Current Methods for Recombination Detection in Bacteria.

The role of genetic exchanges, i.e., homologous recombination (HR) and horizontal gene transfer (HGT), in bacteria cannot be overestimated for it is a pivotal mechanism leading to their evolution and adaptation, thus, tracking the signs of recombination and HGT events is importance both for fundamental and applied science. To date, dozens of bioinformatics tools for revealing recombination signals are available, however, their pros and cons as well as the spectra of solvable tasks have not yet been systematically reviewed. Moreover, there are two major groups of software. One aims to infer evidence of HR, while the other only deals with horizontal gene transfer (HGT). However, despite seemingly different goals, all the methods use similar algorithmic approaches, and the processes are interconnected in terms of genomic evolution influencing each other. In this review, we propose a classification of novel instruments for both HR and HGT detection based on the genomic consequences of recombination. In this context, we summarize available methodologies paying particular attention to the type of traceable events for which a certain program has been designed.

Temporal Control of Seed Development in Dicots: Molecular Bases, Ecological Impact and Possible Evolutionary Ramifications.

In flowering plants, seeds serve as organs of both propagation and dispersal. The developing seed passes through several consecutive stages, following a conserved general outline. The overall time needed for a seed to develop, however, may vary both within and between plant species, and these temporal developmental properties remain poorly understood. In the present paper, we summarize the existing data for seed development alterations in dicot plants. For genetic mutations, the reported cases were grouped in respect of the key processes distorted in the mutant specimens. Similar phenotypes arising from the environmental influence, either biotic or abiotic, were also considered. Based on these data, we suggest several general trends of timing alterations and how respective mechanisms might add to the ecological plasticity of the families considered. We also propose that the developmental timing alterations may be perceived as an evolutionary substrate for heterochronic events. Given the current lack of plausible models describing timing control in plant seeds, the presented suggestions might provide certain insights for future studies in this field.

Dissecting the Environmental Consequences of Bacillus thuringiensis Application for Natural Ecosystems.

Bacillus thuringiensis (Bt), a natural pathogen of different invertebrates, primarily insects, is widely used as a biological control agent. While Bt-based preparations are claimed to be safe for non-target organisms due to the immense host specificity of the bacterium, the growing evidence witnesses the distant consequences of their application for natural communities. For instance, upon introduction to soil habitats, Bt strains can affect indigenous microorganisms, such as bacteria and fungi, and further establish complex relationships with local plants, ranging from a mostly beneficial demeanor, to pathogenesis-like plant colonization. By exerting a direct effect on target insects, Bt can indirectly affect other organisms in the food chain. Furthermore, they can also exert an off-target activity on various soil and terrestrial invertebrates, and the frequent acquisition of virulence factors unrelated to major insecticidal toxins can extend the Bt host range to vertebrates, including humans. Even in the absence of direct detrimental effects, the exposure to Bt treatment may affect non-target organisms by reducing prey base and its nutritional value, resulting in delayed alleviation of their viability. The immense phenotypic plasticity of Bt strains, coupled with the complexity of ecological relationships they can engage in, indicates that further assessment of future Bt-based pesticides' safety should consider multiple levels of ecosystem organization and extend to a wide variety of their inhabitants.

The Distribution of Several Genomic Virulence Determinants Does Not Corroborate the Established Serotyping Classification of Bacillus thuringiensis.

Bacillus thuringiensis, commonly referred to as Bt, is an object of the lasting interest of microbiologists due to its highly effective insecticidal properties, which make Bt a prominent source of biologicals. To categorize the exuberance of Bt strains discovered, serotyping assays are utilized in which flagellin serves as a primary seroreactive molecule. Despite its convenience, this approach is not indicative of Bt strains' phenotypes, neither it reflects actual phylogenetic relationships within the species. In this respect, comparative genomic and proteomic techniques appear more informative, but their use in Bt strain classification remains limited. In the present work, we used a bottom-up proteomic approach based on fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) coupled with liquid chromatography/tandem mass spectrometry(LC-MS/MS) protein identification to assess which stage of Bt culture, vegetative or spore, would be more informative for strain characterization. To this end, the proteomic differences for the israelensis-attributed strains were assessed to compare sporulating cultures of the virulent derivative to the avirulent one as well as to the vegetative stage virulent bacteria. Using the same approach, virulent spores of the israelensis strain were also compared to the spores of strains belonging to two other major Bt serovars, namely darmstadiensis and thuringiensis. The identified proteins were analyzed regarding the presence of the respective genes in the 104 Bt genome assemblies available at open access with serovar attributions specified. Of 21 proteins identified, 15 were found to be encoded in all the present assemblies at 67% identity threshold, including several virulence factors. Notable, individual phylogenies of these core genes conferred neither the serotyping nor the flagellin-based phylogeny but corroborated the reconstruction based on phylogenomics approaches in terms of tree topology similarity. In its turn, the distribution of accessory protein genes was not confined to the existing serovars. The obtained results indicate that neither gene presence nor the core gene sequence may serve as distinctive bases for the serovar attribution, undermining the notion that the serotyping system reflects strains' phenotypic or genetic similarity. We also provide a set of loci, which fit in with the phylogenomics data plausibly and thus may serve for draft phylogeny estimation of the novel strains.

Accumulation of storage proteins in plant seeds is mediated by amyloid formation.

Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved ß-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds.

No More Tears: Mining Sequencing Data for Novel Bt Cry Toxins with CryProcessor.

