Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools.

The CRISPR/Cas9 systems have been developed as tools for genetic engineering and metabolic engineering in various organisms. In this review, various aspects of CRISPR/Cas9 in Saccharomyces cerevisiae, from basic principles to practical applications, have been summarized. First, a comprehensive review has been conducted on the history of CRISPR/Cas9, successful cases of gene disruptions, and efficiencies of multiple DNA fragment insertions. Such advanced systems have accelerated the development of microbial engineering by reducing time and labor, and have enhanced the understanding of molecular genetics. Furthermore, the research progress of the CRISPR/Cas9-based systems in the production of high-value-added chemicals and the improvement of stress tolerance in S. cerevisiae have been summarized, which should have an important reference value for genetic and synthetic biology studies based on S. cerevisiae.

RiboGrove: a database of full-length prokaryotic 16S rRNA genes derived from completely assembled genomes.

16S rRNA gene is frequently used for the identification of prokaryotic organisms and for phylogeny inference. Several specialized public databases exist that contain complete and partial sequences of 16S rRNA genes. In this paper, we present RiboGrove: the first publicly available database that comprises only full-length sequences of 16S rRNA genes originating from completely assembled prokaryotic genomes deposited in RefSeq. Despite being strongly biased towards frequently sequenced genomes, RiboGrove is a useful complement to existing 16S rRNA resources and allows for analyses that would not be possible using amplicon-derived gene sequences. For instance, the absence of partial gene sequences in RiboGrove allowed us to make a summary of prokaryotic organisms, which lack core anti-Shine-Dalgarno sequence in their 16S rRNA genes. In this study, we describe the collected sequence data and present the results of exploratory data analysis of 16S rRNA gene sequences.

The Psychrotrophic Pseudomonas lundensis, a Non-aeruginosa Pseudomonad, Has a Type III Secretion System of the Ysc Family, Which Is Transcriptionally Active at 37°C.

The type III secretion system (T3SS) is a needle-like structure found in Gram-negative pathogens that directly delivers virulence factors like toxins and effector molecules into eukaryotic cells. The T3SS is classified into different families according to the type of effector and host. Of these, the Ysc family T3SS, found in Yersinia species and Pseudomonas aeruginosa, confers high virulence to bacteria against eukaryotic hosts. Here, we present the first identification and transcriptional analyses of a Ysc T3SS in a non-aeruginosa Pseudomonas species, Pseudomonas lundensis, an environmental psychrotrophic bacterium and important agent of frozen food spoilage. We have identified and sequenced isolates of P. lundensis from three very distinct ecological niches (Antarctic temporary meltwater pond, U.S. supermarket 1% pasteurized milk, and cystic fibrosis lungs) and compared these to previously reported food spoilage isolates in Europe. In this paper, we show that strains of P. lundensis isolated from these diverse environments with ambient temperatures ranging from below freezing to 37°C all possess a Ysc family T3SS secretion system and a T3S effector, ExoU. Using in vitro and in vivo transcriptomics, we show that the T3SS in P. lundensis is transcriptionally active, is expressed more highly at mammalian body temperature (37°C) than 4°C, and has even higher expression levels when colonizing a host environment (mouse intestine). Thus, this Ysc T3SS-expressing psychrotrophic Pseudomonad has an even greater range of growth niches than previously appreciated, including diseased human airways. IMPORTANCE P. lundensis strains have been isolated from environments that are distinct and diverse in both nutrient availability and environmental pressures (cold food spoilage, Antarctic melt ponds, cystic fibrosis lungs). As a species, this bacterium can grow in diverse niches that markedly vary in available nutrients and temperature, and in our study, we show that these various strains share greater than 99% sequence similarity. In addition, all isolates studied here encoded complete homologs of the Ysc family T3SS seen in P. aeruginosa. Until recently, P. aeruginosa has remained as the only Pseudomonas species to have a characterized functional Ysc (Psc) family T3SS. With the identification of a complete Ysc T3SS in P. lundensis that is expressed at 37°C in vivo, it is intriguing to wonder whether this bacterium may indeed have some level of symbiotic activity, of yet unknown type, when consumed by a mammalian host.

Barapost: Binning of Nucleotide Sequences According to Taxonomic Annotation.

