IntroductionMetaviromic sequencing is prone to artifacts, necessitating orthogonal validation. This study evaluates a targeted proteogenomic approach for verifying viral presence initially detected by NGS. MethodsWe analyzed oropharyngeal/anal swabs and feces from hedgehogs, where NGS indicated viruses from the Coronaviridae, Picornaviridae, Flaviviridae, and Astroviridae families. For verification, mass spectrometry was applied using sample-specific databases derived from NGS-assembled contigs. ResultsProteomic analysis confirmed Coronaviridae presence in NGS-positive animals, primarily detecting structural virion proteins in anal and fecal samples. However, proteomic evidence for Picornaviridae, Flaviviridae, and Astroviridae was not obtained despite their genomic detection. DiscussionPhylogenetic analysis of two complete Picornaviridae genomes assembled from the NGS data – one closely related to a UK rodent virus and another distantly related to Chinese bat viruses – supports the authenticity of the NGS findings. The results demonstrate that this two-stage pipeline provides critical orthogonal validation for metaviromic studies but also defines a practical sensitivity limit for the proteomic verification of some viral families. Combining NGS with proteomics significantly enhances the reliability of viral confirmation in complex samples, thereby strengthening ecological and zoonotic inferences.

A clinical strain of the opportunistic pathogen Serratia rubidaea, a known contaminant of healthcare environments and an emerging cause of invasive infections, is described. The studied isolate, recovered from a nurse’s hand skin swab during routine screening, exhibits a broad profile of antibiotic resistance combined with reduced susceptibility to several disinfectants. Phenotypic susceptibility testing using a tablet-based microdilution and disk diffusion method was employed to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents from different classes, while broth microdilution assays with disinfectants revealed high-level tolerance to widely used agents, including 70% C2H5OH, 3% H2O2, 0.05% polyhexamethylene guanidine (PHMG) and others. Whole-genome sequencing identified multiple resistance-associated determinants, such as chromosome-encoded class C β-lactamase (ampC), several efflux systems (sdeXY, macAB, and emrAB) combined with multicopy tolC, and specific transferases (fos and arnT). Shotgun bottom-up HPLC-MS/MS proteomics confirmed baseline expression of these and other stress-tolerance-related proteins under non-inducing conditions. Taken together, these data underscore the importance of surveillance for Serratia spp. in healthcare facilities to detect strains that combine intrinsic or acquired multidrug resistance with robust survival traits such as disinfectant tolerance and biofilm formation. The present study provides a reference-level phenotypic, genomic, and proteomic characterization of a S. rubidaea clinical isolate, contributing to the understanding of the adaptive potential of this resilient opportunistic pathogen in clinical environments.

IntroductionHow living cells regulate their function and adapt to stress remains a fundamental question in cell biology. In bacteria, stress responses are classically described at the genomic and transcriptomic levels; however, this paradigm does not adequately explain adaptation in mycoplasmas, which are widely regarded as a model of a minimal cell.MethodsThis study investigated stress adaptation in mycoplasmas by analyzing cellular volume and morphology changes by SEM, DLS, and EPR. Membrane lipid composition was analyzed by mass spectrometry. Intracellular ATP levels were measured using a luciferase‑based assay. Vesicles were isolated by ultracentrifugation and characterized by proteomics, enzyme activity assays, and ATP measurements. Glycolytic flux was assessed in vitro using recombinant enzymes and liposome encapsulation.ResultsStress adaptation in this minimal cell is primarily achieved through a reduction in cellular volume, mediated by the release of the attachment organelle (“tip”) in the form of vesicles. This process triggers metabolic reorganization within the cell and results in the accumulation of ATP. The metabolic adaptation involves changes in the stoichiometric balance of glycolytic enzymes and alterations in local metabolite concentrations under conditions of reduced cell volume, both of which directly influence glycolytic flux. Concurrently, the released vesicles contain VlhA, lipoproteins, adhesins, hydrogen-peroxide-secreting factors, and exhibit peptidase activity.DiscussionThese findings reveal a novel regulatory mechanism in a minimal cell, where physical volume reduction and biochemical adjustments may be related to an energy-efficient stress response. The dual role of vesicle formation—in cellular remodeling and pathogenesis—deepens our understanding of mycoplasma biology and offers insights for synthetic cell design.

