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Abstract CsqR (YihW) is a local transcription factor that controls expression of yih genes involved in degradation of sulfoquinovose in Escherichia coli. We recently showed that expression of the respective gene cassette might be regulated by lactose. Here, we explore the phylogenetic and functional traits of CsqR. Phylogenetic analysis revealed that CsqR had a conserved Met25. Western blot demonstrated that CsqR was synthesized in the bacterial cell as two protein forms, 28.5 (CsqR-l) and 26 kDa (CsqR-s), the latter corresponding to start of translation at Met25. CsqR-s was dramatically activated during growth with sulfoquinovose as a sole carbon source, and displaced CsqR-l in the stationary phase during growth on rich medium. Molecular dynamic simulations revealed two possible states of the CsqR-s structure, with the interdomain linker being represented by either a disordered loop or an ɑ-helix. This helix allowed the hinge-like motion of the N-terminal domain resulting in a switch of CsqR-s between two conformational states, “open” and “compact”. We then modeled the interaction of both CsqR forms with putative effectors sulfoquinovose, sulforhamnose, sulfoquinovosyl glycerol, and lactose, and revealed that they all preferred the same pocket in CsqR-l, while in CsqR-s there were two possible options dependent on the linker structure.

Abstract Recycling vanadium from alternative sources is essential due to its expanding demand, depletion in natural sources, and environmental issues with terrestrial mining. Here, we present a complexation-precipitation method to selectively recover pentavalent vanadium ions, V(V), from complex metal ion mixtures, using an acid-stable metal binding agent, the cyclic imidedioxime, naphthalimidedioxime (H2CIDIII). H2CIDIII showed high extraction capacity and fast binding towards V(V) with crystal structures showing a 1:1 M:L dimer, [V2(O)3(C12H6N3O2)2]2−, 1, and 1:2 M:L non-oxido, [V(C12H6N3O2)2] ̶ complex, 2. Complexation selectivity studies showed only 1 and 2 were anionic, allowing facile separation of the V(V) complexes by pH-controlled precipitation, removing the need for solid support. The tandem complexation-precipitation technique achieved high recovery selectivity for V(V) with a selectivity coefficient above 3 × 105 from synthetic mixed metal solutions and real oil sand tailings. Zebrafish toxicity assay confirmed the non-toxicity of 1 and 2, highlighting H2CIDIII’s potential for practical and large-scale V(V) recovery.

Enterotypes of the human gut microbiome have been proposed to be a powerful prognostic tool to evaluate the correlation between lifestyle, nutrition, and disease. However, the number of enterotypes suggested in the literature ranged from two to four. The growth of available metagenome data and the use of exact, non-linear methods of data analysis challenges the very concept of clusters in the multidimensional space of bacterial microbiomes. Using several published human gut microbiome datasets of variable 16S rRNA regions, we demonstrate the presence of a lower-dimensional structure in the microbiome space, with high-dimensional data concentrated near a low-dimensional non-linear submanifold, but the absence of distinct and stable clusters that could represent enterotypes. This observation is robust with regard to diverse combinations of dimensionality reduction techniques and clustering algorithms.

Abstract Holometabolous insects are predominantly motionless during metamorphosis, when no active feeding is observed and the body is enclosed in a hardened cuticle. These physiological properties as well as undergoing processes resemble embryogenesis, since at the pupal stage organs and systems of the imago are formed. Therefore, recapitulation of the embryonic expression program during metamorphosis could be hypothesized. To assess this hypothesis at the transcriptome level, we have performed a comprehensive analysis of the developmental datasets available in the public domain. Indeed, for most datasets, the pupal gene expression resembles the embryonic rather than the larval pattern, interrupting gradual changes in the transcriptome. Moreover, changes in the transcriptome profile during the pupa-to-imago transition are positively correlated with those at the embryo-to-larvae transition, suggesting that similar expression programs are activated. Gene sets that change their expression level during the larval stage and revert it to the embryonic-like state during the metamorphosis are enriched with genes associated with metabolism and development.