Contemporary sequencing technologies, Oxford Nanopore in particular, provide a way to sequence multiple samples during single run using molecular barcodes. Specific circumstances, however, can make barcoding undesirable or unaffordable. Here, we introduce Barapost: a command-line toolkit that demultiplexes long nucleotide sequences relying on taxonomic annotation instead of barcoding.

Complete Genome Sequence of Pseudomonas syringae BIM B-268, a Plant Pathogen Strain Used for In Vitro Testing of Agricultural Bacteriophage Efficiency.

Pseudomonas syringae BIM B-268 is the strain used for in vitro testing of the efficiency of Multiphage, a bacteriophage-based biopesticide produced in Belarus. The genome sequence of this strain consists of a single circular chromosome harboring the genes encoding the ice nucleation protein, syringopeptin biosynthesis, and types III and VI secretion systems.

The Bacteriophage Pf-10-A Component of the Biopesticide "Multiphage" Used to Control Agricultural Crop Diseases Caused by Pseudomonas syringae.

Phytopathogenic pseudomonads are widespread in the world and cause a wide range of plant diseases. In this work, we describe the Pseudomonas phage Pf-10, which is a part of the biopesticide "Multiphage" used for bacterial diseases of agricultural crops caused by Pseudomonas syringae. The Pf-10 chromosome is a dsDNA molecule with two direct terminal repeats (DTRs). The phage genomic DNA is 39,424 bp long with a GC-content of 56.5%. The Pf-10 phage uses a packaging mechanism based on T7-like short DTRs, and the length of each terminal repeat is 257 bp. Electron microscopic analysis has shown that phage Pf-10 has the podovirus morphotype. Phage Pf-10 is highly stable at pH values from 5 to 10 and temperatures from 4 to 60 °C and has a lytic activity against Pseudomonas strains. Phage Pf-10 is characterized by fast adsorption rate (80% of virions attach to the host cells in 10 min), but has a relatively small number of progeny (37 ± 8.5 phage particles per infected cell). According to the phylogenetic analysis, phage Pf-10 can be classified as a new phage species belonging to the genus Pifdecavirus, subfamily Studiervirinae, family Autographiviridae, order Caudovirales.

SARS-CoV-2 transmission dynamics in Belarus revealed by genomic and incidence data analysis.

Since the emergence of COVID-19, a series of non-pharmaceutical interventions (NPIs) has been implemented by governments and public health authorities world-wide to control and curb the ongoing pandemic spread. From that perspective, Belarus is one of a few countries with a relatively modern healthcare system, where much narrower NPIs have been put in place. Given the uniqueness of this Belarusian experience, the understanding its COVID-19 epidemiological dynamics is essential not only for the local assessment, but also for a better insight into the impact of different NPI strategies globally. In this work, we integrate genomic epidemiology and surveillance methods to investigate the emergence and spread of SARS-CoV-2 in the country. The observed Belarusian SARS-CoV-2 genetic diversity originated from at least eighteen separate introductions, at least five of which resulted in on-going domestic transmissions. The introduction sources represent a wide variety of regions, although the proportion of regional virus introductions and exports from/to geographical neighbors appears to be higher than for other European countries. Phylodynamic analysis indicates a moderate reduction in the effective reproductive number ℛ e after the introduction of limited NPIs, with the reduction magnitude generally being lower than for countries with large-scale NPIs. On the other hand, the estimate of the Belarusian ℛ e at the early epidemic stage is comparable with this number for the neighboring ex-USSR country of Ukraine, where much broader NPIs have been implemented. The actual number of cases by the end of May, 2020 was predicted to be 2-9 times higher than the detected number of cases.

SARS-CoV-2 transmission dynamics in Belarus in 2020 revealed by genomic and incidence data analysis.

