Study of the binding of ΔFN3.1 fragments of the Bifidobacterium longum GT15 with TNFα and prevalence of domain-containing proteins in groups of bacteria of the human gut microbiota.

Aim: This study is mainly devoted to determining the ability of ∆FN3.1 protein fragments of Bifidobacterium (B.) longum subsp. longum GT15, namely two FN3 domains (2D FN3) and a C-terminal domain (CD FN3), to bind to tumor necrosis factor-alpha (TNF-α). Methods: Fragments of the fn3 gene encoding the 2D FN3 and CD FN3 were cloned in Escherichia (E.) coli. In order to assess the binding specificity between 2D FN3 and CD FN3 to TNFα, we employed the previously developed sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. The trRosetta software was used to build 3D models of the ∆FN3.1, 2D FN3, and CD FN3 proteins. The detection of polymorphism in the amino acid sequences of the studied proteins and the analysis of human gut-derived bacterial proteins carrying FN3 domains were performed in silico. Results: We experimentally showed that neither 2D FN3 nor CD FN3 alone can bind to TNFα. Prediction of the 3D structures of ΔFN3.1, 2D FN3, and CD FN3 suggested that only ΔFN3.1 can form a pocket allowing binding with TNFα to occur. Polymorphism analysis of amino acid sequences of ΔFN3.1 proteins in B. longum strains uncovered substitutions that can alter the conformation of the spatial structure of the ΔFN3.1 protein. We also analyzed human gut-derived bacterial proteins harboring FN3 domains which allowed us to differentiate between those containing motifs of cytokine receptors (MCRs) in their FN3 domains and those lacking them. Conclusion: Only the complete ∆FN3.1 protein can selectively bind to TNFα. Analysis of 3D models of the 2D FN3, CD FN3, and ΔFN3.1 proteins showed that only the ΔFN3.1 protein is potentially capable of forming a pocket allowing TNFα binding to occur. Only FN3 domains containing MCRs exhibited sequence homology with FN3 domains of human proteins.

Unveiling the Connection between Microbiota and Depressive Disorder through Machine Learning.

In the last few years, investigation of the gut-brain axis and the connection between the gut microbiota and the human nervous system and mental health has become one of the most popular topics. Correlations between the taxonomic and functional changes in gut microbiota and major depressive disorder have been shown in several studies. Machine learning provides a promising approach to analyze large-scale metagenomic data and identify biomarkers associated with depression. In this work, machine learning algorithms, such as random forest, elastic net, and You Only Look Once (YOLO), were utilized to detect significant features in microbiome samples and classify individuals based on their disorder status. The analysis was conducted on metagenomic data obtained during the study of gut microbiota of healthy people and patients with major depressive disorder. The YOLO method showed the greatest effectiveness in the analysis of the metagenomic samples and confirmed the experimental results on the critical importance of a reduction in the amount of Faecalibacterium prausnitzii for the manifestation of depression. These findings could contribute to a better understanding of the role of the gut microbiota in major depressive disorder and potentially lead the way for novel diagnostic and therapeutic strategies.

Novel Derivatives of Quinoxaline-2-carboxylic Acid 1,4-Dioxides as Antimycobacterial Agents: Mechanistic Studies and Therapeutic Potential.

The World Health Organization (WHO) reports that tuberculosis (TB) is one of the top 10 leading causes of global mortality. The increasing incidence of multidrug-resistant TB highlights the urgent need for an intensified quest to discover innovative anti-TB medications In this study, we investigated four new derivatives from the quinoxaline-2-carboxylic acid 1,4-dioxide class. New 3-methylquinoxaline 1,4-dioxides with a variation in substituents at positions 2 and 6(7) were synthesized via nucleophilic aromatic substitution with amines and assessed against a Mycobacteria spp. Compound 4 showed high antimycobacterial activity (1.25 µg/mL against M. tuberculosis) and low toxicity in vivo in mice. Selection and whole-genomic sequencing of spontaneous drug-resistant M. smegmatis mutants revealed a high number of single-nucleotide polymorphisms, confirming the predicted mode of action of the quinoxaline-2-carboxylic acid 1,4-dioxide 4 as a DNA-damaging agent. Subsequent reverse genetics methods confirmed that mutations in the genes MSMEG_4646, MSMEG_5122, and MSMEG_1380 mediate resistance to these compounds. Overall, the derivatives of quinoxaline-2-carboxylic acid 1,4-dioxide present a promising scaffold for the development of innovative antimycobacterial drugs.

