Lytic Capsule-Specific Acinetobacter Bacteriophages Encoding Polysaccharide-Degrading Enzymes.

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

Genetic Alternatives for Experimental Adaptation to Colistin in Three Pseudomonas aeruginosa Lineages.

Pseudomonas aeruginosa is characterized by a high adaptive potential, developing resistance in response to antimicrobial pressure. We employed a spatiotemporal evolution model to disclose the pathways of adaptation to colistin, a last-resort polymyxin antimicrobial, among three unrelated P. aeruginosa lineages. The P. aeruginosa ATCC-27833 reference strain (Pa_ATCC), an environmental P. aeruginosa isolate (Pa_Environment), and a clinical isolate with multiple drug resistance (Pa_MDR) were grown over an increasing 5-step colistin concentration gradient from 0 to 400 mg/L. Pa_Environment demonstrated the highest growth pace, achieving the 400 mg/L band in 15 days, whereas it took 37 and 60 days for Pa_MDR and Pa_ATCC, respectively. To identify the genome changes that occurred during adaptation to colistin, the isolates selected during the growth of the bacteria (n = 185) were subjected to whole genome sequencing. In total, 17 mutation variants in eight lipopolysaccharide-synthesis-associated genes were detected. phoQ and lpxL/PA0011 were affected in all three lineages, whereas changes in pmrB were found in Pa_Environment and Pa_MDR but not in Pa_ATCC. In addition, mutations were detected in 34 general metabolism genes, and each lineage developed mutations in a unique set of such genes. Thus, the three examined distinct P. aeruginosa strains demonstrated different capabilities and genetic pathways of colistin adaptation.

Xanthomonas Phage PBR31: Classifying the Unclassifiable.

The ability of bacteriophages to destroy bacteria has made them the subject of extensive research. Interest in bacteriophages has recently increased due to the spread of drug-resistant bacteria, although genomic research has not kept pace with the growth of genomic data. Genomic analysis and, especially, the taxonomic description of bacteriophages are often difficult due to the peculiarities of the evolution of bacteriophages, which often includes the horizontal transfer of genes and genomic modules. The latter is particularly pronounced for temperate bacteriophages, which are capable of integration into the bacterial chromosome. Xanthomonas phage PBR31 is a temperate bacteriophage, which has been neither described nor classified previously, that infects the plant pathogen Xanthomonas campestris pv. campestris. Genomic analysis, including phylogenetic studies, indicated the separation of phage PBR31 from known classified bacteriophages, as well as its distant relationship with other temperate bacteriophages, including the Lederbervirus group. Bioinformatic analysis of proteins revealed distinctive features of PBR31, including the presence of a protein similar to the small subunit of D-family DNA polymerase and advanced lysis machinery. Taxonomic analysis showed the possibility of assigning phage PBR31 to a new taxon, although the complete taxonomic description of Xanthomonas phage PBR31 and other related bacteriophages is complicated by the complex evolutionary history of the formation of its genome. The general biological features of the PBR31 phage were analysed for the first time. Due to its presumably temperate lifestyle, there is doubt as to whether the PBR31 phage is appropriate for phage control purposes. Bioinformatics analysis, however, revealed the presence of cell wall-degrading enzymes that can be utilised for the treatment of bacterial infections.

Phytopathogenic Curtobacterium flaccumfaciens Strains Circulating on Leguminous Plants, Alternative Hosts and Weeds in Russia.

Many bacterial plant pathogens have a broad host range important for their life cycle. Alternate hosts from plant families other than the main (primary) host support the survival and dissemination of the pathogen population even in absence of main host plants. Metabolic peculiarities of main and alternative host plants can affect genetic diversity within and between the pathogen populations isolated from those plants. Strains of Gram-positive bacterium Curtobacterium flaccumfaciens were identified as being causal agents of bacterial spot and wilt diseases on leguminous plants, and other crop and weed plants, collected in different regions of Russia. Their biochemical properties and susceptibility to copper compounds have been found to be relatively uniform. According to conventional PCR assays, all of the isolates studied were categorised as pathovar Curtobacterim flaccumfaciens pv. flaccumfaciens, a pathogen of legumes. However, the strains demonstrated a substantial diversity in terms of virulence on several tested host plants and different phylogenetic relationships were revealed by BOX-PCR and alanine synthase gene (alaS) sequencing.