Background: Non-pharmaceutical interventions (NPIs) have been implemented worldwide to curb COVID-19 spread. Belarus is a rare case of a country with a relatively modern healthcare system, where highly limited NPIs have been enacted. Thus, investigation of Belarusian COVID-19 dynamics is essential for the local and global assessment of the impact of NPI strategies. Methods: We integrate genomic epidemiology and surveillance methods to investigate the spread of SARS-CoV-2 in Belarus in 2020. We utilize phylodynamics, phylogeography, and probabilistic bias inference to study the virus import and export routes, the dynamics of the effective reproduction number, and the incidence of SARS-CoV-2 infection. Results: Here we show that the estimated cumulative number of infections by June 2020 exceeds the confirmed case number by a factor of ~4 (95% confidence interval (2; 9)). Intra-country SARS-CoV-2 genomic diversity originates from at least 18 introductions from different regions, with a high proportion of regional transmissions. Phylodynamic analysis indicates a moderate reduction of the effective reproductive number after the introduction of limited NPIs, but its magnitude is lower than for developed countries with large-scale NPIs. On the other hand, the effective reproduction number estimate is comparable with that for the neighboring Ukraine, where NPIs were broader. Conclusions: The example of Belarus demonstrates how countries with relatively low outward population mobility continue to be integral parts of the global epidemiological environment. Comparison of the effective reproduction number dynamics for Belarus and other countries reveals the effect of different NPI strategies but also emphasizes the role of regional Eastern European sociodemographic factors in the virus spread.

Isolation and characterization of Hena1 - a novel Erwinia amylovora bacteriophage.

Fire blight, caused by plant pathogenic bacterium Erwinia amylovora, is one of the most important diseases of Rosaceae plants. Due to the lack of effective control measures, fire blight infections pose a recurrent threat on agricultural production worldwide. Recently, bacterial viruses, or bacteriophages, have been proposed as environmentally friendly natural antimicrobial agents for fire blight control. Here, we isolated a novel bacteriophage Hena1 with activity against E. amylovora. Further analysis revealed that Hena1 is a narrow-host-range lytic phage belonging to Myoviridae family. Its genome consists of a linear 148,842 bp dsDNA (48.42% GC content) encoding 240 ORFs and 23 tRNA genes. Based on virion structure and genomic composition, Hena1 was classified as a new species of bacteriophage subfamily Vequintavirinae. The comprehensive analysis of Hena1 genome may provide further insights into evolution of bacteriophages infecting plant pathogenic bacteria.

Characterization and genomic analysis of highly efficient thermotolerant oil-degrading bacterium Gordonia sp. 1D.

A thermotolerant bacterial strain 1D isolated from refinery oil-contaminated soil was identified as Gordonia sp. based on the analysis of 16S rRNA and gyrB gene sequences. The strain was found to utilize crude oil, diesel fuel, and a wide spectrum of alkanes at temperatures up to 50 °C. Strain 1D is the first representative of Gordonia amicalis capable of utilizing alkanes of chain length up to С36 at a temperature of 45-50 °C. The degree of crude oil degradation by Gordonia sp. 1D at 45 °C was 38% in liquid medium and 40% in soil (with regard to abiotic loss). There are no examples of so effective hydrocarbon-oxidizing thermotolerant Gordonia in the world literature. The 1D genome analysis revealed the presence of two alkane hydroxylase gene clusters, genes of dibenzothiophene cleavage, and the cleavage of salicylate and gentisate - naphthalene metabolism intermediates. The highly efficient thermotolerant strain Gordonia sp. 1D can be used in remediation of oil-contaminated soils in hot climates.

Characterization of a new ViI-like Erwinia amylovora bacteriophage phiEa2809.

Erwinia amylovora is a Gram-negative plant pathogenic bacteria causing fire blight disease in many Rosaceae species. A novel E. amylovora bacteriophage, phiEa2809, was isolated from symptomless apple leaf sample collected in Belarus. This phage was also able to infect Pantoea agglomerans strains. The genome of phiEa2809 is a double-stranded linear DNA 162,160 bp in length, including 145 ORFs and one tRNA gene. The phiEa2809 genomic sequence is similar to the genomes of the Serratia plymutica phage MAM1, Shigella phage AG-3, Dickeya phage vB DsoM LIMEstone1 and Salmonella phage ViI and lacks similarity to described E. amylovora phage genomes. Based on virion morphology (an icosahedral head, long contractile tail) and genome structure, phiEa2809 was classified as a member of Myoviridae, ViI-like bacteriophages group. PhiEa2809 is the firstly characterized ViI-like bacteriophage able to lyse E. amylovora.