Oxidative Stress Response of Probiotic Strain Bifidobacterium longum subsp. longum GT15.

Bifidobacterium is a predominant and important genus in the bacterial population of the human gut microbiota. Despite the increasing number of studies on the beneficial functionality of bifidobacteria for human health, knowledge about their antioxidant potential is still insufficient. Several in vivo and in vitro studies of Bifidobacterium strains and their cellular components have shown good antioxidant capacity that provided a certain protection of their own and the host's cells. Our work presents the data of transcriptomic, proteomic, and metabolomic analyses of the growing and stationary culture of the probiotic strain B. longum subsp. longum GT15 after exposure to hydrogen peroxide for 2 h and oxygen for 2 and 4 h. The results of the analysis of the sequenced genome of B. longum GT15 showed the presence of 16 gene-encoding proteins with known antioxidant functions. The results of the full transcriptomic analysis demonstrated a more than two-fold increase of levels of transcripts for eleven genes, encoding proteins with antioxidant functions. Proteomic data analysis showed an increased level of more than two times for glutaredoxin and thioredoxin after the exposure to oxygen, which indicates that the thioredoxin-dependent antioxidant system may be the major redox homeostasis system in B. longum bacteria. We also found that the levels of proteins presumably involved in global stress, amino acid metabolism, nucleotide and carbohydrate metabolism, and transport had significantly increased in response to oxidative stress. The metabolic fingerprint analysis also showed good discrimination between cells responding to oxidative stress and the untreated controls. Our results provide a greater understanding of the mechanism of oxidative stress response in B. longum and the factors that contribute to its survival in functional food products.

Multi-level analysis of the gut-brain axis shows autism spectrum disorder-associated molecular and microbial profiles.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in ASD although with limited reproducibility across studies. In this study, we developed a Bayesian differential ranking algorithm to identify ASD-associated molecular and taxa profiles across 10 cross-sectional microbiome datasets and 15 other datasets, including dietary patterns, metabolomics, cytokine profiles and human brain gene expression profiles. We found a functional architecture along the GBA that correlates with heterogeneity of ASD phenotypes, and it is characterized by ASD-associated amino acid, carbohydrate and lipid profiles predominantly encoded by microbial species in the genera Prevotella, Bifidobacterium, Desulfovibrio and Bacteroides and correlates with brain gene expression changes, restrictive dietary patterns and pro-inflammatory cytokine profiles. The functional architecture revealed in age-matched and sex-matched cohorts is not present in sibling-matched cohorts. We also show a strong association between temporal changes in microbiome composition and ASD phenotypes. In summary, we propose a framework to leverage multi-omic datasets from well-defined cohorts and investigate how the GBA influences ASD.

Kanamycin and Ofloxacin Activate the Intrinsic Resistance to Multiple Antibiotics in Mycobacterium smegmatis.

Drug resistance (DR) in Mycobacterium tuberculosis is the main problem in fighting tuberculosis (TB). This pathogenic bacterium has several types of DR implementation: acquired and intrinsic DR. Recent studies have shown that exposure to various antibiotics activates multiple genes, including genes responsible for intrinsic DR. To date, there is evidence of the acquisition of resistance at concentrations well below the standard MICs. In this study, we aimed to investigate the mechanism of intrinsic drug cross-resistance induction by subinhibitory concentrations of antibiotics. We showed that pretreatment of M. smegmatis with low doses of antibiotics (kanamycin and ofloxacin) induced drug resistance. This effect may be caused by a change in the expression of transcriptional regulators of the mycobacterial resistome, in particular the main transcriptional regulator whiB7.

Genetic Relationships and Signatures of Adaptation to the Climatic Conditions in Populations of Apis cerana Based on the Polymorphism of the Gene Vitellogenin.