Abstract Background The histidine metabolism and transport (his) genes are controlled by a variety of RNA-dependent regulatory systems among diverse taxonomic groups of bacteria including T-box riboswitches in Firmicutes and Actinobacteria and RNA attenuators in Proteobacteria. Using a comparative genomic approach, we previously identified a novel DNA-binding transcription factor (named HisR) that controls the histidine metabolism genes in diverse Gram-positive bacteria from the Firmicutes phylum. Results Here we report the identification of HisR-binding sites within the regulatory regions of the histidine metabolism and transport genes in 395 genomes representing the Bacilli, Clostridia, Negativicutes, and Tissierellia classes of Firmicutes, as well as in 97 other HisR-encoding genomes from the Actinobacteria, Proteobacteria, and Synergistetes phyla. HisR belongs to the TrpR family of transcription factors, and their predicted DNA binding motifs have a similar 20-bp palindromic structure but distinct lineage-specific consensus sequences. The predicted HisR-binding motif was validated in vitro using DNA binding assays with purified protein from the human gut bacterium Ruminococcus gnavus. To fill a knowledge gap in the regulation of histidine metabolism genes in Firmicutes genomes that lack a hisR repressor gene, we systematically searched their upstream regions for potential RNA regulatory elements. As result, we identified 158 T-box riboswitches preceding the histidine biosynthesis and/or transport genes in 129 Firmicutes genomes. Finally, novel candidate RNA attenuators were identified upstream of the histidine biosynthesis operons in six species from the Bacillus cereus group, as well as in five Eubacteriales and six Erysipelotrichales species. Conclusions The obtained distribution of the HisR transcription factor and two RNA-mediated regulatory mechanisms for histidine metabolism genes across over 600 species of Firmicutes is discussed from functional and evolutionary points of view.

ABSTRACT: Objectives: Eravacycline is a novel, fully-synthetic tetracycline approved by the FDA for treatment of complicated intra-abdominal infections in August 2018. This study sought to characterise early clinical experience with this novel antibiotic. Methods: Eravacycline utilisation for 66 patients was retrospectively evaluated. Results: Eravacycline was used as monotherapy in 62.1% of cases. Mean duration of therapy was 13.1 ± 9.9 days. The majority (68.2%) of treatment was for off-label indications, including 34.8% for pulmonary and 28.8% for skin/soft tissue infections. A number of difficult-to-treat organisms were encountered: 50% of identified Gram-negative pathogens were resistant to carbapenems in vitro; and 48% of identified Gram-positive pathogens were resistant to vancomycin in vitro. The patient population had a high illness acuity, with 42.4% requiring ICU admission, 59.1% having ≥2 co-morbidities and 33.3% having ≥3 co-morbidities. Nevertheless, 95.5% experienced clinical improvement, with 86.4% achieving full infection resolution following eravacycline. Three patients who did not experience clinical improvement had an intra-abdominal source of infection without adequate source control. The remaining six who did not experience full infection resolution died from unrelated non-infectious causes during hospital admission. Adverse events were uncommon (4.5%), limited to nausea/vomiting, and not leading to eravacycline discontinuation. Although two patients had a history of Clostridioides difficile infection (CDI), no patients developed CDI while receiving eravacycline. Conclusion: These results illustrate the potential versatility of eravacycline with a broad activity spectrum, good safety and tolerability profile, flexibility for use in patients with renal injury or antibiotic allergies, and positive clinical outcomes in this real-world cohort.

Abstract Until recently, Shigella and enteroinvasive Escherichia coli were thought to be primate-restricted pathogens. The base of their pathogenicity is the type 3 secretion system (T3SS) encoded by the pINV virulence plasmid, which facilitates host cell invasion and subsequent proliferation. A large family of T3SS effectors, E3 ubiquitin-ligases encoded by the ipaH genes, have a key role in the Shigella pathogenicity through the modulation of cellular ubiquitination that degrades host proteins. However, recent genomic studies identified ipaH genes in the genomes of Escherichia marmotae, a potential marmot pathogen, and an E. coli extracted from fecal samples of bovine calves, suggesting that non-human hosts may also be infected by these strains, potentially pathogenic to humans. We performed a comparative genomic study of the functional repertoires in the ipaH gene family in Shigella and enteroinvasive Escherichia from human and predicted non-human hosts. We found that fewer than half of Shigella genomes had a complete set of ipaH genes, with frequent gene losses and duplications that were not consistent with the species tree and nomenclature. Non-human host IpaH proteins had a diverse set of substrate-binding domains and, in contrast to the Shigella proteins, two variants of the NEL C-terminal domain. Inconsistencies between strains phylogeny and composition of effectors indicate horizontal gene transfer between E. coli adapted to different hosts. These results provide a framework for understanding of ipaH-mediated host-pathogens interactions and suggest a need for a genomic study of fecal samples from diseased animals.

Abstract RNA editing in the form of substituting adenine with inosine (A-to-I editing) is the most frequent type of RNA editing in many metazoan species. In most species, A-to-I editing sites tend to form clusters and editing at clustered sites depends on editing of the adjacent sites. Although functionally important in some specific cases, A-to-I editing usually is rare. The exception occurs in soft-bodied coleoid cephalopods, where tens of thousands of potentially important A-to-I editing sites have been identified, making coleoids an ideal model for studying of properties and evolution of A-to-I editing sites. Here, we apply several diverse techniques to demonstrate a strong tendency of coleoid RNA editing sites to cluster along the transcript. We show that clustering of editing sites and correlated editing substantially contribute to the transcriptome diversity that arises due to extensive RNA editing. Moreover, we identify three distinct types of editing site clusters, varying in size, and describe RNA structural features and mechanisms likely underlying formation of these clusters. In particular, these observations may explain sequence conservation at large distances around editing sites and the observed dependency of editing on mutations in the vicinity of editing sites.

