Search Results (8)

The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS. Full article

The alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most commonly used virus in the Baculovirus Expression Vector System (BEVS) and has been utilized for the production of many human and veterinary biologics. AcMNPV has a large dsDNA genome that remains understudied, and relatively unmodified from the wild-type, especially considering how extensively utilized it is as an expression vector. Previously, our group utilized CRISPR-Cas9 genome engineering that revealed phenotypic changes when baculovirus genes are targeted using either co-expressed sgRNA or transfected sgRNA into a stable insect cell line that produced the Cas9 protein. Here, we describe a pipeline to sequence the recombinant AcMNPV expression vectors using shotgun sequencing, provide a set of primers for tiled-amplicon sequencing, show that untargeted baculovirus vector genomes remain relatively unchanged when amplified in Sf9-Cas9 cells, and confirm that AcMNPV gp64 gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing in process genome editing of baculoviruses. Full article

Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important Solanaceae crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5′ and 3′ ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples. Full article

Brevundimonas sp. is a bacteria able to grow in metal(loid) contaminated soil from Puchuncaví Valley, central Chile. This study has isolated a bacterial strain capable of growth under high doses of arsenic (As) (6000 mg L⁻¹), and a draft genome sequence was generated. Additionally, real-time PCR was performed to examine the effect of As on some genes related to As resistance. Results demonstrated a total of 3275 predicted annotated genes with several genes related to the ars operon, metal(loid) resistance-related genes, metal efflux pumps, and detoxifying enzymes. Real-time PCR showed that the arsB involved in the efflux of As was down-regulated, whereas arsR, arsH, and ACR3 did not show differences with the addition of As. Our study provides novel evidence of diverse As regulating systems in tolerant bacteria that will lead to a better understanding of how microorganisms overcome toxic elements and colonize As contaminated soils and to the possible use of their specific properties in bioremediation. Full article

The rhizosphere microbiome is key in survival, development, and stress tolerance in plants. Salinity, drought, and extreme temperatures are frequent events in the Atacama Desert, considered the driest in the world. However, little information of the rhizosphere microbiome and its possible contribution to the adaptation and tolerance of plants that inhabit the desert is available. We used a high-throughput Illumina MiSeq sequencing approach to explore the composition, diversity, and functions of fungal and bacterial communities of the rhizosphere of Baccharis scandens and Solanum chilense native plants from the Atacama Desert. Our results showed that the fungal phyla Ascomycota and Basidiomycota and the bacterial phyla Actinobacteria and Proteobacteria were the dominant taxa in the rhizosphere of both plants. The linear discriminant analysis (LDA) effect size (LefSe) of the rhizosphere communities associated with B. scandens showed the genera Penicillium and Arthrobacter were the preferential taxa, whereas the genera Oidiodendron and Nitrospirae was the preferential taxa in S. chilense. Both plant showed similar diversity, richness, and abundance according to Shannon index, observed OTUs, and evenness. Our results indicate that there are no significant differences (p = 0.1) between the fungal and bacterial communities of both plants, however through LefSe, we find taxa associated with each plant species and the PCoA shows a separation between the samples of each species. This study provides knowledge to relate the assembly of the microbiome to the adaptability to drought stress in desert plants. Full article