Apis cerana and Apis mellifera are important honey bee species in Asia. A. cerana populations are distributed from a cold, sharply continental climate in the north to a hot, subtropical climate in the south. Due to the Sacbrood virus, almost all A. cerana populations in Asia have declined significantly in recent decades and have recovered over the past five years. This could lead to a shift in the gene pool of local A. cerana populations that could affect their sustainability and adaptation. It was assumed that adaptation of honey bees could be observed by comparative analysis of the sequences of genes involved in development, labor division, and caste differentiation, such as the gene Vitellogenin VG. The VG gene nucleotide sequences were used to assess the genetic structure and signatures of adaptation of local populations of A. cerana from Korea, Russia, Japan, Nepal, and China. A. mellifera samples from India and Poland were used as the outgroup. The signatures of adaptive selection were found in the local population of A. cerana using VG gene sequence analysis based on Jukes−Cantor genetic distances, cluster analysis, dN/dS ratio evaluation, and Tajima's D neutrality test. Based on analysis of the VG gene sequences, Apis cerana koreana subspecies in the Korean Peninsula were subdivided into three groups in accordance with their geographic localization from north to south. The VG gene sequences are acceptable tools to study the sustainability and adaptation of A. cerana populations.

Alterations of the Composition and Neurometabolic Profile of Human Gut Microbiota in Major Depressive Disorder.

Major depressive disorder (MDD) is among the most prevalent mental disorders worldwide. Factors causing the pathogenesis of MDD include gut microbiota (GM), which interacts with the host through the gut-brain axis. In previous studies of GM in MDD patients, 16S rRNA sequencing was used, which provided information about composition but not about function. In our study, we analyzed whole metagenome sequencing data to assess changes in both the composition and functional profile of GM. We looked at the GM of 36 MDD patients, compared with that of 38 healthy volunteers. Comparative taxonomic analysis showed decreased abundances of Faecalibacterium prausnitzii, Roseburia hominis, and Roseburia intestinalis, and elevated abundances of Escherichia coli and Ruthenibacterium lactatiformans in the GM of MDD patients. We observed decreased levels of bacterial genes encoding key enzymes involved in the production of arginine, asparagine, glutamate, glutamine, melatonin, acetic, butyric and conjugated linoleic acids, and spermidine in MDD patients. These genes produced signature pairs with Faecalibacterium prausntizii and correlated with decreased levels of this species in the GM of MDD patients. These results show the potential impact of the identified biomarker bacteria and their metabolites on the pathogenesis of MDD, and should be confirmed in future metabolomic studies.

What are the prospects for using complexes of copper(ii) and zinc(ii) to suppress the vital activity of Mycolicibacterium smegmatis?

New complexes of zinc(ii) and copper(ii) with 2-furoic acid (Hfur), acetic acids and N-donor ligands with the compositions [Zn2(fur)4] n (1), [Zn2(fur)4(NH2py)2] (2, NH2py = 3-aminopyridine), [Zn(fur)2(neoc)] (3, neoc = 2,9-dimethyl-1,10-phenantroline), [Zn(OAc)2(neoc)] (4, OAc = acetat-anion), and [Cu(fur)2(neoc)(H2O)] (5) were synthesized. The structures of the compounds were established by single crystal X-ray diffraction analysis. Complexes 1 and 2 are binuclear; whereas 3-5 are mononuclear. The stabilization of supramolecular architectures in crystals for compounds 1-5 occurs due to π-π-bonding between heterocycles and hydrogen interactions that provide good solubility in aqueous solutions. The stability of the complexes upon dissolution in 5% dextrose and 0.9% NaCl was confirmed by UV-vis spectroscopic and NMR (1H) data. The study of in vitro biological activity was carried out against the non-pathogenic strain of Mycolicibacterium smegmatis that is a model for M. tuberculosis. The synergistic effect of ligands is observed for complexes 3-5 and is characterized by an increase in the biological activity values. On passage from Zn2+ to Cu2+ complexes, the biological activity increases and the maximum effect is observed for compound [Cu(fur)2(phen)]. Analysis of the transcriptomic profiles of the M. smegmatis mc 2 155 strain under the pressure of the copper complex [Cu(fur)2(phen)] made it possible to isolate 185 genes, one quarter of which are associated with the compensation of iron deficiency in the bacterial strain. Genes associated with the transport and metabolism of heavy metals, biosynthesis of fatty and amino acids, biodegradation and transport of urea were also isolated.

CRISPR-Cas Systems in Gut Microbiome of Children with Autism Spectrum Disorders.

