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17 pages, 2699 KiB

Open AccessArticle

Isolation, Characterization, and Genome Engineering of a Lytic Pseudomonas aeruginosa Phage

by Xiaomei Cong, Shuang Zhao, Qing Zhang, Shuo Liu, Youming Zhang and Fu Yan

Microorganisms 2024, 12(11), 2346; https://doi.org/10.3390/microorganisms12112346 - 16 Nov 2024

Cited by 1 | Viewed by 2562

Antibiotic-resistant bacterial infections have become one of the leading causes of human mortality. Bacteriophages presented great potential for combating antibiotic-resistant infections in the post-antibiotic era due to their high host specificity and safety profile. Pseudomonas aeruginosa, an opportunistic pathogenic bacterium, has shown a surge in multidrug-resistant strains, severely impacting both human health and livestock. In this study, we successfully isolated and purified a P. aeruginosa-specific phage, PpY1, from feces collected from a breeding farm. This phage harbors a short tail and a 43,787 bp linear genome, and exhibited potent lytic activity against several pathogenic P. aeruginosa strains. Leveraging Transformation-associated recombination (TAR) cloning and phage assembly techniques in a P. aeruginosa host lacking a restriction–modification system, we developed a genome engineering platform for PpY1. Through a systematic gene knockout approach, we identified and eliminated 21 nonessential genes from the PpY1 genome, resulting in a series of phages with reduced genomes. This research not only enhances our understanding of the phage genome but also paves the way for the functional optimization of phages, e.g., broadening the host spectrum and elevating the lytic capacity, dedicated towards the treatment of bacterial infections. Full article

(This article belongs to the Section Molecular Microbiology and Immunology)

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13 pages, 1620 KiB

Open AccessArticle

Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion

by Lin Ding, David M. Brown and John I. Glass

Viruses 2021, 13(4), 603; https://doi.org/10.3390/v13040603 - 1 Apr 2021

Cited by 6 | Viewed by 3729

Abstract

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2. Full article

(This article belongs to the Section Invertebrate Viruses)

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10 pages, 2030 KiB

Open AccessCommunication

Heterologous Expression of the Core Genes in the Complex Fusarubin Gene Cluster of Fusarium Solani

by Tobias Bruun Pedersen, Mikkel Rank Nielsen, Sebastian Birkedal Kristensen, Eva Mie Lang Spedtsberg, Wafaa Yasmine, Rikke Matthiesen, Samba Evelyne Kabemba Kaniki, Trine Sørensen, Celine Petersen, Jens Muff, Teis Esben Sondergaard, Kåre Lehmann Nielsen, Reinhard Wimmer and Jens Laurids Sørensen

Int. J. Mol. Sci. 2020, 21(20), 7601; https://doi.org/10.3390/ijms21207601 - 14 Oct 2020

Cited by 16 | Viewed by 4069

Abstract

Through stepwise recreation of the biosynthetic gene cluster containing PKS3 from Fusarium solani, it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in Saccharomyces cerevisiae. This was achieved through sequential transformation associated recombination (TAR) cloning of FvPPT, fsr1, fsr2, and fsr3 into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in S. cerevisiae. The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1–4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L. Full article

(This article belongs to the Special Issue Molecular Biology and Chemistry of Mycotoxins and Phytotoxins)

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