IntroductionBacteroides fragilis (Bf)’s T6SS locus has been characterized and shown to have functional activity in competition experiments. It has been demonstrated that symbiont nontoxigenic Bf strains have a more effective “weapon” effect on pathogenic Bf, which is realized through the activity of effector-immune (E-I) protein pairs. Intensive study of the T6SS structure has led to an understanding of certain issues related to its functional activity, but the exact regulatory mechanisms of E-I protein pair activity remain unclear. Proteomic annotation of T6SS components and detailed descriptions of all immune-effector pairs are currently available. In this research, we performed detailed proteogenomic analysis and subsequent proteomic annotation of the T6SS components of the toxigenic Bf BOB25.Material and methodsFractionated cells, cultivated media and vesicles were prepared for proteome analysis by HPLC-MS/MS. Proteogenomic annotation and comparative genomic study of the T6SS loci of the toxigenic Bf BOB25 were carried out by comparison with the reference genomes of the following Bf strains: JIM10, NCTC 9343 and 638R.ResultsAccording to the data obtained, T6SS components were represented in all types of the analysed samples. The following components of the T6SS were identified in culture media and cells: ClpV (TssH), TssK, TssC, TssB, Hcp (TssD), and TetR. The predicted effector protein AKA51715.1 (VU15_08315) was also detected in media. The greatest amount of T6SS proteins, including the Hcp protein, was detected in the vesicle samples, which was also observed by TEM. Potential effectors, including AKA51715.1 (VU15_08315), AKA51716.1 (VU15_08320), AKA51728.1 (VU15_08385) and the immune protein AKA51727.1 (VU15_08380), were detected in vesicles.DiscussionThe presence of the immune and effector proteins in the Bf secretome indicates the high activity of the T6SS without bacterial competition. It is possible that the T6SS is also used by bacteria to regulate population size by altering the activity of different repertoires of E-I pairs.

The COVID-19 pandemic was the most dramatic in the newest history with nearly 7 million deaths and global impact on mankind. Here, we report binding index of 305 human leukocyte antigen (HLA) class I molecules from 18,771 unique haplotypes of 28,104 individuals to 821 peptides experimentally observed from spike protein receptor binding domain (RBD) of five main SARS-CoV-2 strains hydrolyzed by human proteasomes with constitutive and immune catalytic phenotypes. Our data read that mutations in the human angiotensin-converting enzyme 2 (hACE2)-binding region RBD496-513 of Omicron B.1.1.529 strain results in a dramatic increase of proteasome-mediated release of two public HLA class I epitopes. Global population analysis of HLA class I haplotypes, specific to these peptides, demonstrated decreased mortality of human populations enriched in these haplotypes from COVID-19 after but not before December, 2021, when Omicron became dominant SARS-CoV-2 strain. Noteworthy, currently circulating BA.2.86 and JN.1 strains contain same amino acid substitutions at key proteasomal cleavage sites, thus preserving identified core epitopes.

IntroductionBats are considered the natural reservoirs of several viruses including coronaviruses (CoVs), two genera of which, alpha- and betacoronaviruses, infect humans. Despite widespread screening of bat samples for novel viruses, information on the diversity of coronaviruses in bats inhabiting Russian territory remains scarce. Here, we analyzed the presence and diversity of alphacoronaviruses (Alpha-CoVs) in bats from European Russia.MethodsFourty three fecal samples from bats of 8 species: P. nathusii, P. kuhlii, M. brandtii, M. daubentonii, N. noctula, V. murinus, M. dasycneme, and P. auritus were taken to study.Results and discussionWe detected Alpha-CoV RdRp gene fragments in 30% (13/43) of samples examined in 75% (6/8) of species sampled. Phylogenetic analysis of RdRp showed that most of the identified Alpha-CoV sequences fall into clades within the Pedacovirus subgenus, with minor clusters of nyctacoviruses or myotacoviruses. We assert that closely related pedacoviruses have been circulating for a long time (from 2015 to 2021) in a large region from European Russia to Northern Europe. We propose that closely related pedacoviruses collected from common areas represent a separate species, which we name NE-Alpha coronavirus, with its host being bats of the genus Pipistrellus that inhabit a region from the European part of Russia to Northern Europe. Among the animals sampled, 4.6% (2/43) carried two Alpha-CoVs related to different subgenera (pedacovirus/myotacovirus or pedacovirus/nyctacovirus) simultaneously. We confirmed the presence of two different Alpha-CoV subgenera related to pedacovirus and nyctacovirus in P. kuhlii captured in 2021 and kept in captivity using whole genome sequencing of these viruses. The presence of two or more coronaviruses in one individual animal host is an essential prerequisite for recombination to occur. We also obtained two Alpha-CoV whole genomes from two specimens of P. nathusii captured in 2015. The genomic organization of BatCoV/MOW15-21 and BatCoV/MOW15-23 was similar to other Alpha-CoVs, but the assembled genomes contained a long insertion in the ORF1ab gene which has not been described in other Alpha-CoVs, except for a single sequence from P. nathusii captured in the Netherlands. We propose that the insertion encodes a previously undescribed domain of unknown function, probably related to the SEA domain superfamily.