The soil response to a jet-fuel contamination is uncertain. In this article, original data on the influence of a jet-fuel spillage on the topsoil properties are presented. The data set is obtained during a one-year long pot and field experiments with Dystric Arenosols, Fibric Histosols and Albic Luvisols. Kerosene loads were 1, 5, 10, 25 and 100 g/kg. The data set includes information about temporal changes in kerosene concentration; physicochemical properties, such as рН, moisture, cation exchange capacity, content of soil organic matter, available P and K, exchangeable NH4+, and water-soluble NO3–; and biological properties, such as biological consumption of oxygen, and cellulolytic activity. Also, we provide sequencing data on variable regions of 16S ribosomal RNA of microbial communities from the respective soil samples.

Small non-coding and antisense RNAs are widespread in all kingdoms of life, however, the diversity of their functions in bacteria is largely unknown. Here, we study RNAs synthesised from divergent promoters located in the 3′-end of the uxuR gene, encoding transcription factor regulating hexuronate metabolism in Escherichia coli. These overlapping promoters were predicted in silico with rather high scores, effectively bound RNA polymerase in vitro and in vivo and were capable of initiating transcription in sense and antisense directions. The genome-wide correlation between in silico promoter scores and RNA polymerase binding in vitro and in vivo was higher for promoters located on the antisense strands of the genes, however, sense promoters within the uxuR gene were more active. Both regulatory RNAs synthesised from the divergent promoters inhibited expression of genes associated with the E. coli motility and chemotaxis independent of a carbon source on which bacteria had been grown. Direct effects of these RNAs were confirmed for the fliA gene encoding σ28 subunit of RNA polymerase. In addition to intracellular sRNAs, promoters located within the uxuR gene could initiate synthesis of transcripts found in the fraction of RNAs secreted in the extracellular medium. Their profile was also carbon-independent suggesting that intragenic uxuR transcripts have a specific regulatory role not directly related to the function of the protein in which gene they are encoded.

Deep learning is a class of machine learning techniques capable of creating internal representation of data without explicit preprogramming. Hence, in addition to practical applications, it is of interest to analyze what features of biological data may be learned by such models. Here, we describe PredPair, a deep learning neural network trained to predict base pairs in RNA structure from sequence alone, without any incorporated prior knowledge, such as the stacking energies or possible spatial structures. PredPair learned the Watson-Crick and wobble base-pairing rules and created an internal representation of the stacking energies and helices. Application to independent experimental (DMS-Seq) data on nucleotide accessibility in mRNA showed that the nucleotides predicted as paired indeed tend to be involved in the RNA structure. The performance of the constructed model was comparable with the state-of-the-art method based on the thermodynamic approach, but with a higher false positives rate. On the other hand, it successfully predicted pseudoknots. t-SNE clusters of embeddings of RNA sequences created by PredPair tend to contain embeddings from particular Rfam families, supporting the predictions of PredPair being in line with biological classification.

Genomes are partitioned into topologically associating domains (TADs). Here the authors present single-nucleus Hi-C maps in Drosophila at 10 kb resolution, demonstrating the presence of chromatin compartments in individual nuclei, and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which resembles population TAD profiles.

Summary Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next‐generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100‐fold range.

Abstract Background Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. Results We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Conclusions Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

Intravenous (IV) drugs are administered through infusion pumps and IV administration sets for patients who are seen in healthcare settings. There are multiple areas of the medication administration process that can influence the amount of a drug a patient receives. For example, IV administration sets that deliver a drug from an infusion bag to a patient vary in terms of length and bore. In addition, fluid manufacturers report that the total acceptable volume range for a 250 mL bag of normal saline can be anywhere from 265 to 285 mL. At the institution chosen for our study, each 50 mg vial of eravacycline is reconstituted using 5 mL of diluent, and the total dose is administered as a 250 mL admixture. This single-center, retrospective, quasi-experimental study evaluated the residual medication volume after the completion of an IV eravacycline infusion in patients admitted during the pre-intervention study period compared to those in the post-intervention study period. The primary outcome of the study was to compare the residual antibiotic volume remaining in the bags following IV infusions of eravacycline before and after the implementation of interventions. The secondary outcomes included the following: comparing the amount of the drug lost in the pre- and post-intervention periods, determining whether the amount of residual volume was affected by nursing shifts (day versus night), and lastly assessing the cost of facility drug waste. On average, approximately 15% of the total bag volume was not infused during the pre-intervention period, which was reduced to less than 5% in the post-intervention period. Clinically, the average estimated amount of eravacycline discarded decreased from 13.5 mg to 4.7 mg in the pre- and post-intervention periods, respectively. Following the statistically significant results of this study, the interventions were expanded at this facility to include all admixed antimicrobials. Further studies are needed to determine the potential clinical impact when patients do not receive complete antibiotic infusions.