The human gut microbiome is associated with various diseases, including autism spectrum disorders (ASD). Variations of the taxonomical composition in the gut microbiome of children with ASD have been observed repeatedly. However, features and parameters of the microbiome CRISPR-Cas systems in ASD have not been investigated yet. Here, we demonstrate such an analysis in order to describe the overall changes in the microbiome CRISPR-Cas systems during ASD as well as to reveal their potential to be used in diagnostics and therapy. For the systems identification, we used a combination of the publicly available tools suited for completed genomes with subsequent filtrations. In the considered data, the microbiomes of children with ASD contained fewer arrays per Gb of assembly than the control group, but the arrays included more spacers on average. CRISPR arrays from the microbiomes of children with ASD differed from the control group neither in the fractions of spacers with protospacers from known genomes, nor in the sets of known bacteriophages providing protospacers. Almost all bacterial protospacers of the gut microbiome systems for both children with ASD and the healthy ones were located in prophage islands, leaving no room for the systems to participate in the interspecies competition.

Repurposing Based Identification of Novel Inhibitors against MmpS5-MmpL5 Efflux Pump of Mycobacterium smegmatis: A Combined In Silico and In Vitro Study.

In the current era of a pandemic, infections of COVID-19 and Tuberculosis (TB) enhance the detrimental effects of both diseases in suffering individuals. The resistance mechanisms evolving in Mycobacterium tuberculosis are limiting the efficiency of current therapeutic measures and pressurizing the stressed medical infrastructures. The bacterial efflux pumps enable the development of resistance against recently approved drugs such as bedaquiline and clofazimine. Consequently, the MmpS5-MmpL5 protein system was selected because of its role in efflux pumping of anti-TB drugs. The MmpS5-MmpL5 systems of Mycobacterium smegmatis were modelled and the virtual screening was performed using an ASINEX library of 5968 anti-bacterial compounds. The inhibitors with the highest binding affinities and QSAR based highest predicted inhibitory concentration were selected. The MmpS5-MmpL5 associated systems with BDE_26593610 and BDD_27860195 showed highest inhibitory parameters. These were subjected to 100 ns Molecular Dynamics simulations and provided the validation regarding the interaction studies. The in vitro studies demonstrated that the BDE_26593610 and BDD_27860195 can be considered as active inhibitors for M. smegmatis MmpS5-MmpL5. The outcomes of this study can be utilized in other experimentation aimed at drug design and discovery against the drug resistance strains of M. tuberculosis.

Synthesis and Characterization of Novel 2-Acyl-3-trifluoromethylquinoxaline 1,4-Dioxides as Potential Antimicrobial Agents.

The emergence of drug resistance in pathogens leads to a loss of effectiveness of antimicrobials and complicates the treatment of bacterial infections. Quinoxaline 1,4-dioxides represent a prospective scaffold for search of new compounds with improved chemotherapeutic characteristics. Novel 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides with alteration of substituents at position 2 and 6 were synthesized via nucleophilic substitution with piperazine moiety and evaluated against a broad panel of bacteria and fungi by measuring their minimal inhibitory concentrations. Their mode of action was assessed by whole-genomic sequencing of spontaneous drug-resistant Mycobacterium smegmatis mutants, followed by comparative genomic analysis, and on an original pDualrep2 system. Most of the 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides showed high antibacterial properties against Gram-positive strains, including mycobacteria, and the introduction of a halogen atom in the position 6 of the quinoxaline ring further increased their activity, with 13c being the most active compound. The mode of action studies confirmed the DNA-damaging nature of the obtained quinoxaline 1,4-dioxides, while drug-resistance may be provided by mutations in redox homeostasis genes, encoding enzymes potentially involved in the activation of the compounds. This study extends views about the antimicrobial and antifungal activities of the quinoxaline 1,4-dioxides and can potentially lead to the discovery of new antibacterial drugs.

Biomarkers and Utility of the Antioxidant Potential of Probiotic Lactobacilli and Bifidobacteria as Representatives of the Human Gut Microbiota.