Abstract Background The previously underestimated effects of commensal gut microbiota on the human body are increasingly being investigated using omics. The discovery of active molecules of interaction between the microbiota and the host may be an important step towards elucidating the mechanisms of symbiosis. Results Here, we show that in the bloodstream of healthy people, there are over 900 peptides that are fragments of proteins from microorganisms which naturally inhabit human biotopes, including the intestinal microbiota. Absolute quantitation by multiple reaction monitoring has confirmed the presence of bacterial peptides in the blood plasma and serum in the range of approximately 0.1 nM to 1 μM. The abundance of microbiota peptides reaches its maximum about 5 h after a meal. Most of the peptides correlate with the bacterial composition of the small intestine and are likely obtained by hydrolysis of membrane proteins with trypsin, chymotrypsin and pepsin – the main proteases of the gastrointestinal tract. The peptides have physicochemical properties that likely allow them to selectively pass the intestinal mucosal barrier and resist fibrinolysis. Conclusions The proposed approach to the identification of microbiota peptides in the blood, after additional validation, may be useful for determining the microbiota composition of hard-to-reach intestinal areas and monitoring the permeability of the intestinal mucosal barrier.

Agricultural and industrial activities are increasing pollution of water bodies with low doses of xenobiotics that have detrimental effects on aquaculture. The aim of this work was to determine the possibility of using Levilactobacillus brevis 47f culture in fish aquaculture under the influence of low doses of xenobiotics as an adaptogen. An increase in the survival of Danio rerio individuals exposed to the xenobiotic bisphenol A solution and fed with the L. brevis 47f was shown compared to control groups and, at the same time, the cytokine profile in the intestinal tissues of Danio rerio was also investigated. Analysis of differential gene expression of the L. brevis 47f grown under the action of high concentrations of bisphenol A showed changes in mRNA levels of a number of genes, including genes of various transport proteins, genes involved in fatty acid synthesis, genes of transcriptional regulators, genes of the arabinose operon, and the oppA gene. The identification of L. brevis 47f proteins from polyacrylamide gel by mass spectrometry revealed L-arabinose isomerase, Clp chaperone subunit, ATP synthase subunits, pentose phosphate pathway and glycolysis enzyme proteins, which are likely part of the L. brevis 47f strain’s anti-stress response, but probably do not affect its adaptogenic activity toward Danio rerio.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance. Ovarian cancers frequently develop resistance to therapy. Here, using transcriptomics, proteomics, and preclinical models to analyse paired ascitic fluids before and after chemotherapy in ovarian cancer patients, the authors discover that extracellular secretion and spliceosomal components contribute to therapy resistance, enhancing the DNA damage response in recipient cancer cells.

Motivation: The Oxford Nanopore technology has a great potential for the analysis of methylated motifs in genomes, including whole-genome methylome profiling. However, we found that there are no methylation motifs detection algorithms, which would be sensitive enough and return deterministic results. Thus, the MEME suit does not extract all Helicobacter pylori methylation sites de novo even using the iterative approach implemented in the most up-to-date methylation analysis tool Nanodisco. Results: We present Snapper, a new highly sensitive approach, to extract methylation motif sequences based on a greedy motif selection algorithm. Snapper does not require manual control during the enrichment process and has enrichment sensitivity higher than MEME coupled with Tombo or Nanodisco instruments that was demonstrated on H.pylori strain J99 studied earlier by the PacBio technology and on four external datasets representing different bacterial species. We used Snapper to characterize the total methylome of a new H.pylori strain A45. At least four methylation sites that have not been described for H.pylori earlier were revealed. We experimentally confirmed the presence of a new CCAG-specific methyltransferase and inferred a gene encoding a new CCAAK-specific methyltransferase. Availability and implementation: Snapper is implemented using Python and is freely available as a pip package named "snapper-ont." Also, Snapper and the demo dataset are available in Zenodo (10.5281/zenodo.10117651).

Introduction.Mycoplasma hominis is a bacterium belonging to the class Mollicutes. It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517-622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection.Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division.Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms.Methodology. LC-MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer.Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC-MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis.Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms' ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA's action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected-due to their notable expression differences-for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and β (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes; BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.