Construction of chromosomes 3D models based on single cell Hi-C data constitute an important challenge. We present a reconstruction approach, DPDchrom, that incorporates basic knowledge whether the reconstructed conformation should be coil-like or globular and spring relaxation at contact sites. In contrast to previously published protocols, DPDchrom can naturally form globular conformation due to the presence of explicit solvent. Benchmarking of this and several other methods on artificial polymer models reveals similar reconstruction accuracy at high contact density and DPDchrom advantage at low contact density. To compare 3D structures insensitively to spatial orientation and scale, we propose the Modified Jaccard Index. We analyzed two sources of the contact dropout: contact radius change and random contact sampling. We found that the reconstruction accuracy exponentially depends on the number of contacts per genomic bin allowing to estimate the reconstruction accuracy in advance. We applied DPDchrom to model chromosome configurations based on single-cell Hi-C data of mouse oocytes and found that these configurations differ significantly from a random one, that is consistent with other studies.

Abstract Background Chlamydia are ancient intracellular pathogens with reduced, though strikingly conserved genome. Despite their parasitic lifestyle and isolated intracellular environment, these bacteria managed to avoid accumulation of deleterious mutations leading to subsequent genome degradation characteristic for many parasitic bacteria. Results We report pan-genomic analysis of sixteen species from genus Chlamydia including identification and functional annotation of orthologous genes, and characterization of gene gains, losses, and rearrangements. We demonstrate the overall genome stability of these bacteria as indicated by a large fraction of common genes with conserved genomic locations. On the other hand, extreme evolvability is confined to several paralogous gene families such as polymorphic membrane proteins and phospholipase D, and likely is caused by the pressure from the host immune system. Conclusions This combination of a large, conserved core genome and a small, evolvable periphery likely reflect the balance between the selective pressure towards genome reduction and the need to adapt to escape from the host immunity.

Abstract Background The genus Streptococcus comprises pathogens that strongly influence the health of humans and animals. Genome sequencing of multiple Streptococcus strains demonstrated high variability in gene content and order even in closely related strains of the same species and created a newly emerged object for genomic analysis, the pan-genome. Here we analysed the genome evolution of 25 strains of Streptococcus suis, 50 strains of Streptococcus pyogenes and 28 strains of Streptococcus pneumoniae. Results Fractions of the pan-genome, unique, periphery, and universal genes differ in size, functional composition, the level of nucleotide substitutions, and predisposition to horizontal gene transfer and genomic rearrangements. The density of substitutions in intergenic regions appears to be correlated with selection acting on adjacent genes, implying that more conserved genes tend to have more conserved regulatory regions. The total pan-genome of the genus is open, but only due to strain-specific genes, whereas other pan-genome fractions reach saturation. We have identified the set of genes with phylogenies inconsistent with species and non-conserved location in the chromosome; these genes are rare in at least one species and have likely experienced recent horizontal transfer between species. The strain-specific fraction is enriched with mobile elements and hypothetical proteins, but also contains a number of candidate virulence-related genes, so it may have a strong impact on adaptability and pathogenicity. Mapping the rearrangements to the phylogenetic tree revealed large parallel inversions in all species. A parallel inversion of length 15 kB with breakpoints formed by genes encoding surface antigen proteins PhtD and PhtB in S. pneumoniae leads to replacement of gene fragments that likely indicates the action of an antigen variation mechanism. Conclusions Members of genus Streptococcus have a highly dynamic, open pan-genome, that potentially confers them with the ability to adapt to changing environmental conditions, i.e. antibiotic resistance or transmission between different hosts. Hence, integrated analysis of all aspects of genome evolution is important for the identification of potential pathogens and design of drugs and vaccines.

The role of the nuclear lamina (NL) in chromatin architecture is still poorly understood. Here, the authors provide evidence that disruption of the NL in Drosophila cells leads to overall chromatin compaction and repositioning from the nuclear envelope, whereas lamina-associated regions become less compacted and transcription within them is increased.