Lactobacilli and bifidobacteria are an important part of human gut microbiota. Among numerous benefits, their antioxidant properties are attracting more and more attention. Multiple in vivo and in vitro studies have demonstrated that lactobacilli and bifidobacteria, along with their cellular components, possess excellent antioxidant capacity, which provides a certain degree of protection to the human body against diseases associated with oxidative stress. Recently, lactobacilli and bifidobacteria have begun to be considered as a new source of natural antioxidants. This review summarizes the current state of research on various antioxidant properties of lactobacilli and bifidobacteria. Special emphasis is given to the mechanisms of antioxidant activity of these bacteria in the human gut microbiota, which involve bacterial cell components and metabolites. This review is also dedicated to the genes involved in the antioxidant properties of lactobacilli and bifidobacteria strains as indicators of their antioxidant potential in human gut microbiota. Identification of the antioxidant biomarkers of the gut microbiota is of great importance both for creating diagnostic systems for assessing oxidative stress and for choosing strategies aimed at restoring the normal functioning of the microbiota and, through it, restoring human health. In this review, the practical application of probiotic strains with proven antioxidant properties to prevent oxidative stress is also considered.

The Role of the PFNA Operon of Bifidobacteria in the Recognition of Host's Immune Signals: Prospects for the Use of the FN3 Protein in the Treatment of COVID-19.

Bifidobacteria are some of the major agents that shaped the immune system of many members of the animal kingdom during their evolution. Over recent years, the question of concrete mechanisms underlying the immunomodulatory properties of bifidobacteria has been addressed in both animal and human studies. A possible candidate for this role has been discovered recently. The PFNA cluster, consisting of five core genes, pkb2, fn3, aaa-atp, duf58, tgm, has been found in all gut-dwelling autochthonous bifidobacterial species of humans. The sensory region of the species-specific serine-threonine protein kinase (PKB2), the transmembrane region of the microbial transglutaminase (TGM), and the type-III fibronectin domain-containing protein (FN3) encoded by the I gene imply that the PFNA cluster might be implicated in the interaction between bacteria and the host immune system. Moreover, the FN3 protein encoded by one of the genes making up the PFNA cluster, contains domains and motifs of cytokine receptors capable of selectively binding TNF-α. The PFNA cluster could play an important role for sensing signals of the immune system. Among the practical implications of this finding is the creation of anti-inflammatory drugs aimed at alleviating cytokine storms, one of the dire consequences resulting from SARS-CoV-2 infection.

Transcriptomic Profile of Mycobacterium smegmatis in Response to an Imidazo[1,2-b][1,2,4,5]tetrazine Reveals Its Possible Impact on Iron Metabolism.

Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex bacteria, is one of the most pressing health problems. The development of new drugs and new therapeutic regimens effective against the pathogen is one of the greatest challenges in the way of tuberculosis control. Imidazo[1,2-b][1,2,4,5]tetrazines have shown promising activity against M. tuberculosis and M. smegmatis strains. Mutations in MSMEG_1380 lead to mmpS5-mmpL5 operon overexpression, which provides M. smegmatis with efflux-mediated resistance to imidazo[1,2-b][1,2,4,5]tetrazines, but the exact mechanism of action of these compounds remains unknown. To assess the mode of action of imidazo[1,2-b][1,2,4,5]tetrazines, we analyzed the transcriptomic response of M. smegmatis to three different concentrations of 3a compound: 1/8×, 1/4×, and 1/2× MIC. Six groups of genes responsible for siderophore synthesis and transport were upregulated in a dose-dependent manner, while virtual docking revealed proteins involved in siderophore synthesis as possible targets for 3a. Thus, we suggest that imidazo[1,2-b][1,2,4,5]tetrazines may affect mycobacterial iron metabolism.

Identification of Mutations Conferring Tryptanthrin Resistance to Mycobacterium smegmatis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a global burden, responsible for over 1 million deaths annually. The emergence and spread of drug-resistant M. tuberculosis strains (MDR-, XDR- and TDR-TB) is the main challenge in global TB-control, requiring the development of novel drugs acting on new biotargets, thus able to overcome the drug-resistance. Tryptanthrin is a natural alkaloid, with great therapeutic potential due to its simple way of synthesis and wide spectrum of biological activities including high bactericidal activity on both drug-susceptible and MDR M. tuberculosis strains. InhA was suggested as the target of tryptanthrins by in silico modeling, making it a promising alternative to isoniazid, able to overcome drug resistance provided by katG mutations. However, neither the mechanism of action of tryptanthrin nor the mechanism of resistance to tryptanthrins was ever confirmed in vitro. We show that the MmpS5-MmpL5 efflux system is able to provide resistance to tryptanthrins using an in-house test-system. Comparative genomic analysis of spontaneous tryptanthrin-resistant M. smegmatis mutants showed that mutations in MSMEG_1963 (EmbR transcriptional regulator) lead to a high-level resistance, while those in MSMEG_5597 (TetR transcriptional regulator) to a low-level one. Mutations in an MFS transporter gene (MSMEG_4427) were also observed, which might be involved in providing a basal level of tryptanthrins-resistance.