Characterisation of New Foxunavirus Phage Murka with the Potential of Xanthomonas campestris pv. campestris Control.

Phages of phytopathogenic bacteria are considered to be promising agents for the biological control of bacterial diseases in plants. This paper reports on the isolation and characterisation of a new Xanthomonas campestris pv. campestris phage, Murka. Phage morphology and basic kinetic characteristics of the infection were determined, and a phylogenomic analysis was performed. The phage was able to lyse a reasonably broad range (64%, 9 of the 14 of the Xanthomonas campestris pv. campestris strains used in the study) of circulating strains of the cabbage black rot pathogen. This lytic myovirus has a DNA genome of 44,044 bp and contains 83 predicted genes. Taxonomically, it belongs to the genus Foxunavirus. This bacteriophage is promising for use as a possible means of biological control of cabbage black rot.

New Obolenskvirus Phages Brutus and Scipio: Biology, Evolution, and Phage-Host Interaction.

Two novel virulent phages of the genus Obolenskvirus infecting Acinetobacter baumannii, a significant nosocomial pathogen, have been isolated and studied. Phages Brutus and Scipio were able to infect A. baumannii strains belonging to the K116 and K82 capsular types, respectively. The biological properties and genomic organization of the phages were characterized. Comparative genomic, phylogenetic, and pangenomic analyses were performed to investigate the relationship of Brutus and Scipio to other bacterial viruses and to trace the possible origin and evolutionary history of these phages and other representatives of the genus Obolenskvirus. The investigation of enzymatic activity of the tailspike depolymerase encoded in the genome of phage Scipio, the first reported virus infecting A. baumannii of the K82 capsular type, was performed. The study of new representatives of the genus Obolenskvirus and mechanisms of action of depolymerases encoded in their genomes expands knowledge about the diversity of viruses within this taxonomic group and strategies of Obolenskvirus-host bacteria interaction.

Depolymerisation of the Klebsiella pneumoniae Capsular Polysaccharide K21 by Klebsiella Phage K5.

Klebsiella pneumoniae is a pathogen associated with various infection types, which often exhibits multiple antibiotic resistance. Phages, or bacterial viruses, have an ability to specifically target and destroy K. pneumoniae, offering a potential means of combatting multidrug-resistant infections. Phage enzymes are another promising therapeutic agent that can break down bacterial capsular polysaccharide, which shields K. pneumoniae from the immune response and external factors. In this study, Klebsiella phage K5 was isolated; this phage is active against Klebsiella pneumoniae with the capsular type K21. It was demonstrated that the phage can effectively lyse the host culture. The adsorption apparatus of the phage has revealed two receptor-binding proteins (RBPs) with predicted polysaccharide depolymerising activity. A recombinant form of both RBPs was obtained and experiments showed that one of them depolymerised the capsular polysaccharide K21. The structure of this polysaccharide and its degradation fragments were analysed. The second receptor-binding protein showed no activity on capsular polysaccharide of any of the 31 capsule types tested, so the substrate for this enzyme remains to be determined in the future. Klebsiella phage K5 may be considered a useful agent against Klebsiella infections.

Analysis of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Revealed Novel Groups of Filamentous Bacteriophages.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages.

Limnofasciculus baicalensis gen. et sp. nov. (Coleofasciculaceae, Coleofasciculales): A New Genus of Cyanobacteria Isolated from Sponge Fouling in Lake Baikal, Russia.