Human multiforme glioblastoma is characterized by an unfavorable prognosis, low survival rate and extremely limited possibilities for therapy. Rat C6 glioma is an experimental model for the study of glioblastoma growth and invasion. It has been shown that the growth and development of the tumor is accompanied by changes in the surrounding normotypic tissues [1]. These changes create a favorable environment for the development of the tumor and give it an evolutionary advantage [2]. Description of changes occurring in normotypic cells of the body upon their contact with tumor cells is of great interest. We have grown C6 glioma cells and rat astrocytes, as well as astrocyte cells co-cultured together with C6 glioma. We performed proteome-wide LC-MS analysis of these experimental groups. The data includes LC-MS/MS raw files and exported MaxQuant and ProteinPilot search results with fasta. Dataset published in the PRIDE repository project accession PXD026776.

Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is a bioactive molecule synthesized by various phytoplankton species, such as cyanobacteria, diatoms and dinoflagellates, and is known to be a causative agent of human neurodegeneration diseases. The ability of different microalgae to synthesize BMAA may be an indicator of the importance of this molecule in the interaction of phytoplankton organisms in nature. We were interested in the question: what kinds of mechanisms underline BMAA’s action on cyanobacterial cells under different nitrogen supply conditions. To answer this question we have performed molecular studies using a model cyanobacterial strain Nostoc (Anabaena) sp. PCC 7120. We have experimentally shown that the action of BMAA on nitrogen-fixing filamentous cyanobacteria changes nitrogen-carbon balance regulation, and differs under nitrogen starvation and in nitrogen-replete conditions. The primary main targets of BMAA’s action in cyanobacteria cells are, apparently, metabolic processes, such as nitrogen fixation, photosynthesis, carbon fixation and different biosynthetic processes, the regulation of which involves 2-oxyglutarate and glutamate. Our proteomic study has demonstrated that under BMAA-treatment the most significant difference lies in the expression change of a key nitrogen regulatory protein PII. This protein is downregulated in nitrogen-starving conditions and it is upregulated in nitrogen-replete conditions in the presence of BMAA. This could be the main reason behind a specific regulatory effect on heterocyst formation and heterocyst- and nitrogenase-related gene expression that this amino acid causes in Nostoc sp. PCC 7120. Due to the fact that all metabolic processes are interconnected and well balanced in cyanobacteria cells, the disturbance in nitrogen metabolism leads to changes in carbon metabolism and photosynthesis. This explains the severe changes of CO2 fixation proteins and photosystem reaction centre proteins that were found in our proteomics studies. BMAA addition leads to disorder in both amino acid synthesis and in purine synthesis, as well as disturbs DNA transcription and protein translation. Finally, many enzymes of oxidative stress, chaperones and SOS-response proteins are upregulated under such metabolic stress conditions. Therefore we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as: starvation-inducible DNA-binding protein, stress-response enzymes, proteases and SOS-response and DNA repair enzymes. It can be hypothesized that BMAA could be used by phytoplankton representatives (cyanobacteria, diatom, dinoflagelates) as a possible allelopatic tool to control cyanobacteria cell populations during their competition for nitrogen and other resources.

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. We studied the mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila . In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. We found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction.

Symbiotic interaction between the human body and its microbiota is an important issue of modern biomedicine and personalized medicine. However, little is known on molecular mechanisms of that relationship. Bearing in mind the ubiquitous participation of peptides in biomolecular interactions and regulatory processes we attempted direct search of blood peptides originated from microbial proteins. LC-MS/MS analysis was carried out of blood serum and plasma samples taken from 20 healthy donors on Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass-spectrometer. Sample preparation was carried out based on our previously developed method of peptide desorption from the surface of major blood plasma proteins followed by standard chromatographic steps. Mascot and X! Tandem search engines were used for peptide identification. Human protein sequences were taken from UniProt Knowledgebase and sequences of human microbiota proteins – from NIH Human Microbiome Project (HMP). As a result, out of 13625 identified peptides 912 were unique fragments of microbial precursors, which is 6.69% of the total amount of detected bloodstream peptides. In 30 cases peptide identification was confirmed by mass-spectral study of individual synthetic samples. Absolute quantification by the mass-spectrometric method of multiple reaction monitoring (MRM) confirmed the presence of bacterial peptides in plasma and serum in the range of approximately 0.1 nMol/L to 1 mkMol/L, which is comparable to physiologically significant hormone concentrations in human blood in normal conditions. Analysis of the in silico obtained hydrolyzates of microbiotic proteins showed that significant number of the identified peptides are derived from the precursor proteins as a result of hydrolysis with trypsin, chymotrypsin and pepsin, the main proteases of the gastrointestinal system. 60% of the identified “microbial” peptides are derived from the intestine flora, about 20% - from oral microbiota and 20% fall on the remaining microbiotic communities. Most of the precursor proteins refer to intracellular, cytoplasmic proteins. The isolated fraction of peripheral blood mononuclear cells showed increase secretion of proinflammatory cytokines, colony stimulating factors and chemoattractants as the response to the addition of some of the identified microbiotic peptides. The data obtained serve as a basis for the ongoing study of the functional properties of microbiome derived peptides.