Bacillus thuringiensis (Bt) is a natural pathogen of insects and some other groups of invertebrates that produces three-domain Cry (3d-Cry) toxins, which are highly host-specific pesticidal proteins. These proteins represent the most commonly used bioinsecticides in the world and are used for commercial purposes on the market of insecticides, being convergent with the paradigm of sustainable growth and ecological development. Emerging resistance to known toxins in pests stresses the need to expand the list of known toxins to broaden the horizons of insecticidal approaches. For this purpose, we have elaborated a fast and user-friendly tool called CryProcessor, which allows productive and precise mining of 3d-Cry toxins. The only existing tool for mining Cry toxins, called a BtToxin_scanner, has significant limitations such as limited query size, lack of accuracy and an outdated database. In order to find a proper solution to these problems, we have developed a robust pipeline, capable of precise 3d-Cry toxin mining. The unique feature of the pipeline is the ability to search for Cry toxins sequences directly on assembly graphs, providing an opportunity to analyze raw sequencing data and overcoming the problem of fragmented assemblies. Moreover, CryProcessor is able to predict precisely the domain layout in arbitrary sequences, allowing the retrieval of sequences of definite domains beyond the bounds of a limited number of toxins presented in CryGetter. Our algorithm has shown efficiency in all its work modes and outperformed its analogues on large amounts of data. Here, we describe its main features and provide information on its benchmarking against existing analogues. CryProcessor is a novel, fast, convenient, open source (https://github.com/lab7arriam/cry_processor), platform-independent, and precise instrument with a console version and elaborated web interface (https://lab7.arriam.ru/tools/cry_processor). Its major merits could make it possible to carry out massive screening for novel 3d-Cry toxins and obtain sequences of specific domains for further comprehensive in silico experiments in constructing artificial toxins.

Transcriptomic Insights into Mechanisms of Early Seed Maturation in the Garden Pea (Pisum sativum L.).

The garden pea (Pisum sativum L.) is a legume crop of immense economic value. Extensive breeding has led to the emergence of numerous pea varieties, of which some are distinguished by accelerated development in various stages of ontogenesis. One such trait is rapid seed maturation, which, despite novel insights into the genetic control of seed development in legumes, remains poorly studied. This article presents an attempt to dissect mechanisms of early maturation in the pea line Sprint-2 by means of whole transcriptome RNA sequencing in two developmental stages. By using a de novo assembly approach, we have obtained a reference transcriptome of 25,756 non-redundant entries expressed in pea seeds at either 10 or 20 days after pollination. Differential expression in Sprint-2 seeds has affected 13,056 transcripts. A comparison of the two pea lines with a common maturation rate demonstrates that while at 10 days after pollination, Sprint-2 seeds show development retardation linked to intensive photosynthesis, morphogenesis, and cell division, and those at 20 days show a rapid onset of desiccation marked by the cessation of translation and cell anabolism and accumulation of dehydration-protective and -storage moieties. Further inspection of certain transcript functional categories, including the chromatin constituent, transcription regulation, protein turnover, and hormonal regulation, has revealed transcriptomic trends unique to specific stages and cultivars. Among other remarkable features, Sprint-2 demonstrated an enhanced expression of transposable element-associated open reading frames and an altered expression of major maturation regulators and DNA methyltransferase genes. To the best of our knowledge, this is the first comparative transcriptomic study in which the issue of the seed maturation rate is addressed.

Repertoire of the Bacillus thuringiensis Virulence Factors Unrelated to Major Classes of Protein Toxins and Its Role in Specificity of Host-Pathogen Interactions.

Bacillus thuringiensis (Bt) is a Gram-positive soil bacteria that infects invertebrates, predominantly of Arthropoda phylum. Due to its immense host range Bt has become a leading producer of biopesticides applied both in biotechnology and agriculture. Cytotoxic effect of Bt, as well as its host specificity, are commonly attributed either to proteinaceous crystal parasporal toxins (Cry and Cyt) produced by bacteria in a stationary phase or to soluble toxins of Vip and Sip families secreted by vegetative cells. At the same time, numerous non-toxin virulence factors of Bt have been discovered, including metalloproteases, chitinases, aminopolyol antibiotics and nucleotide-mimicking moieties. These agents act at each stage of the B. thuringiensis invasion and contribute to cytotoxic properties of Bt strains enhancing toxin activity, ensuring host immune response evasion and participating in extracellular matrix degeneration. In this review we attempt to classify Bt virulence factors unrelated to major groups of protein toxins and discuss their putative role in the establishment of Bt specificity to various groups of insects.

RNA Sequencing Reveals Specific TranscriptomicSignatures Distinguishing Effects of the [SWI⁺] Prion and SWI1 Deletion in Yeast Saccharomyces cerevisiae.

Prions are infectious, self-perpetuating protein conformers. In mammals, pathological aggregation of the prion protein causes incurable neurodegenerative disorders, while in yeast Saccharomyces cerevisiae, prion formation may be neutral or even beneficial. According to the prevailing contemporary point of view, prion formation is considered to be a functional inactivation of the corresponding protein whose conformational state shifts from the functional monomeric one to the infectious aggregated one. The Swi1 protein forms the [SWI⁺] prion and belongs to the nucleosome remodeler complex SWI/SNF controlling the expression of a significant part of the yeast genome. In this work, we performed RNA sequencing of isogenic S. cerevisiae strains grown on the media containing galactose as the sole carbon source. These strains bore the [SWI⁺] prion or had its structural gene SWI1 deleted. The comparative analysis showed that [SWI⁺] affects genome expression significantly weaker as compared to the SWI1 deletion. Moreover, in contrast to [SWI⁺], the SWI1 deletion causes the general inhibition of translation-related genes expression and chromosome I disomy. At the same time, the [SWI⁺] prion exhibits a specific pattern of modulation of the metabolic pathways and some biological processes and functions, as well as the expression of several genes. Thus, the [SWI⁺] prion only partially corresponds to the loss-of-function of SWI1 and demonstrates several gain-of-function traits.