The proliferation of benthic cyanobacteria has been observed in Lake Baikal since 2011 and is a vivid manifestation of the ecological crisis occurring in the littoral zone. The cyanobacterium Symplocastrum sp. has formed massive fouling on all types of benthic substrates, including endemic Baikal sponges. The strain BBK-W-15 (=IPPAS B-2062T), which was isolated from sponge fouling in 2015, was used for further taxonomic determination. A polyphasic approach revealed that it is a cryptic taxon of cyanobacteria. Morphological evaluation of the strain indicated the presence of cylindrical filaments with isodiametric cells enclosed in individual sheaths and coleodesmoid false branching. Strain ultrastructure (fascicular thylakoids and type C cell division) is characteristic of the Microcoleaceae and Coleofasciculaceae families. An integrated analysis that included 16S rRNA gene phylogeny, conserved protein phylogeny and whole-genome comparisons indicated the unique position of BBK-W-15, thus supporting the proposed delineation of the new genus Limnofasciculus. Through characterisation by morphology, 16S, ITS and genomic analysis, a new cyanobacterium of the family Coleofasciculaceae Limnofasciculus baicalensis gen. et sp. nov. was described.

The structure of Klebsiella pneumoniae K108 capsular polysaccharide is similar to Escherichia coli colanic acid.

The clinical isolate of Klebsiella pneumoniae 1333/P225 was revealed as containing a KL108 K. pneumoniae K locus for capsule biosynthesis. The gene cluster demonstrated a high level of sequence and arrangement similarity with that of the E. coli colanic acid biosynthesis gene cluster. The KL108 gene cluster includes a gene of WcaD polymerase responsible for joining oligosaccharide K units into capsular polysaccharide (CPS), acetyltransferase, pyruvyltransferasefive and genes for glycosyltransferases (Gtrs), four of which have homologues in genetic units of the colanic acid synthesis. The fifth Gtr is specific to this cluster. The work involved the use of sugar analysis, Smith degradation and one- and two-dimensional 1H and 13C NMR spectroscopy to establish the structure of the K108 CPS. The CPS repetitive K unit is composed of branched pentasaccharide with three monosaccharides in the backbone and a disaccharide side chain. The main chain is the same as for colanic acid but the side chain differs. Two bacteriophages infecting K. pneumoniae strain 1333/P225 were isolated and structural depolymerase genes were determined; depolymerases Dep108.1 and Dep108.2 were cloned, expressed and purified. It was demonstrated that both depolymerases specifically cleave the ß-Glcp-(1→4)-α-Fucp linkage between K108 units in the CPS.

Friunavirus Phage-Encoded Depolymerases Specific to Different Capsular Types of Acinetobacter baumannii.

Acinetobacter baumannii is a critical priority nosocomial pathogen that produces a variety of capsular polysaccharides (CPSs), the primary receptors for specific depolymerase-carrying phages. In this study, the tailspike depolymerases (TSDs) encoded in genomes of six novel Friunaviruses, APK09, APK14, APK16, APK86, APK127v, APK128, and one previously described Friunavirus phage, APK37.1, were characterized. For all TSDs, the mechanism of specific cleavage of corresponding A. baumannii capsular polysaccharides (CPSs) was established. The structures of oligosaccharide fragments derived from K9, K14, K16, K37/K3-v1, K86, K127, and K128 CPSs degradation by the recombinant depolymerases have been determined. The crystal structures of three of the studied TSDs were obtained. A significant reduction in mortality of Galleria mellonella larvae infected with A. baumannii of K9 capsular type was shown in the example of recombinant TSD APK09_gp48. The data obtained will provide a better understanding of the interaction of phage-bacterial host systems and will contribute to the formation of principles of rational usage of lytic phages and phage-derived enzymes as antibacterial agents.

Using AlphaFold Predictions in Viral Research.

Elucidation of the tertiary structure of proteins is an important task for biological and medical studies. AlphaFold, a modern deep-learning algorithm, enables the prediction of protein structure to a high level of accuracy. It has been applied in numerous studies in various areas of biology and medicine. Viruses are biological entities infecting eukaryotic and procaryotic organisms. They can pose a danger for humans and economically significant animals and plants, but they can also be useful for biological control, suppressing populations of pests and pathogens. AlphaFold can be used for studies of molecular mechanisms of viral infection to facilitate several activities, including drug design. Computational prediction and analysis of the structure of bacteriophage receptor-binding proteins can contribute to more efficient phage therapy. In addition, AlphaFold predictions can be used for the discovery of enzymes of bacteriophage origin that are able to degrade the cell wall of bacterial pathogens. The use of AlphaFold can assist fundamental viral research, including evolutionary studies. The ongoing development and improvement of AlphaFold can ensure that its contribution to the study of viral proteins will be significant in the future.