The IclR-family is a large group of transcription factors (TFs) regulating various biological processes in diverse bacteria. Using comparative genomics techniques, we have identified binding motifs of IclR-family TFs, reconstructed regulons and analyzed their content, finding co-occurrences between the regulated COGs (clusters of orthologous genes), useful for future functional characterizations of TFs and their regulated genes. We describe two main types of IclR-family motifs, similar in sequence but different in the arrangement of the half-sites (boxes), with GKTYCRYW3–4RYGRAMC and TGRAACAN1–2TGTTYCA consensuses, and also predict that TFs in 32 orthologous groups have binding sites comprised of three boxes with alternating direction, which implies two possible alternative modes of dimerization of TFs. We identified trends in site positioning relative to the translational gene start, and show that TFs in 94 orthologous groups bind tandem sites with 18–22 nucleotides between their centers. We predict protein–DNA contacts via the correlation analysis of nucleotides in binding sites and amino acids of the DNA-binding domain of TFs, and show that the majority of interacting positions and predicted contacts are similar for both types of motifs and conform well both to available experimental data and to general protein–DNA interaction trends.

Shigella are pathogens originating within the Escherichia lineage but frequently classified as a separate genus. Shigella genomes contain numerous insertion sequences (ISs) that lead to pseudogenisation of affected genes and an increase of non-homologous recombination. Here, we study 414 genomes of E. coli and Shigella strains to assess the contribution of genomic rearrangements to Shigella evolution. We found that Shigella experienced exceptionally high rates of intragenomic rearrangements and had a decreased rate of homologous recombination compared to pathogenic and non-pathogenic E. coli. The high rearrangement rate resulted in independent disruption of syntenic regions and parallel rearrangements in different Shigella lineages. Specifically, we identified two types of chromosomally encoded E3 ubiquitin-protein ligases acquired independently by all Shigella strains that also showed a high level of sequence conservation in the promoter and further in the 5′-intergenic region. In the only available enteroinvasive E. coli (EIEC) strain, which is a pathogenic E. coli with a phenotype intermediate between Shigella and non-pathogenic E. coli, we found a rate of genome rearrangements comparable to those in other E. coli and no functional copies of the two Shigella-specific E3 ubiquitin ligases. These data indicate that the accumulation of ISs influenced many aspects of genome evolution and played an important role in the evolution of intracellular pathogens. Our research demonstrates the power of comparative genomics-based on synteny block composition and an important role of non-coding regions in the evolution of genomic islands.

The emerging human enteropathogen Clostridioides difficile is the main cause of diarrhea associated with antibiotherapy. Regulatory pathways underlying the adaptive responses remain understudied and the global view of C. difficile promoter structure is still missing. In the genome of C. difficile 630, 22 genes encoding sigma factors are present suggesting a complex pattern of transcription in this bacterium. We present here the first transcriptional map of the C. difficile genome resulting from the identification of transcriptional start sites (TSS), promoter motifs and operon structures. By 5′-end RNA-seq approach, we mapped more than 1000 TSS upstream of genes. In addition to these primary TSS, this analysis revealed complex structure of transcriptional units such as alternative and internal promoters, potential RNA processing events and 5′ untranslated regions. By following an in silico iterative strategy that used as an input previously published consensus sequences and transcriptomic analysis, we identified candidate promoters upstream of most of protein-coding and non-coding RNAs genes. This strategy also led to refine consensus sequences of promoters recognized by major sigma factors of C. difficile. Detailed analysis focuses on the transcription in the pathogenicity locus and regulatory genes, as well as regulons of transition phase and sporulation sigma factors as important components of C. difficile regulatory network governing toxin gene expression and spore formation. Among the still uncharacterized regulons of the major sigma factors of C. difficile, we defined the SigL regulon by combining transcriptome and in silico analyses. We showed that the SigL regulon is largely involved in amino-acid degradation, a metabolism crucial for C. difficile gut colonization. Finally, we combined our TSS mapping, in silico identification of promoters and RNA-seq data to improve gene annotation and to suggest operon organization in C. difficile. These data will considerably improve our knowledge of global regulatory circuits controlling gene expression in C. difficile and will serve as a useful rich resource for scientific community both for the detailed analysis of specific genes and systems biology studies.

Regulation of gene transcription is a complex process controlled by many factors, including the conformation of chromatin in the nucleus. Insights into chromatin conformation on both local and global scales can be provided by the Hi-C (high-throughput chromosomes conformation capture) method. One of the drawbacks of Hi-C analysis and interpretation is the presence of systematic biases, such as different accessibility to enzymes, amplification, and mappability of DNA regions, which all result in different visibility of the regions. Iterative correction (IC) is one of the most popular techniques developed for the elimination of these systematic biases. IC is based on the assumption that all chromatin regions have an equal number of observed contacts in Hi-C. In other words, the IC procedure is equalizing the experimental visibility approximated by the cumulative contact frequency (CCF) for all genomic regions. However, the differences in experimental visibility might be explained by biological factors such as chromatin openness, which is characteristic of distinct chromatin states. Here we show that CCF is positively correlated with active transcription. It is associated with compartment organization, since compartment A demonstrates higher CCF and gene expression levels than compartment B. Notably, this observation holds for a wide range of species, including human, mouse, and Drosophila. Moreover, we track the CCF state for syntenic blocks between human and mouse and conclude that active state assessed by CCF is an intrinsic property of the DNA region, which is independent of local genomic and epigenomic context. Our findings establish a missing link between Hi-C normalization procedures removing CCF from the data and poorly investigated and possibly relevant biological factors contributing to CCF.

