scholarly journals Attenuation of a Pathogenic Mycoplasma Strain by Modification of the obg Gene by Using Synthetic Biology Approaches

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Carole Lartigue ◽  
Yanina Valverde Timana ◽  
Fabien Labroussaa ◽  
Elise Schieck ◽  
Anne Liljander ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Recipient Cell ◽  
Single Point ◽  
Chemical Mutagenesis ◽  
Economic Losses ◽  
Temperature Sensitive ◽  
Content Type ◽  
Mycoplasma Mycoides ◽  
Virulent Phenotype

ABSTRACT Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety. IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri. Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.

scholarly journals Development of oriC-Based Plasmids for Mesoplasma florum

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Dominick Matteau ◽  
Marie-Eve Pepin ◽  
Vincent Baby ◽  
Samuel Gauthier ◽  
Mélissa Arango Giraldo ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Genetic Engineering ◽  
Genome Engineering ◽  
Antibiotic Resistance Genes ◽  
Viable Cell ◽  
Pathogenic Potential ◽  
Strong Basis ◽  
Content Type ◽  
Basic Biology

ABSTRACT The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10−6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10−6 and 8.44 × 10−7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

scholarly journals Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

2015 ◽  
Vol 89 (12) ◽  
pp. 6312-6327 ◽  
Author(s):  
Kathleen A. Boyle ◽  
Matthew D. Greseth ◽  
Paula Traktman
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Cell Line ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Chemical Mutagenesis ◽  
Temperature Sensitive ◽  
Genome Replication ◽  
Content Type ◽  
Physical Autonomy

ABSTRACTThe duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant istsfor function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed thets57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1–H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vΔH5). Analysis of vΔH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis.IMPORTANCEVariola virus, the causative agent of smallpox, is the most notorious member of thePoxviridaefamily. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.

scholarly journals Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

2016 ◽  
Vol 82 (17) ◽  
pp. 5421-5427 ◽  
Author(s):  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Genome Editing ◽  
Genome Engineering ◽  
Shuttle Vector ◽  
Temperature Sensitive ◽  
Target Sequence ◽  
Target Specificity ◽  
Content Type ◽  
A Genome ◽  
Single Plasmid

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system fromStreptococcus pyogeneshas recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing ofBacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication forEscherichia coli, the temperature-sensitive replication origin of plasmid pE194tsforB. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries thecas9gene under the control of theB. subtilismannose-inducible promoter PmanPand a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5′ end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in theB. subtilischromosome and by repair of thetrpC2mutation ofB. subtilis168.IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing ofB. subtilisovercomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

The European Library – history, technique and user expectations

2007 ◽  
Vol 35 (3) ◽  
pp. 154-156 ◽  
Author(s):  
Eric van der Meulen
Keyword(s):  
Design Methodology ◽  
Single Point ◽  
General Description ◽  
Library History ◽  
Content Type ◽  
User Expectations ◽  
National Libraries

PurposeThe purpose of this paper is to describe the single point of access to the collections of the European National Libraries via The European Library.Design/methodology/approachThe paper presents a description of The European Library.FindingsThe emphasis in this article is on user expectations with regards to access, but more importantly to the content behind the record. It describes how the European Library is responding to these expectations, rather than a general description of developments.Originality/valueThe paper provides a useful overview.

Enabling plant synthetic biology through genome engineering

2015 ◽  
Vol 33 (2) ◽  
pp. 120-131 ◽  
Author(s):  
Nicholas J. Baltes ◽  
Daniel F. Voytas
Keyword(s):  
Synthetic Biology ◽  
Genome Engineering ◽  
Plant Synthetic Biology

scholarly journals Low-Molecular-Weight Metabolites Secreted by Paenibacillus larvae as Potential Virulence Factors of American Foulbrood

2014 ◽  
Vol 80 (8) ◽  
pp. 2484-2492 ◽  
Author(s):  
Hedwig-Annabell Schild ◽  
Sebastian W. Fuchs ◽  
Helge B. Bode ◽  
Bernd Grünewald
Keyword(s):  
Molecular Weight ◽  
Virulence Factors ◽  
Gene Clusters ◽  
Paenibacillus Larvae ◽  
Economic Losses ◽  
Low Molecular Weight ◽  
American Foulbrood ◽  
Toxic Activity ◽  
Content Type

ABSTRACTThe spore-forming bacteriumPaenibacillus larvaecauses a severe and highly infective bee disease, American foulbrood (AFB). Despite the large economic losses induced by AFB, the virulence factors produced byP. larvaeare as yet unknown. To identify such virulence factors, we experimentally infected young, susceptible larvae of the honeybee,Apis mellifera carnica, with differentP. larvaeisolates. Honeybee larvae were rearedin vitroin 24-well plates in the laboratory after isolation from the brood comb. We identified genotype-specific differences in the etiopathology of AFB between the tested isolates ofP. larvae, which were revealed by differences in the median lethal times. Furthermore, we confirmed that extracts ofP. larvaecultures contain low-molecular-weight compounds, which are toxic to honeybee larvae. Our data indicate thatP. larvaesecretes metabolites into the medium with a potent honeybee toxic activity pointing to a novel pathogenic factor(s) ofP. larvae. Genome mining ofP. larvaesubsp.larvaeBRL-230010 led to the identification of several biosynthesis gene clusters putatively involved in natural product biosynthesis, highlighting the potential ofP. larvaeto produce such compounds.