Tychonema sp. BBK16 Characterisation: Lifestyle, Phylogeny and Related Phages.

Cyanobacterial expansion is harmful to the environment, the ecology of Lake Baikal and the economy of nearby regions and can be dangerous to people and animals. Since 2011, the process of colonisation of the lake with potentially toxic cyanobacteria belonging to the genus Tychonema has continued. An understanding of the mechanism of successful expansion of Tychonema requires scrutiny of biological and genomic features. Tychonema sp. BBK16 was isolated from the coastal zone of Lake Baikal. The morphology of BBK16 biofilm was studied with light, scanning electron and confocal microscopy. The biofilm is based on filaments of cyanobacteria, which are intertwined like felt; there are also dense fascicles of rope-like twisted filaments that impart heterogeneity to the surface of the biofilm. Genome sequencing, intergenomic comparisons and phylogenetic analyses indicated that Tychonema sp. BBK16 represent a new species related to planktic cyanobacterium Tychonema bourrellyi, isolated from Alpine lentic freshwater. Genome investigation revealed the genes possibly responsible for the mixotrophic lifestyle. The presence of CRISPR-Cas and restriction modification defence mechanisms allowed to suggest the existence of phages infecting Tychonema sp. BBK16. Analysis of CRISPR spacers and prophage-derived regions allowed to suggest related cyanophages. Genomic analysis supported the assumption that mobile elements and horizontal transfer participate in shaping the Tychonema sp. BBK16 genome. The findings of the current research suggest that the aptitude of Tychonema sp. BBK16 for biofilm formation and, possibly, its mixotrophic lifestyle provide adaptation advantages that lead to the successful expansion of this cyanobacterium in the Baikal's conditions of freshwater lake environments.

Use of an Integrated Approach Involving AlphaFold Predictions for the Evolutionary Taxonomy of Duplodnaviria Viruses.

The evaluation of the evolutionary relationships is exceptionally important for the taxonomy of viruses, which is a rapidly expanding area of research. The classification of viral groups belonging to the realm Duplodnaviria, which include tailed bacteriophages, head-tailed archaeal viruses and herpesviruses, has undergone many changes in recent years and continues to improve. One of the challenging tasks of Duplodnaviria taxonomy is the classification of high-ranked taxa, including families and orders. At the moment, only 17 of 50 families have been assigned to orders. The evaluation of the evolutionary relationships between viruses is complicated by the high level of divergence of viral proteins. However, the development of structure prediction algorithms, including the award-winning AlphaFold, encourages the use of the results of structural predictions to clarify the evolutionary history of viral proteins. In this study, the evolutionary relationships of two conserved viral proteins, the major capsid protein and terminase, representing different viruses, including all classified Duplodnaviria families, have been analysed using AlphaFold modelling. This analysis has been undertaken using structural comparisons and different phylogenetic methods. The results of the analyses mainly indicated the high quality of AlphaFold modelling and the possibility of using the AlphaFold predictions, together with other methods, for the reconstruction of the evolutionary relationships between distant viral groups. Based on the results of this integrated approach, assumptions have been made about refining the taxonomic classification of bacterial and archaeal Duplodnaviria groups, and problems relating to the taxonomic classification of Duplodnaviria have been discussed.

Prophage-Derived Regions in Curtobacterium Genomes: Good Things, Small Packages.

