White mulberry (Morus alba L.) fruit-associated bacterial and fungal microbiota

Morus alba L. has been worldwide cultivated and commercially exploited plant with profound potential in environmental management, food and medicinal industries. Plant-associated microbial communities are playing an essential role in sustainable plant development. In the present study, the bacterial and fungal microorganism populations distributed on the white mulberry fruits harvested in the Czech Republic for the first time were characterized by metagenomics approach. A total of 62 bacterial and 37 fungal families were identified on white mulberry. Bacterial population was represented by the genera Tatumella, Leuconostoc, Frateuria and Pseudomonas, while fungal microorganisms – by Hanseniaspora, Cryptococcus, Cladosporium and Phoma. Both potentially beneficial, inducing resistance in the hosting plant, and pathogenic, responsible for disease development, microorganisms were detected. The information on the prevalence of bacterial and fungal microorganisms on the carposphere of M. alba is highly relevant for the development of strategies for environment-friendly plant cultivation, disease management and prevention

Saccharomyces paradoxus Transcriptional Alterations in Cells of Distinct Phenotype and Viral dsRNA Content

Killer yeasts are attractive antifungal agents with great potential applications in the food industry. Natural Saccharomyces paradoxus isolates provide new dsRNA-based killer systems available for investigation. The presence of viral dsRNA may alter transcriptional profile of S. paradoxus. To test this possibility, a high-throughput RNA sequencing was employed to compare the transcriptomes of S. paradoxus AML 15-66 K66 killer strains after curing them of either M-66 alone or both M-66 and L-A-66 dsRNA viruses. The S. paradoxus cells cured of viral dsRNA(s) showed respiration deficient or altered sporulation patterns. We have identified numerous changes in the transcription profile of genes including those linked to ribosomes and amino acid biosynthesis, as well as mitochondrial function. Our work advance studies of transcriptional adaptations of Saccharomyces spp. induced by changes in phenotype and set of dsRNA viruses, reported for the first time.This article belongs to the Special Issue Recent Advances in the Yeast Killer Systems ResearchThis research was partly funded by European Regional Funds (CZ.02.1.01/16_019/0000759)

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the β-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.This article belongs to the Special Issue MycovirusesThis research was funded by a grant from the Lithuanian Research Council (No. SIT-7/2015

Saccharomyces cerevisiae K2 biocidinė sistema: žudymo ir atsparumo ryšys

The natural distribution of killer yeasts implies a strong competitive advantage of toxin within the ecological niche. Numerous yeast species in many genera as Candida, Kluyveromyces, Pichia, Williopsis, Ustilago etc. are able to produce and secrete extracellular toxic proteins (mycocins) (Schmitt & Breinig, 2006). Such proteins are able to kill the nonkiller yeast as well as yeast of other killer types, while the toxinproducing cells remain immune to their own or the same type of killers. Genetic and molecular studies have shown that the killer proteins are encoded by extrachromosomal genetic elements such as double-stranded RNA or linear DNA, or by nuclear genes (Meinhart & Klassen, 2009)

Different Metabolic Pathways Are Involved in Response of Saccharomyces cerevisiae to L-A and M Viruses

Competitive and naturally occurring yeast killer phenotype is governed by coinfection with dsRNA viruses. Long-term relationship between the host cell and viruses appear to be beneficial and co-adaptive; however, the impact of viral dsRNA on the host gene expression has barely been investigated. Here, we determined the transcriptomic profiles of the host Saccharomyces cerevisiae upon the loss of the M-2 dsRNA alone and the M-2 along with the L-A-lus dsRNAs. We provide a comprehensive study based on the high-throughput RNA-Seq data, Gene Ontology and the analysis of the interaction networks. We identified 486 genes differentially expressed after curing yeast cells of the M-2 dsRNA and 715 genes affected by the elimination of both M-2 and L-A-lus dsRNAs. We report that most of the transcriptional responses induced by viral dsRNAs are moderate. Differently expressed genes are related to ribosome biogenesis, mitochondrial functions, stress response, biosynthesis of lipids and amino acids. Our study also provided insight into the virus–host and virus–virus interplays

Saccharomyces cerevisiae K2 killer system: interplay between killing and resistance

The natural distribution of killer yeasts implies a strong competitive advantage of toxin within the ecological niche. Numerous yeast species in many genera as Candida, Kluyveromyces, Pichia, Williopsis, Ustilago etc. are able to produce and secrete extracellular toxic proteins (mycocins) (Schmitt & Breinig, 2006). Such proteins are able to kill the nonkiller yeast as well as yeast of other killer types, while the toxinproducing cells remain immune to their own or the same type of killers. Genetic and molecular studies have shown that the killer proteins are encoded by extrachromosomal genetic elements such as double-stranded RNA or linear DNA, or by nuclear genes (Meinhart & Klassen, 2009)

Association of ScV-LA virus with host protein metabolism determined by proteomics analysis and cognate RNA sequencing

Saccharomyces yeasts are highly dispersed in the environment and microbiota of higher organisms. The yeast killing phenotype, encoded by the viral system, was discovered to be a significant property for host survival. Minor alterations in transcription patterns underpin the reciprocal relationship between LA and M viruses and their hosts, suggesting the fine-tuning of the transcriptional landscape. To uncover the principal targets of both viruses, we performed proteomics analysis of virus-enriched subsets of host proteins in virus type-specific manner. The essential pathways of protein metabolism–from biosynthesis and folding to degradation–were found substantially enriched in virus-linked subsets. The fractionation of viruses allowed separation of virus-linked host RNAs, investigated by high-content RNA sequencing. Ribosomal RNA was found to be inherently associated with LA-lus virus, along with other RNAs essential for ribosome biogenesis. This study provides a unique portrayal of yeast virions through the characterization of the associated proteome and cognate RNAs, and offers a background for understanding ScV-LA viral infection persistency

The bacterial microbiota of edible insects Acheta domesticus and Gryllus assimilis revealed by high content analysis

In the concept of novel food, insects reared under controlled conditions are considered mini livestock. Mass-reared edible insect production is an economically and ecologically beneficial alternative to conventional meat gain. Regarding food safety, insect origin ingredients must comply with food microbial requirements. House crickets (Acheta domesticus) and Jamaican field crickets (Gryllus assimilis) are preferred insect species that are used commercially as food. In this study, we examined cricket-associated bacterial communities using amplicon-based sequencing of the 16S ribosomal RNA gene region (V3–V4). The high taxonomic richness of the bacterial populations inhabiting both tested cricket species was revealed. According to the analysis of alpha and beta diversity, house crickets and Jamaican field crickets displayed significantly different bacterial communities. Investigation of bacterial amplicon sequence variants (ASVs) diversity revealed cricket species as well as surface and entire body-associated bacterial assemblages. The efficiency of crickets processing and microbial safety were evaluated based on viable bacterial counts and identified bacterial species. Among the microorganisms inhabiting both tested cricket species, the potentially pathogenic bacteria are documented. Some bacteria representing identified genera are inhabitants of the gastrointestinal tract of animals and humans, forming a normal intestinal microflora and performing beneficial probiotic functions. The novel information on the edible insect-associated microbiota will contribute to developing strategies for cricket processing to avoid bacteria-caused risks and reap the benefits

Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility

Background. Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal findings. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action