Abstract Background The genus Burkholderia consists of species that occupy remarkably diverse ecological niches. Its best known members are important pathogens, B. mallei and B. pseudomallei, which cause glanders and melioidosis, respectively. Burkholderia genomes are unusual due to their multichromosomal organization, generally comprised of 2-3 chromosomes. Results We performed integrated genomic analysis of 127 Burkholderia strains. The pan-genome is open with the saturation to be reached between 86,000 and 88,000 genes. The reconstructed rearrangements indicate a strong avoidance of intra-replichore inversions that is likely caused by selection against the transfer of large groups of genes between the leading and the lagging strands. Translocated genes also tend to retain their position in the leading or the lagging strand, and this selection is stronger for large syntenies. Integrated reconstruction of chromosome rearrangements in the context of strains phylogeny reveals parallel rearrangements that may indicate inversion-based phase variation and integration of new genomic islands. In particular, we detected parallel inversions in the second chromosomes of B. pseudomallei with breakpoints formed by genes encoding membrane components of multidrug resistance complex, that may be linked to a phase variation mechanism. Two genomic islands, spreading horizontally between chromosomes, were detected in the B. cepacia group. Conclusions This study demonstrates the power of integrated analysis of pan-genomes, chromosome rearrangements, and selection regimes. Non-random inversion patterns indicate selective pressure, inversions are particularly frequent in a recent pathogen B. mallei, and, together with periods of positive selection at other branches, may indicate adaptation to new niches. One such adaptation could be a possible phase variation mechanism in B. pseudomallei.

Introduction. Originality and independence is the first, absolute and general requirement to the content of dissertations, regardless of an academic degree and research direction. At the same time, recently a considerable fraction of different types of plagiarism have been found out in the Russian dissertation works. The aim is an analysis of the most frequent plagiarism in candidate and doctoral dissertations. Results. We consider the established practices in the evaluation of independence in academic works. Unethical use of someone else’s work and materials by the authors of dissertations include, in particular, copying from official documents and abstracts without acknowledgement the source and with no citation; replication of existing reviews and chapters from already defended dissertations; falsification by refreshing of outdated data with substituted dates, and or wordfor-word copy-pasting with substitution of the research object. The latter type of the plagiarism involving fraud in experimental and statistical data constitutes a special peril for the science and for the society in general. The primary principle for assessment of independence in dissertations has to be proper citing and referencing that should allow a reader to distinguish the author’s contribution from someone else’s text. Undocumented verbatim quotations in dissertations are inadmissible, whether the author claims scientific novelty or not. At that, it does not matter whether well-known or unique data are provided, and whether the source is protected by a copyright. Practical significance. The qualitative analysis of the factual material, based on the author’s observations, can serve as a starting point for the subsequent quantitative analysis of plagiarism in scientific texts.

One of the most important challenges for soil science is to determine the limits for the sustainable functioning of contaminated ecosystems. The response of soil microbiomes to kerosene pollution is still poorly understood. Here, we model the impact of kerosene leakage on the composition of the topsoil microbiome in pot and field experiments with different loads of added kerosene (loads up to 100 g/kg; retention time up to 360 days). At four time points we measured kerosene concentration and sequenced variable regions of 16S ribosomal RNA in the microbial communities. Mainly alkaline Dystric Arenosols with low content of available phosphorus and soil organic matter had an increased fraction of Actinobacteriota, Firmicutes, Nitrospirota, Planctomycetota, and, to a lesser extent, Acidobacteriota and Verrucomicobacteriota. In contrast, in highly acidic Fibric Histosols, rich in soil organic matter and available phosphorus, the fraction of Acidobacteriota was higher, while the fraction of Actinobacteriota was lower. Albic Luvisols occupied an intermediate position in terms of both physicochemical properties and microbiome composition. The microbiomes of different soils show similar response to equal kerosene loads. In highly contaminated soils, the proportion of anaerobic bacteria-metabolizing hydrocarbons increased, whereas the proportion of aerobic bacteria decreased. During the field experiment, the soil microbiome recovered much faster than in the pot experiments, possibly due to migration of microorganisms from the polluted area. The microbial community of Fibric Histosols recovered in 6 months after kerosene had been loaded, while microbiomes of Dystric Arenosols and Albic Luvisols did not restore even after a year.

