Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.
Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3′ polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae. The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast.
The injection of laboratory animals with pathogenic microorganisms poses a significant safety risk because of the potential for injury by accidental needlestick. This is especially true for researchers using invertebrate models of disease due to the required precision and accuracy of the injection. The restraint of the greater wax moth larvae (Galleria mellonella) is often achieved by grasping a larva firmly between finger and thumb. Needle resistant gloves or forceps can be used to reduce the risk of a needlestick but can result in animal injury, a loss of throughput, and inconsistencies in experimental data. Restraint devices are commonly used for the manipulation of small mammals, and in this manuscript, we describe the construction of two devices that can be used to entrap and restrain G. mellonella larvae prior to injection with pathogenic microbes. These devices reduce the manual handling of larvae and provide an engineering control to protect against accidental needlestick injury while maintaining a high rate of injection.
By characterizing the trajectories of antibiotic resistance gene transfer in bacterial communities such as the gut microbiome, we will better understand the factors that influence this spread of resistance. Our aim was to investigate the host network of a multi-drug resistance broad-host-range plasmid in the culturable gut microbiome of zebrafish. This was done through in vitro and in vivo conjugation experiments with Escherichia coli as donor of the plasmid pB10::gfp. When this donor was mixed with the extracted gut microbiome, only transconjugants of Aeromonas veronii were detected. In separate matings between the same donor and four prominent isolates from the gut microbiome, the plasmid transferred to two of these four isolates, A. veronii and Plesiomonas shigelloides, but not to Shewanella putrefaciens and Vibrio mimicus. When these A. veronii and P. shigelloides transconjugants were the donors in matings with the same four isolates, the plasmid now also transferred from A. veronii to S. putrefaciens. P. shigelloides was unable to donate the plasmid and V. mimicus was unable to acquire it. Finally, when the E. coli donor was added in vivo to zebrafish through their food, plasmid transfer was observed in the gut but only to Achromobacter sp., a rare member of the gut microbiome. This work shows that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome depends on the donor-recipient species combinations and therefore their spatial arrangement. It also suggests that rare gut microbiome members should not be ignored as potential reservoirs of multi-drug resistance plasmids from food. Importance: To understand how antibiotic resistance plasmids end up in human pathogens it is crucial to learn how, where and when they are transferred and maintained in members of bacterial communities such as the gut microbiome. To gain insight into the network of plasmid-mediated antibiotic resistance sharing in the gut microbiome, we investigated the transferability and maintenance of a multi-drug resistance plasmid among the culturable bacteria of the zebrafish gut. We show that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome can depend on which species are involved, as some are important nodes in the plasmid-host network and others dead-ends. Our findings also suggest that rare gut microbiome members should not be ignored as potential reservoirs of multi-drug resistance plasmids from food.
Mycoviruses are widely distributed across fungi, including the yeasts of the Saccharomycotina subphylum. This manuscript reports the first double-stranded RNA (dsRNA) virus isolated from Pichia membranifaciens. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is a member of the Totiviridae. PmV-L-A is 4579 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses that infect Saccharomycotina yeasts. PmV-L-A was found to be part of a monophyletic group within the I-A totiviruses, implying a shared ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy-minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the Gag protein of Saccharomyces cerevisiae virus L-A (ScV-L-A). The predicted tertiary structure of the PmV-L-A Pol and other homologs provided a possible mechanism for totivirus RNA replication due to structural similarities with the RNA-dependent RNA polymerases of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts are essential because of their emerging role in animal disease and their parallels with mammalian viruses.
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