Endophytic bacteria are relevant symbionts that contribute to plant growth and development. However, the diversity of bacteria associated with the roots of terrestrial orchids colonizing Andean ecosystems is limited. This study identifies and examines the capabilities of endophytic bacteria associated with peloton-containing roots of six terrestrial orchid species from southern Chile. To achieve our goals, we placed superficially disinfected root fragments harboring pelotons on oatmeal agar (OMA) with no antibiotic addition and cultured them until the bacteria appeared. Subsequently, they were purified and identified using molecular tools and examined for plant growth metabolites production and antifungal activity. In total, 168 bacterial strains were isolated and assigned to 8 OTUs. The orders Pseudomonadales, Burkholderiales, and Xanthomonadales of phylum Proteobacteria were the most frequent. The orders Bacillales and Flavobacteriales of the phylla Firmicutes and Bacteroidetes were also obtained. Phosphate solubilization was detected in majority of isolates; however, it was significantly higher in Collimonas pratensis and Chryseobacterium sp. (PSI = 1.505 ± 0.09 and 1.405 ± 0.24, respectively). Siderophore production was recorded only for C. pratensis (0.657 ± 0.14 mm day⁻¹), Dyella marensis (0.131 ± 0.02 mm day⁻¹), and Luteibacter rhizovicinus (0.343 ± 0.12 mm day⁻¹). Indole acetic acid production was highly influenced by the isolate identity; however, the significantly higher activity was recorded for Pseudomonas spp. (ranging from 5.507 ± 1.57 µg mL⁻¹ to 7.437 ± 0.99 µg mL⁻¹). Additionally, six bacterial isolates were able to inhibit the growth of some potential plant pathogenic fungi. Our findings demonstrate the potential for plant growth promoting capabilities and some antifungal activities of endophytic bacteria inhabiting the mycorrhizal tissue of terrestrial orchids, which may contribute especially at early developmental stages of orchid seedlings. Full article

In soils multi-contaminated with heavy metal and metalloids, the establishment of plant species is often hampered due to toxicity. This may be overcome through the inoculation of beneficial soil microorganisms. In this study, two arsenic-resistant bacterial isolates, classified as Pseudomonas gessardii and Brevundimonas intermedia, and two arsenic-resistant fungi, classified as Fimetariella rabenhortii and Hormonema viticola, were isolated from contaminated soil from the Puchuncaví valley (Chile). Their ability to produce indoleacetic acid and siderophores and mediate phosphate solubilization as plant growth-promoting properties were evaluated, as well as levels of arsenic resistance. A real time PCR applied to Triticum aestivum that grew in soil inoculated with the bacterial and fungal isolates was performed to observe differences in the relative expression of heavy metal stress defense genes. The minimum inhibitory concentration of the bacterial strains to arsenate was up to 7000 mg·L⁻¹ and that of the fungal strains was up to 2500 mg·L⁻¹. P. gessardi was able to produce siderophores and solubilize phosphate; meanwhile, B. intermedia and both fungi produced indoleacetic acid. Plant dry biomass was increased and the relative expression of plant metallothionein, superoxide dismutase, ascorbate peroxidase and phytochelatin synthase genes were overexpressed when P. gessardii plus B. intermedia were inoculated. Full article

A recombinant of Pseudomonas putida LS461 (deletion of the phaC1phaZphaC2 genes) was constructed by introducing cosmid JC123 carrying a novel phaC1₁₆ gene from a metagenomic clone. The resulting strain, P. putida LS46123, was able to synthesize polyhydroxyalkanoate (PHA) polymers with novel monomer compositions when cultured on glucose or free fatty acids, and accumulated PHAs from 9.24% to 27.09% of cell dry weight. The PHAs synthesized by P. putida LS46123 contained up to 50 mol % short chain length subunits (3-hydroxybutyrate and 3-hydroxyvalerate), with the remaining monomers consisting of various medium chain length subunits. The PhaC1₁₆ protein expressed by P. putida LS46123 had an amino acid sequence similarity of 45% with the PhaC1 protein of the parent strain, P. putida LS46. Predicted 3D structures of the PhaC1₁₆ proteins from P. putida LS46123 and P. putida LS46 revealed several differences in the numbers and locations of protein secondary structures. The physical and thermal properties of the novel polymers synthesized by P. putida LS46123 cultured with glucose or free fatty acids differed significantly from those produced by P. putida LS46 grown on the same substrates. PHA polymers with different subunit compositions, and hence different physical and thermal properties, can be tailor-made using novel PHA synthase for specific applications. Full article