Abstract Background Bacterial carbohydrate metabolism is extremely diverse, since carbohydrates serve as a major energy source and are involved in a variety of cellular processes. Bacterial genes belonging to same metabolic pathway are often co-localized in the chromosome, but it is not a strict rule. Gene co-localization in linked to co-evolution and co-regulation. This study focuses on a large-scale analysis of bacterial genomic loci related to the carbohydrate metabolism. Results We demonstrate that only 53% of 148,000 studied genes from over six hundred bacterial genomes are co-localized in bacterial genomes with other carbohydrate metabolism genes, which points to a significant role of singleton genes. Co-localized genes form cassettes, ranging in size from two to fifteen genes. Two major factors influencing the cassette-forming tendency are gene function and bacterial phylogeny. We have obtained a comprehensive picture of co-localization preferences of genes for nineteen major carbohydrate metabolism functional classes, over two hundred gene orthologous clusters, and thirty bacterial classes, and characterized the cassette variety in size and content among different species, highlighting a significant role of short cassettes. The preference towards co-localization of carbohydrate metabolism genes varies between 40 and 76% for bacterial taxa. Analysis of frequently co-localized genes yielded forty-five significant pairwise links between genes belonging to different functional classes. The number of such links per class range from zero to eight, demonstrating varying preferences of respective genes towards a specific chromosomal neighborhood. Genes from eleven functional classes tend to co-localize with genes from the same class, indicating an important role of clustering of genes with similar functions. At that, in most cases such co-localization does not originate from local duplication events. Conclusions Overall, we describe a complex web formed by evolutionary relationships of bacterial carbohydrate metabolism genes, manifested as co-localization patterns. Reviewers This article was reviewed by Daria V. Dibrova (A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia), nominated by Armen Mulkidjanian (University of Osnabrück, Germany), Igor Rogozin (NCBI, NLM, NIH, USA) and Yuri Wolf (NCBI, NLM, NIH, USA).

Abstract Background In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. Results We found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation. Conclusions Our findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.

Abstract Background Escherichia coli (E. coli) has been increasingly implicated in the pathogenesis of Crohn’s disease (CD). The phylogeny of E. coli isolated from Crohn’s disease patients (CDEC) was controversial, and while genotyping results suggested heterogeneity, the sequenced strains of E. coli from CD patients were closely related. Results We performed the shotgun genome sequencing of 28 E. coli isolates from ten CD patients and compared genomes from these isolates with already published genomes of CD strains and other pathogenic and non-pathogenic strains. CDEC was shown to belong to A, B1, B2 and D phylogenetic groups. The plasmid and several operons from the reference CD-associated E. coli strain LF82 were demonstrated to be more often present in CDEC genomes belonging to different phylogenetic groups than in genomes of commensal strains. The operons include carbon-source induced invasion GimA island, prophage I, iron uptake operons I and II, capsular assembly pathogenetic island IV and propanediol and galactitol utilization operons. Conclusions Our findings suggest that CDEC are phylogenetically diverse. However, some strains isolated from independent sources possess highly similar chromosome or plasmids. Though no CD-specific genes or functional domains were present in all CD-associated strains, some genes and operons are more often found in the genomes of CDEC than in commensal E. coli. They are principally linked to gut colonization and utilization of propanediol and other sugar alcohols.

An amendment to this paper has been published and can be accessed via the original article.

Aromatic compounds are a common carbon and energy source for many microorganisms, some of which can even degrade toxic chloroaromatic xenobiotics. This comparative study of aromatic metabolism in 32 Betaproteobacteria species describes the links between several transcription factors (TFs) that control benzoate (BenR, BenM, BoxR, BzdR), catechol (CatR, CatM, BenM), chlorocatechol (ClcR), methylcatechol (MmlR), 2,4-dichlorophenoxyacetate (TfdR, TfdS), phenol (AphS, AphR, AphT), biphenyl (BphS), and toluene (TbuT) metabolism. We characterize the complexity and variability in the organization of aromatic metabolism operons and the structure of regulatory networks that may differ even between closely related species. Generally, the upper parts of pathways, rare pathway variants, and degradative pathways of exotic and complex, in particular, xenobiotic compounds are often controlled by a single TF, while the regulation of more common and/or central parts of the aromatic metabolism may vary widely and often involves several TFs with shared and/or dual, or cascade regulation. The most frequent and at the same time variable connections exist between AphS, AphR, AphT, and BenR. We have identified a novel LysR-family TF that regulates the metabolism of catechol (or some catechol derivative) and either substitutes CatR(M)/BenM, or shares functions with it. We have also predicted several new members of aromatic metabolism regulons, in particular, some COGs regulated by several different TFs.

