a cosmopolitan member of the marine phytoplankton. This species’ capacities for carbon sequestration and sulfur mobilization make it a key player in oceanic biogeochemical cycles that influence climate on a planetary scale. Seasonal E. huxleyi blooms are abruptly terminated by viral epidemics caused by a clade of large DNA viruses collectively known as coccolithoviruses (EhVs). EhVs thereby mediate a significant part of material and energy fluxes associated with E. huxleyi population dynamics. In this study, we use spontaneous Raman microspectroscopy to perform label-free and non-invasive measurements of the macromolecular composition of individual virions and E. huxleyi host cells. Our novel autofluorescence suppression protocol enabled spectroscopic visualization of evolving macromolecular redistributions in individual E. huxleyi cells at different stages of EhV infection. Material transfer from E. huxleyi hosts to single EhV-163 virions was confirmed by combining stable isotope probing (SIP) experiments with Raman microspectroscopy. Inheritance of the host cells’ 13C-enriched isotopic signature was quantified based on red shifts of Raman peaks characteristic of phenylalanine’s phenyl ring. Two-dimensional Raman mapping of EhV-infected E. huxleyi cells revealed that the compact region producing an intense Raman DNA signal (i.e., the nucleus) in healthy E. huxleyi cells becomes diffuse during the first hours of infection. Raman DNA emissions integrated throughout individual cells decreased during the infection cycle. Our observations are consistent with EhV-163 degrading the host’s nuclear DNA, scavenging released nucleotides for its own genome replication, and shedding newly-produced virions prior to host lysis via budding.

Label-Free Determination of the Cell Cycle Phase in Human Embryonic Stem Cells by Raman Microspectroscopy

2013 ◽  
Vol 85 (19) ◽  
pp. 8996-9002 ◽  
Author(s):  
Stanislav O. Konorov ◽  
H. Georg Schulze ◽  
James M. Piret ◽  
Michael W. Blades ◽  
Robin F. B. Turner
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Raman Microspectroscopy ◽  
Cell Cycle Phase ◽  
Cycle Phase ◽  
Label Free

scholarly journals Label-Free Discrimination of Rhizobial Bacteroids and Mutants by Single-Cell Raman Microspectroscopy

2017 ◽  
Vol 89 (12) ◽  
pp. 6336-6340 ◽  
Author(s):  
Jiabao Xu ◽  
Isabel Webb ◽  
Philip Poole ◽  
Wei E. Huang
Keyword(s):  
Single Cell ◽  
Raman Microspectroscopy ◽  
Label Free

scholarly journals Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 288 ◽  
Author(s):  
Raquel Tenorio ◽  
Isabel Fernández de Castro ◽  
Jonathan J. Knowlton ◽  
Paula F. Zamora ◽  
Danica M. Sutherland ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Host Cells ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Particle Assembly ◽  
The Matrix ◽  
Viral Genome Replication ◽  
Pathogenic Viruses ◽  
Infected Cells ◽  
Reovirus Replication

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses.

scholarly journals Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired

2017 ◽  
Vol 114 (47) ◽  
pp. 12466-12471 ◽  
Author(s):  
Paulina H. Wanrooij ◽  
Martin K. M. Engqvist ◽  
Josefin M. E. Forslund ◽  
Clara Navarrete ◽  
Anna Karin Nilsson ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Dna ◽  
Excision Repair ◽  
Genome Replication ◽  
Dntp Pool ◽  
Frequent Exchange ◽  
Repair Mechanisms ◽  
Ribonucleoside Triphosphate ◽  
Deoxyribonucleoside Triphosphate ◽  
Mitochondrial Dna Polymerase

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.

scholarly journals Structural and phenotypic analysis of Chikungunya virus RNA replication elements

2019 ◽  
Vol 47 (17) ◽  
pp. 9296-9312 ◽  
Author(s):  
Catherine Kendall ◽  
Henna Khalid ◽  
Marietta Müller ◽  
Dominic H Banda ◽  
Alain Kohl ◽  
...  
Keyword(s):  
Rational Design ◽  
Chikungunya Virus ◽  
Rna Replication ◽  
Host Cells ◽  
Genome Replication ◽  
Reverse Genetic ◽  
Genetic Approach ◽  
Phenotypic Analysis ◽  
Efficient Replication ◽  
Reverse Genetic Approach

