Global biochemical profiling of fast-growing Antarctic bacteria isolated from meltwater ponds by high-throughput FTIR spectroscopy.

Fourier transform infrared (FTIR) spectroscopy is a biophysical technique used for non-destructive biochemical profiling of biological samples. It can provide comprehensive information about the total cellular biochemical profile of microbial cells. In this study, FTIR spectroscopy was used to perform biochemical characterization of twenty-nine bacterial strains isolated from the Antarctic meltwater ponds. The bacteria were grown on two forms of brain heart infusion (BHI) medium: agar at six different temperatures (4, 10, 18, 25, 30, and 37°C) and on broth at 18°C. Multivariate data analysis approaches such as principal component analysis (PCA) and correlation analysis were used to study the difference in biochemical profiles induced by the cultivation conditions. The observed results indicated a strong correlation between FTIR spectra and the phylogenetic relationships among the studied bacteria. The most accurate taxonomy-aligned clustering was achieved with bacteria cultivated on agar. Cultivation on two forms of BHI medium provided biochemically different bacterial biomass. The impact of temperature on the total cellular biochemical profile of the studied bacteria was species-specific, however, similarly for all bacteria, lipid spectral region was the least affected while polysaccharide region was the most affected by different temperatures. The biggest temperature-triggered changes of the cell chemistry were detected for bacteria with a wide temperature tolerance such Pseudomonas lundensis strains and Acinetobacter lwoffii BIM B-1558.

Whole genome analysis, thermal and UV-tolerance of Lactococcus phage BIM BV-114 isolated from cheese brine.

Lactococcus phages that belong to the genus Ceduovirus are among the three most frequently isolated phage groups infecting Lactococcus lactis starter strains in dairy plants. In this study, we characterized virulent Lactococcus phage BIM BV-114 isolated from industrial cheese brine in Belarus and identified as Ceduovirus. The bacteriophage demonstrated a relatively short lytic cycle (latent period of 23 ± 5 min, lysis time of 90 ± 5 min), high thermal stability (inactivation after 7 min at 95 °C in skimmed milk) and tolerance to UV radiation (inactivation time - 15 min), indicating adaptation for better persistence in dairy facilities. The genome of the phage BIM BV-114 (21 499 bp; 37 putative open reading frames) has a similar organization to that of other Ceduovirus phages. RLf1_00140 and RLf_00050 gene products, found in the early genes region, may be involved in the sensitivity of phage to the lactococcal abortive infection mechanisms AbiV and AbiQ, respectively. Furthermore, nucleotide deletion, observed in the middle region of the gene encoding putative tape measure protein (RLf1_00300), is possibly responsible for increased thermal tolerance of phage BIM BV-114. Together, these findings will contribute to a better knowledge of virulent Lactococcus phages and the development of effective methods of their control for dairy technologies.

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.

Explorative characterization and taxonomy-aligned comparison of alterations in lipids and other biomolecules in Antarctic bacteria grown at different temperatures.

Temperature significantly impacts bacterial physiology, metabolism and cell chemistry. In this study, we analysed lipids and the total cellular biochemical profile of 74 fast-growing Antarctic bacteria grown at different temperatures. Fatty acid diversity and temperature-induced alterations aligned with bacterial classification-Gram-groups, phylum, genus and species. Total lipid content, varied from 4% to 19% of cell dry weight, was genus- and species-specific. Most bacteria increased lipid content at lower temperatures. The effect of temperature on the profile was complex and more species-specific, while some common for all bacteria responses were recorded. Gram-negative bacteria adjusted unsaturation and acyl chain length. Gram-positive bacteria adjusted methyl branching (anteiso-/iso-), chain length and unsaturation. Fourier transform infrared spectroscopy analysis revealed Gram-, genus- and species-specific changes in the total cellular biochemical profile triggered by temperature fluctuations. The most significant temperature-related alterations detected on all taxonomy levels were recorded for mixed region 1500-900 cm-1 , specifically the band at 1083 cm-1 related to phosphodiester groups mainly from phospholipids (for Gram-negative bacteria) and teichoic/lipoteichoic acids (for Gram-positive bacteria). Some changes in protein region were detected for a few genera, while the lipid region remained relatively stable despite the temperature fluctuations.

Roseateles amylovorans sp. nov., isolated from freshwater.

A Gram-stain-negative, rod-shaped, amylolytic bacterial strain, designated as bsSlp3-1T, was isolated from the Slepian water system, a freshwater reservoir. Strain bsSlp3-1T was found to be aerobic, oxidase-positive and catalase-negative, grew at 5-37 °C (optimum, 28 °C), pH 5.0-9.5 (optimum, pH 7.0) and low NaCl concentration (up to 1.0 %). Comparative analysis of 16S rRNA gene sequence similarity revealed that strain bsSlp3-1T clustered with Roseateles species and is closely related to Roseateles depolymerans KCTC 42856T (98.7 %) and Roseateles terrae CCUG 52222T (98.6 %). Whole-genome comparisons using average nucleotide identity and digital DNA-DNA hybridization values suggested that strain bsSlp3-1T represents a novel species within the genus Roseateles and is most closely related to Roseateles aquatilis CCUG 48205T (81.2 and 25.6 %, respectively). The genome of strain bsSlp3-1T consisted of a single circular chromosome with size 6 289 366 bp and DNA G+C content of 66.8 mol%. The predominant cellular fatty acids of bsSlp3-1T were cis-9-hexadecanoic and hexadecenoic acids. According to the data obtained in this work, strain bsSlp3-1T represents a novel Roseateles species for which the name Roseateles amylovorans sp. nov. is proposed. The type strain is bsSlp3-1T (=BIM B-1768T=NBIMCC 9098T=VKM B-3671T).