Holospora-like bacteria (HLB) are obligate intracellular Alphaproteobacteria, inhabiting nuclei of Paramecium and other ciliates such as “Candidatus Hafkinia” is in Frontonia. The HLB clade is comprised of four genera, Holospora, Preeria, “Candidatus Gortzia”, and “Candidatus Hafkinia”. These bacteria have a peculiar life cycle with two morphological forms and some degree of specificity to the host species and the type of nucleus they inhabit. Here we describe a novel species of HLB—“Candidatus Gortzia yakutica” sp. nov.—a symbiont from the macronucleus of Paramecium putrinum, the first described HLB for this Paramecium species. The new endosymbiont shows morphological similarities with other HLB. The phylogenetic analysis of the SSU rRNA gene places it into the “Candidatus Gortzia” clade.

While most endosymbiotic bacteria are transmitted only vertically, Holospora spp., an alphaproteobacterium from the Rickettsiales order, can desert its host and invade a new one. All bacteria from the genus Holospora are intranuclear symbionts of ciliates Paramecium spp. with strict species and nuclear specificity. Comparative metabolic reconstruction based on the newly sequenced genome of Holospora curviuscula, a macronuclear symbiont of Paramecium bursaria, and known genomes of other Holospora species shows that even though all Holospora spp. can persist outside the host, they cannot synthesize most of the essential small molecules, such as amino acids, and lack some central energy metabolic pathways, including glycolysis and the citric acid cycle. As the main energy source, Holospora spp. likely rely on nucleotides pirated from the host. Holospora-specific genes absent from other Rickettsiales are possibly involved in the lifestyle switch from the infectious to the reproductive form and in cell invasion.

The pangenome is the collection of all groups of orthologous genes (OGGs) from a set of genomes. We apply the pangenome analysis to propose a definition of prokaryotic species based on identification of lineage-specific gene sets. While being similar to the classical biological definition based on allele flow, it does not rely on DNA similarity levels and does not require analysis of homologous recombination. Hence this definition is relatively objective and independent of arbitrary thresholds. A systematic analysis of 110 accepted species with the largest numbers of sequenced strains yields results largely consistent with the existing nomenclature. However, it has revealed that abundant marine cyanobacteria Prochlorococcus marinus should be divided into two species. As a control we have confirmed the paraphyletic origin of Yersinia pseudotuberculosis (with embedded, monophyletic Y. pestis) and Burkholderia pseudomallei (with B. mallei). We also demonstrate that by our definition and in accordance with recent studies Escherichia coli and Shigella spp. are one species.

Genome rearrangements have played an important role in the evolution of Yersinia pestis from its progenitor Yersinia pseudotuberculosis. Traditional phylogenetic trees for Y. pestis based on sequence comparison have short internal branches and low bootstrap supports as only a small number of nucleotide substitutions have occurred. On the other hand, even a small number of genome rearrangements may resolve topological ambiguities in a phylogenetic tree. We reconstructed phylogenetic trees based on genome rearrangements using several popular approaches such as Maximum likelihood for Gene Order and the Bayesian model of genome rearrangements by inversions. We also reconciled phylogenetic trees for each of the three CRISPR loci to obtain an integrated scenario of the CRISPR cassette evolution. Analysis of contradictions between the obtained evolutionary trees yielded numerous parallel inversions and gain/loss events. Our data indicate that an integrated analysis of sequence-based and inversion-based trees enhances the resolution of phylogenetic reconstruction. In contrast, reconstructions of strain relationships based on solely CRISPR loci may not be reliable, as the history is obscured by large deletions, obliterating the order of spacer gains. Similarly, numerous parallel gene losses preclude reconstruction of phylogeny based on gene content.

Invasive fungal infections (IFI) are a heterogeneous group of diseases, whose clinical significance is increased due to large number of cancerpatients, patients with aplastic anemia, congenital or acquired immunity defects. Morbidity and mortality due to fungal infection remain high, and the available diagnostic methods do not always allow timely detect and isolate infection pathogen. Microbiological and histological examination of biopsy material is the “gold standard” of IFI diagnostic, but in practice it is often associated with high risk for patients. Reliable nonculture methods of early IFI detection needed to improve current diagnostic and allowing an earlier decision of specific antifungal therapy. This article presents the advantages and limitations of one of these tests. In recent years, many articles and reviews about circulating (1→3)β-D-glucan (BDG) detection (major cell wall component of most pathogenic fungi) has appeared in the available literature. Despite that this test has a high diagnostic value, to recommend it for routine use in children with febrile neutropenia is premature. This test is less useful in making timely decision about diagnosis and start of antifungal therapy because of moderate sensitivity and specificity, the lack of evaluation criteria in children, late results (if detected in reference laboratory) and cost. BDG detection results can be interpreted only in conjunction with clinical course and data of routine laboratory and radiological methods.