Curtobacterium is a genus of Gram-positive bacteria within the order Actinomycetales. Some Curtobacterium species (C. flaccumfaciens, C. plantarum) are harmful pathogens of agricultural crops such as soybean, dry beans, peas, sugar beet and beetroot, which occur throughout the world. Bacteriophages (bacterial viruses) are considered to be potential curative agents to control the spread of harmful bacteria. Temperate bacteriophages integrate their genomes into bacterial chromosomes (prophages), sometimes substantially influencing bacterial lifestyle and pathogenicity. About 200 publicly available genomes of Curtobacterium species, including environmental metagenomic sequences, were inspected for the presence of sequences of possible prophage origin using bioinformatic methods. The comparison of the search results with several ubiquitous bacterial groups showed the relatively low level of the presence of prophage traces in Curtobacterium genomes. Genomic and phylogenetic analyses were undertaken for the evaluation of the evolutionary and taxonomic positioning of predicted prophages. The analyses indicated the relatedness of Curtobacterium prophage-derived sequences with temperate actinophages of siphoviral morphology. In most cases, the predicted prophages can represent novel phage taxa not described previously. One of the predicted temperate phages was induced from the Curtobacterium genome. Bioinformatic analysis of the modelled proteins encoded in prophage-derived regions led to the discovery of some 100 putative glycopolymer-degrading enzymes that contained enzymatic domains with predicted cell-wall- and cell-envelope-degrading activity; these included glycosidases and peptidases. These proteins can be considered for the experimental design of new antibacterials against Curtobacterium phytopathogens.

First report of Curtobacterium flaccumfaciens pv. flaccumfaciens causing bacterial tan spot of soybean in Russia.

Curtobacterium flaccumfaciens pv. flaccumfaciens (H.) Collins & Jones is known as a pathogen of different legume crops, including soybean (Glycine max (L.) Merr.) (Hedges 1922; Dunleavy 1983). OEPP/EPPO (2011) considers C. flaccumfaciens pv. flaccumfaciens as present in Russia based on reports of the disease on common beans in two regions of Russia (North Caucasus and Far East) made without proper pathogen identification. During the summer of 2020 and the spring of 2021, soybean plants with tan spot disease (10-40% of plants) were reported during routine assays of several fields in Stavropol Krai (44.72°N, 43.29°E). After harvest in 2021, we inspected 48 soybean seed lots collected in different regions of Russia for the presence of C. flaccumfaciens pv. flaccumfaciens. Seed testing was performed using the OEPP/EPPO (2011) protocol. For bacteria isolation, seed extracts were spread on MSCFF agar plates (Maringoni et al. 2006). After 5 days of incubation at 28°C potential, C. flaccumfaciens pv. flaccumfaciens colonies were used for further tests on NSA and SSM agar (Tegli et al. 2017, Maringoni et al. 2016). Six seed lots produced in Stavropol, Ryazan (53.95°N, 40.62°E), Orel (52.39°N, 37.69°E) and Amur (51.31°N, 128.22°E) regions were suspect. Ten isolates (SB1 to SB4 from Stavropol, F-125-1 to F-125-3 from Ryazan, and F-30-1 to F-30-3 from Amur) were selected, and further identified by morphological, physiological, and biochemical properties, MALDI TOF MS, 16S rRNA sequences, and specific primers CffFOR2 and CffREV4 (Tegli et al. 2017). Isolates consistently formed yellow, circular, smooth colonies on agar, and were identical to C. flaccumfaciens pv. flaccumfaciens type strain DSM 20129T in diagnostic physiological properties (Tegli et al. 2017). DNA was isolated from the bacteria by the CytoSorb Kit (Sintol, Moscow). All tested strains were positive in the PCR assay (Fig. 1). 16S rRNA fragments were amplified using primers 27F/1492R (Marchesi et al. 1998) and PCR products were sequenced (Evrogen, Moscow, Russia). The obtained 16S rRNA sequences (1473 bp, Accession No. OL539808.1-OL539817.1) were 100% identical to DSM 20129T (AM410688.1) according to a BLAST NCBI search. A pathogenicity test was done by leaf-cutting with scissors wetted with inoculum (for soybeans) or by injecting 5 microliters of the bacterial suspension (108 CFU/ml) into the stem (for common beans). All ten isolates for the inoculum were grown on nutrient agar for 72 h at 28°C. Soybean cv. Kasatka plants (stage V1) were used for inoculation, and common bean (cv. Purpurnaya) plants were inoculated as well to confirm multi-host virulence. Sterile water served as a control. Ten plantlets were used as replicates for each treatment. The plants were incubated at 24°C, 80% RH, and a 14 hour light/10 hour dark cycle. Tan spots (soybean) and wilt (beans) have developed 7-21 d.p.i (Fig. 2.1-2.6). Control plants remained asymptomatic. Seed inoculation by soaking them in the same bacterial suspension repeatedly produced twisted primary root (Fig. 2.7-2.8), but typical disease symptoms on leaves developed in 4-5 weeks only. The pathogen was successfully reisolated from all infected plants and not from the controls, thus fulfilling Koch's postulates. The identity of the reisolated strains was confirmed using morphological and physiological characteristics and the DNA sequence data for the 16S rRNA. These results indicated that a causal agent of the tan spot is present on soybean in three important agricultural areas of Russia (South, Central, and the Far East). To the best of our knowledge, this is the first report of C. flaccumfaciens pv. flaccumfaciens causing a bacterial tan spot of soybean in Russia.