Isolation, Physiological Characterization, and Antibiotic Susceptibility Testing of Fast-Growing Bacteria from the Sea-Affected Temporary Meltwater Ponds in the Thala Hills Oasis (Enderby Land, East Antarctica).

In this study, for the first time, we report the identification and characterization of culturable fast-growing bacteria isolated from the sea-affected temporary meltwater ponds (MPs) in the East Antarctica area of the Vecherny region (-67.656317, 46.175058) of the Thala Hills Oasis, Enderby Land. Water samples from the studied MPs showed alkaline pH (from 8.0 to 10.1) and highly varied total dissolved solids (86-94,000 mg/L). In total, twenty-nine bacterial isolates were retrieved from the studied MPs. The phylogenetic analysis based on 16S rRNA gene sequence similarities showed that the isolated bacteria belong to the phyla Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes and the twelve genera Pseudomonas, Shewanella, Acinetobacter, Sporosarcina, Facklamia, Carnobacterium, Arthrobacter, Brachybacterium, Micrococcus, Agrococcus, Leifsonia, and Flavobacterium. Most of the isolated bacteria were psychrotrophs and showed the production of one or more extracellular enzymes. Lipolytic and proteolytic activities were more prevalent among the isolates. Five isolates from the Actinobacteria phylum and one isolate from the Bacteroidetes phylum had strong pigmentation. Antibiotic susceptibility testing revealed that most of the isolates are resistant to at least one antibiotic, and seven isolates showed multi-resistance.

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.

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.

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.

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 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.

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.

Isolation and characterization of fast-growing green snow bacteria from coastal East Antarctica.

Snow microorganisms play a significant role in climate change and affecting the snow melting rate in the Arctic and Antarctic regions. While research on algae inhabiting green and red snow has been performed extensively, bacteria dwelling in this biotope have been studied to a much lesser extent. In this study, we performed 16S rRNA gene amplicon sequencing of two green snow samples collected from the coastal area of the eastern part of Antarctica and conducted genotypic and phenotypic profiling of 45 fast-growing bacteria isolated from these samples. 16S rRNA gene amplicon sequencing of two green snow samples showed that bacteria inhabiting these samples are mostly represented by families Burkholderiaceae (46.31%), Flavobacteriaceae (22.98%), and Pseudomonadaceae (17.66%). Identification of 45 fast-growing bacteria isolated from green snow was performed using 16S rRNA gene sequencing. We demonstrated that they belong to the phyla Actinobacteria and Proteobacteria, and are represented by the genera Arthrobacter, Cryobacterium, Leifsonia, Salinibacterium, Paeniglutamicibacter, Rhodococcus, Polaromonas, Pseudomonas, and Psychrobacter. Nearly all bacterial isolates exhibited various growth temperatures from 4°C to 25°C, and some isolates were characterized by a high level of enzymatic activity. Phenotyping using Fourier transform infrared (FTIR) spectroscopy revealed a possible accumulation of intracellular polymer polyhydroxyalkanoates (PHA) or lipids in some isolates. The bacteria showed different lipids/PHA and protein profiles. It was shown that lipid/PHA and protein spectral regions are the most discriminative for differentiating the isolates.

Complete Genome Sequence of Pseudomonas putida BS3701, a Promising Polycyclic Aromatic Hydrocarbon-Degrading Strain for Bioremediation Technologies.

The strain Pseudomonas putida BS3701 was isolated from soil contaminated with coke by-product waste (Moscow Region, Russian Federation). It is capable of degrading crude oil and polycyclic aromatic hydrocarbons (PAHs). The P. putida BS3701 genome consists of a 6,337,358-bp circular chromosome and two circular plasmids (pBS1141 with 107,388 bp and pBS1142 with 54,501 bp).

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.

Complete Genome Sequence of Gordonia sp. 135, a Promising Dibenzothiophene- and Hydrocarbon-Degrading Strain.

Gordonia sp. strain 135 is a promising dibenzothiophene-desulfurizing and hydrocarbon-degrading bacterium. It can utilize dibenzothiophene as the sole sulfur source. The genome of strain 135 was completely sequenced; it consists of a 5,039,827-bp circular chromosome and a 164,963-bp circular plasmid.

Complete Genome Sequence of Rhodococcus erythropolis X5, a Psychrotrophic Hydrocarbon-Degrading Biosurfactant-Producing Bacterium.

Rhodococcus erythropolis X5 is a psychrotrophic (cold-adapted) hydrocarbon-degrading bacterium, as it showed effective n-alkane destruction at low positive temperatures. Here, the genome of strain X5 was completely sequenced; it consists of a 6,472,161-bp circular chromosome (62.25% GC content) and a 526,979-bp linear plasmid, pRhX5-526k (62.37% GC content).

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.