Abstract Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.

scholarly journals Genome replication dynamics of a bacteriophage and its satellite reveal strategies for parasitism and viral restriction

2019 ◽  
Author(s):  
Zachary K Barth ◽  
Tania V Silvas ◽  
Angus Angermeyer ◽  
Kimberley D Seed
Keyword(s):  
Dna Replication ◽  
Vibrio Cholerae ◽  
Host Cells ◽  
Initiation Factor ◽  
Cell Populations ◽  
Lytic Phage ◽  
Genome Replication ◽  
Origin Of Replication ◽  
New Host ◽  
Viral Restriction

Abstract Phage-inducible chromosomal island-like elements (PLEs) are bacteriophage satellites found in Vibrio cholerae. PLEs parasitize the lytic phage ICP1, excising from the bacterial chromosome, replicating, and mobilizing to new host cells following cell lysis. PLEs protect their host cell populations by completely restricting the production of ICP1 progeny. Previously, it was found that ICP1 replication was reduced during PLE(+) infection. Despite robust replication of the PLE genome, relatively few transducing units are produced. We investigated if PLE DNA replication itself is antagonistic to ICP1 replication. Here we identify key constituents of PLE replication and assess their role in interference of ICP1. PLE encodes a RepA_N initiation factor that is sufficient to drive replication from the PLE origin of replication during ICP1 infection. In contrast to previously characterized bacteriophage satellites, expression of the PLE initiation factor was not sufficient for PLE replication in the absence of phage. Replication of PLE was necessary for interference of ICP1 DNA replication, but replication of a minimalized PLE replicon was not sufficient for ICP1 DNA replication interference. Despite restoration of ICP1 DNA replication, non-replicating PLE remained broadly inhibitory against ICP1. These results suggest that PLE DNA replication is one of multiple mechanisms contributing to ICP1 restriction.

High Sensitivity Label-Free Imaging with Doppler Raman Microspectroscopy

2019 ◽  
Author(s):  
David R. Smith ◽  
Jeffrey J. Field ◽  
David Winters ◽  
Scott Domingue ◽  
Jesse W. Wilson ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Raman Microspectroscopy ◽  
Label Free

scholarly journals Influence of changing carbonate chemistry on morphology and weight of coccoliths formed by <i>Emiliania huxleyi</i>

2012 ◽  
Vol 9 (8) ◽  
pp. 3449-3463 ◽  
Author(s):  
L. T. Bach ◽  
C. Bauke ◽  
K. J. S. Meier ◽  
U. Riebesell ◽  
K. G. Schulz
Keyword(s):  
Sediment Cores ◽  
Emiliania Huxleyi ◽  
Marine Phytoplankton ◽  
Carbonate Chemistry ◽  
Production Rates ◽  
Organic Part ◽  
Carbonate Ion ◽  
Seawater Carbonate Chemistry ◽  
Decreasing Ph ◽  
Carbon Dioxide Co2

Abstract. The coccolithophore Emiliania huxleyi is a marine phytoplankton species capable of forming small calcium carbonate scales (coccoliths) which cover the organic part of the cell. Calcification rates of E. huxleyi are known to be sensitive to changes in seawater carbonate chemistry. It has, however, not yet been clearly determined how these changes are reflected in size and weight of individual coccoliths and which specific parameter(s) of the carbonate system drive morphological modifications. Here, we compare data on coccolith size, weight, and malformation from a set of five experiments with a large diversity of carbonate chemistry conditions. This diversity allows distinguishing the influence of individual carbonate chemistry parameters such as carbon dioxide (CO2), bicarbonate (HCO3−), carbonate ion (CO32−), and protons (H+) on the measured parameters. Measurements of fine-scale morphological structures reveal an increase of coccolith malformation with decreasing pH suggesting that H+ is the major factor causing malformations. Coccolith distal shield area varies from about 5 to 11 μm2. Changes in size seem to be mainly induced by varying [HCO3−] and [H+] although influence of [CO32−] cannot be entirely ruled out. Changes in coccolith weight were proportional to changes in size. Increasing CaCO3 production rates are reflected in an increase in coccolith weight and an increase of the number of coccoliths formed per unit time. The combined investigation of morphological features and coccolith production rates presented in this study may help to interpret data derived from sediment cores, where coccolith morphology is used to reconstruct calcification rates in the water column.

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