All cyanobacteria produce a neurotoxic non-protein amino acid β-N-methylamino-L-alanine (BMAA). However, the biological function of BMAA in the regulation of cyanobacteria metabolism still remains undetermined. It is known that BMAA suppresses the formation of heterocysts in diazotrophic cyanobacteria under nitrogen starvation conditions, and BMAA induces the formation of heterocyst-like cells under nitrogen excess conditions, by causing the expression of heterocyst-specific genes that are usually “silent” under nitrogen-replete conditions, as if these bacteria receive a nitrogen deficiency intracellular molecular signal. In order to find out the molecular mechanisms underlying this unexpected BMAA effect, we studied the proteome of cyanobacterium Nostoc sp. PCC 7120 grown under BMAA treatment in nitrogen-replete medium. Experiments were performed in two experimental settings: (1) in control samples consisted of cells grown without the BMAA treatment and (2) the treated samples consisted of cells grown with addition of an aqueous solution of BMAA (20 µM). In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by LC-MS/MS spectrometry. Among them, 80 proteins belonging to different functional categories were chosen for further functional analysis and interpretation of obtained proteomic data. Here, we provide the evidence that a pleiotropic regulatory effect of BMAA on the proteome of cyanobacterium was largely different under conditions of nitrogen-excess compared to its effect under nitrogen starvation conditions (that was studied in our previous work). The most significant difference in proteome expression between the BMAA-treated and untreated samples under different growth conditions was detected in key regulatory protein PII (GlnB). BMAA downregulates protein PII in nitrogen-starved cells and upregulates this protein in nitrogen-replete conditions. PII protein is a key signal transduction protein and the change in its regulation leads to the change of many other regulatory proteins, including different transcriptional factors, enzymes and transporters. Complex changes in key metabolic and regulatory proteins (RbcL, RbcS, Rca, CmpA, GltS, NodM, thioredoxin 1, RpbD, ClpP, MinD, RecA, etc.), detected in this experimental study, could be a reason for the appearance of the “starvation” state in nitrogen-replete conditions in the presence of BMAA. In addition, 15 proteins identified in this study are encoded by genes, which are under the control of NtcA—a global transcriptional regulator—one of the main protein partners and transcriptional regulators of PII protein. Thereby, this proteomic study gives a possible explanation of cyanobacterium starvation under nitrogen-replete conditions and BMAA treatment. It allows to take a closer look at the regulation of cyanobacteria metabolism affected by this cyanotoxin.

Antimicrobial peptides (AMPs) are natural antagonistic tools of many bacteria and are considered as attractive antimicrobial agents for the treatment of bacteria with multidrug resistance. Lactic acid bacteria from the gastrointestinal tract of animals and human produce various AMPs inhibiting the growth of pathogens. Here we report the isolation and identification of novel Lactobacillus fermentum strain HF-D1 from the human gut producing AMPs which prevents the growth of P. aeruginosa and S. marcescens. The active fraction of peptides was obtained from the culture liquid by precipitation at 80% saturation of ammonium sulphate. For peptides identification, the precipitate was treated with guanidine hydrochloride to desorb from proteins, separated with ultrafiltration on spin columns with 10,000 MWCO, desalted with a reversed-phase chromatography and subjected to LC–MS/MS analysis. The in silico analysis of the identified 1111 peptides by using ADAM, CAMPR3 and AMPA prediction servers led to identification of the linear peptide with highly probable antimicrobial activity and further investigation of its antibacterial activity mechanism is promising. By using the dereplication algorithm, the peptide highly similar to non-ribosomal cyclic AMPs originally isolated from Staphylococcus epidermidis has been identified. This indicates that L. fermentum HF-D1 represents a novel strain producing antimicrobial peptides targeting P. aeruginosa and S. marcescens.