Pectobacterium versatile Bacteriophage Possum: A Complex Polysaccharide-Deacetylating Tail Fiber as a Tool for Host Recognition in Pectobacterial Schitoviridae.

Novel, closely related phages Possum and Horatius infect Pectobacterium versatile, a phytopathogen causing soft rot in potatoes and other essential plants. Their properties and genomic composition define them as N4-like bacteriophages of the genus Cbunavirus, a part of a recently formed family Schitoviridae. It is proposed that the adsorption apparatus of these phages consists of tail fibers connected to the virion through an adapter protein. Tail fibers possess an enzymatic domain. Phage Possum uses it to deacetylate O-polysaccharide on the surface of the host strain to provide viral attachment. Such an infection mechanism is supposed to be common for all Cbunavirus phages and this feature should be considered when designing cocktails for phage control of soft rot.

First Report of Curtobacterium flaccumfaciens pv. flaccumfaciens Causing Bacterial Wilt and Blight on Sunflower in Russia.

In the summer of 2018, wilt and leaf spots were observed on sunflower (Helianthus annuus L.) plants in fields near Kursk (51.74°N, 36.02°E) in Russia. In the following years, incidence of this disease was 5 to 20% in the inspected fields. Marginal chlorosis on seedling leaves developed into wilt and necrosis about one week later (Fig. 1). Mature plants had leaves with blight and reduced height compared to symptomless plants. Pathogen isolation from seeds was done by the method of Tegli et al. (2002) with modifications. Bacteria from diseased plants were isolated by streaking inoculum from symptomatic tissues on nutrient dextrose agar (NDA) (Schaad et al. 1988). The plates were incubated at 30°C for 7 to 10 days. Isolates consistently formed slow-growing, yellow, circular, smooth colonies without soluble pigment. The isolated bacteria were aerobic, gram-positive, and rod-shaped. Eight strains, CF-20 to CF-26 from plants, and Curt1 and Curt3 from seeds, were identified by MALDI TOF MS analysis as Curtobacterium flaccumfaciens pv. flaccumfaciens or C. flaccumfaciens pv. poinsettiae. All strains had GENIII MicroPlate (BIOLOG) test results identical to C. flaccumfaciens pv. flaccumfaciens strain DSM20129T. Further analysis was done by specific PCR (Tegli et al. 2002) and 16S rDNA, gyrB, and atpD gene sequencing. For PCR amplification, DNA was extracted by the CitoSorb Kit (Syntol Co., Moscow). Primers 27F/1492R (16S rRNA) (Marchesi et al. 1998), 2F/6R (gyrB) (Richert et al. 2005), and aptD2F/aptD2R (Jacques et al. 2012) were used to amplify the target gene sequences. The PCR products were sequenced by Evrogen (Moscow). The 16S rRNA sequences (OL584192.1 to OL584199.1) were identical to that of C. flaccumfaciens pv. flaccumfaciens strain DSM20129T (AM410688.1; 1,477/1,477 bp). The phylogenetic tree of concatenated gyrB (560 bp) and atpD (716 bp) sequences (OL548915.1 to OL548922.1 and OL548923.1 to OL548930.1, respectively) clustered the strains from sunflower among C. flaccumfaciens pv. flaccumfaciens, C. flaccumfaciens pv. betae, and C. flaccumfaciens pv. oortii (Fig. 2) with high genetic similarity to other C. flaccumfaciens strains: 96.3 to 100% for atpD and 95 to 100% for gyrB. A pathogenicity test for each of the strains was performed by injecting 5 µl of a bacterial suspension (108 CFU/ml) grown for 72 h on NDA into the stems of five plantlets (four true leaf stage) of the sunflower cv. Tunka (Limagrain, France) and soybean cv. Kasatka (VIM, Russia). Strain DSM20129T was a positive control, while sterile