The effects of corporate brand symbolism on consumer satisfaction and loyalty

2016 ◽  
Vol 28 (3) ◽  
pp. 481-498 ◽  
Author(s):  
Tatiana Anisimova
Keyword(s):  
Social Life ◽  
Consumer Satisfaction ◽  
Single Point ◽  
Social Aspects ◽  
Consumer Loyalty ◽  
Cross Sectional ◽  
Corporate Brand ◽  
Content Type ◽  
Strategic Role ◽  
The Impact

Purpose – The purpose of this paper is to test the effects of corporate brand symbolism on consumer satisfaction and loyalty on a sample of Australian automobile consumers. Design/methodology/approach – Survey research was employed to test the study hypotheses. The regression analysis was used to evaluate the relationships between an independent variable (corporate brand symbolism) and dependent variables (consumer satisfaction and loyalty). Findings – Support was found for all hypotheses formulated in this study. Regression results reveal consistent favourable and significant effects of corporate brand symbolism on both consumer satisfaction and loyalty. Research limitations/implications – Although this paper makes contributions in international marketing, the cross-sectional nature of the data collection method limits the information gained to the single point in time. This research studied the impact of corporate brand symbolism on consumers of one original equipment manufacturers (OEM). Having a larger number of participating car manufacturers/OEMs would have provided a wider insight. However, time and resources limitation did not allow to study a larger sample. In the future, practitioners are recommended to further understand the relationship between self and social aspects of brand symbolism in order to formulate more targeted communication strategies. Practical implications – The findings of this study point to the strategic role of the brand in generating both satisfaction and loyalty. In the light of increasing advertising costs and decreasing consumer loyalty, strengthening corporate brand symbolism makes a lot of economic sense. The findings suggest that managers need to take into account consumer need for identity expression and consider this in their branding strategies. Social implications – Humans are social beings by nature. However, international brand research has paid relatively little attention to how products are used by consumers in everyday life, including their social life. Consumer behaviours increasingly depend on social meanings they imbue brands with beyond products’ functional utility. It is argued the focus of symbolic consumption needs to be broadened and integrated more with social science concepts. Originality/value – This study captures a construct of corporate brand symbolism by including self and social aspects of symbolism. The current study also comprehensively measures consumer loyalty, including cognitive, affective and behavioural types of loyalty.

Influence of occupational heat stress on labour productivity – a case study from Chennai, India

2016 ◽  
Vol 65 (2) ◽  
pp. 245-255 ◽  
Author(s):  
Jeremiah Chinnadurai ◽  
Vidhya Venugopal ◽  
Kumaravel P ◽  
Paramesh R
Keyword(s):  
Climate Change ◽  
Heart Rate ◽  
Heat Stress ◽  
Pearson Correlation ◽  
Labour Productivity ◽  
Productivity Loss ◽  
Threshold Limit Value ◽  
Control Measures ◽  
Economic Losses ◽  
Content Type

Purpose – Raise in temperatures due to climate change is likely to increase the heat stress in occupations that are physically exerting and performed outdoors which might potentially have adverse health and productivity consequences. The purpose of this paper is to estimate the productivities in construction work under the influence of heat stress using the predicted mean vote (PMV) index. Design/methodology/approach – Field studies were conducted during May 2014 which is summer time in Chennai. Continuous heart rate of workers and wet bulb globe temperature measurements are conducted for workers engaged in different jobs in construction. Metabolic rates and the workload of the workers from heart rate were calculated using the ISO method 8996 and the PMV values are calculated using the tool developed by Malchaire based on the method ISO 7730. Direct observations and personal interviews were conducted to substantiate the productivity estimations. Findings – The results showed that workers working outdoors with moderate and heavy workload exceeded the threshold limit value of 28°C and had adverse productivity impacts (18-35 per cent productivity loss), whereas the workers engaged in light indoor work was not affected by heat stress and consequent productivity losses. The productivity estimations using the PMV index is found to be statistically significant for three types of construction works (Pearson correlation coefficient value of −0.78) and also correlated well with the observations and self-reported productivities of the workers. Originality/value – The method used in this paper provides a scientific and reliable estimation of the productivities which may benefit the industry to set realistic project completion goals in hot weather and also implement interventions and policies to protect workers’ health. Developing adaptive strategies and implementing control measures are the need of the hour to protect worker’s health and economic losses in the face of climate change.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

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