Bacterial Metabolites of Human Gut Microbiota Correlating with Depression.

Depression is a global threat to mental health that affects around 264 million people worldwide. Despite the considerable evolution in our understanding of the pathophysiology of depression, no reliable biomarkers that have contributed to objective diagnoses and clinical therapy currently exist. The discovery of the microbiota-gut-brain axis induced scientists to study the role of gut microbiota (GM) in the pathogenesis of depression. Over the last decade, many of studies were conducted in this field. The productions of metabolites and compounds with neuroactive and immunomodulatory properties among mechanisms such as the mediating effects of the GM on the brain, have been identified. This comprehensive review was focused on low molecular weight compounds implicated in depression as potential products of the GM. The other possible mechanisms of GM involvement in depression were presented, as well as changes in the composition of the microbiota of patients with depression. In conclusion, the therapeutic potential of functional foods and psychobiotics in relieving depression were considered. The described biomarkers associated with GM could potentially enhance the diagnostic criteria for depressive disorders in clinical practice and represent a potential future diagnostic tool based on metagenomic technologies for assessing the development of depressive disorders.

FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3, which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro. We cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain B. longum subsp. longum GT15. After cloning the fragment into the expression vector pET16b and expressing it in E. coli, the protein product was purified to a homogenous state for further analysis. Using the immunoferment method, we tested the purified fragment's ability to bind the following human cytokines: IL-1ß, IL-6, IL-10, TNFα. We developed a sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. We found that the purified protein fragment only binds to TNFα.

Transcriptomic dataset of Mycolicibacterium smegmatis exposed to an imidazo[1,2-b][1,2,4,5]tetrazine.

Deciphering the mechanism of action of novel anti-tuberculosis compounds is a key step in the drug development process. We have previously described a number of imidazo[1,2-b][1,2,4,5]tetrazines with a promising activity on Mycobacterium tuberculosis[1]. These compounds had predicted activity as serine­threonine protein kinase inhibitors, however spontaneous drug resistant Mycolicibacterium smegmatis mc 2 155 (formerly Mycobacterium smegmatis) revealed only the mycobacterial mechanism of resistance to imidazo[1,2-b][1,2,4,5]tetrazines: mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis, thus providing resistance to imidazo[1,2-b][1,2,4,5]tetrazines via enhanced efflux [2]. Here we report the RNA sequencing data of M. smegmatis mc 2  155 culture treated with one of the imidazo[1,2-b][1,2,4,5]tetrazines for 1.5 h and the untreated culture as a control. The mapped reads showed that a total of 1386 genes are differentially expressed in this experiment. A further analysis of these data can shed light of the mechanism of action of imidazo[1,2-b][1,2,4,5]tetrazines. The data generated by RNA-seq (raw reads) have been deposited to NCBI sequence read archive (SRA) and have been assigned a BioProject accession number PRJNA615922.

Toxin-Antitoxin Systems: A Tool for Taxonomic Analysis of Human Intestinal Microbiota.

The human gastrointestinal microbiota (HGM) is known for its rich diversity of bacterial species and strains. Yet many studies stop at characterizing the HGM at the family level. This is mainly due to lack of adequate methods for a high-resolution profiling of the HGM. One way to characterize the strain diversity of the HGM is to look for strain-specific functional markers. Here, we propose using type II toxin-antitoxin systems (TAS). To identify TAS systems in the HGM, we previously developed the software TAGMA. This software was designed to detect the TAS systems, MazEF and RelBE, in lactobacilli and bifidobacteria. In this study, we updated the gene catalog created previously and used it to test our software anew on 1346 strains of bacteria, which belonged to 489 species and 49 genera. We also sequenced the genomes of 20 fecal samples and analyzed the results with TAGMA. Although some differences were detected at the strain level, the results showed no particular difference in the bacterial species between our method and other classic analysis software. These results support the use of the updated catalog of genes encoding type II TAS as a useful tool for computer-assisted species and strain characterization of the HGM.