water was a negative control. The plants were incubated at 24°C, 80% relative humidity, and 14-h light/day. Wilting and blight on sunflower (Fig. 3) and tan spots on soybean were observed in 15 to 20 days after inoculation for all sunflower strains and strain DSM20129T. The negative control plants were asymptomatic. The bacteria re-isolated from the inoculated plants exhibited the same morphological characteristics and 16S rDNA sequence as the original culture, thus fulfilling Koch's postulates. The presence of C. flaccumfaciens pv. flaccumfaciens in sunflower seeds indicated that the bacterium was transmitted via seed. Sunflower has been previously reported as a host for the pathogen (Harveson et al. 2015). The presence of C. flaccumfaciens pv. flaccumfaciens on beans in Russia was suggested from the disease symptoms (Nikitina and Korsakov 1978), but, to our knowledge, this is the first report of the pathogen affecting sunflower in Russia. Phytosanitary categorization placed C. flaccumfaciens pv. flaccumfaciens in the EPPO A2 list (EPPO 2011). Thus, sunflower seeds should be tested to protect pathogen-free areas from introduction of this pathogen.

Ayka, a Novel Curtobacterium Bacteriophage, Provides Protection against Soybean Bacterial Wilt and Tan Spot.

Diseases caused by the Gram-positive bacterium Curtobacteriumflaccumfaciens pv. flaccumfaciens (Cff) inflict substantial economic losses in soybean cultivation. Use of specific bacterial viruses (bacteriophages) for treatment of seeds and plants to prevent the development of bacterial infections is a promising approach for bioprotection in agriculture. Phage control has been successfully tested for a number of staple crops. However, this approach has never been applied to treat bacterial diseases of legumes caused by Cff, and no specific bacteriophages have been known to date. This paper presents detailed characteristics of the first lytic bacteriophage infecting this pathogen. Phage Ayka, related to φ29-like (Salasmaviridae) viruses, but representing a new subfamily, was shown to control the development of bacterial wilt and tan spot in vitro and in greenhouse plants.

Evolution of Phage Tail Sheath Protein.

Sheath proteins comprise a part of the contractile molecular machinery present in bacteriophages with myoviral morphology, contractile injection systems, and the type VI secretion system (T6SS) found in many Gram-negative bacteria. Previous research on sheath proteins has demonstrated that they share common structural features, even though they vary in their size and primary sequence. In this study, 112 contractile phage tail sheath proteins (TShP) representing different groups of bacteriophages and archaeal viruses with myoviral morphology have been modelled with the novel machine learning software, AlphaFold 2. The obtained structures have been analysed and conserved and variable protein parts and domains have been identified. The common core domain of all studied sheath proteins, including viral and T6SS proteins, comprised both N-terminal and C-terminal parts, whereas the other parts consisted of one or several moderately conserved domains, presumably added during phage evolution. The conserved core appears to be responsible for interaction with the tail tube protein and assembly of the phage tail. Additional domains may have evolved to maintain the stability of the virion or for adsorption to the host cell. Evolutionary relations between TShPs representing distinct viral groups have been proposed using a phylogenetic analysis based on overall structural similarity and other analyses.