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Heliyon Mar 2023For decades, transcription of -operon was considered being constitutive. Therefore, this -operon has been used for measurements in non-specific bacterial luminescent...
For decades, transcription of -operon was considered being constitutive. Therefore, this -operon has been used for measurements in non-specific bacterial luminescent biosensors. Here, the expression of -operon under high temperature was studied. The expression was researched in the natural strain and in the heterologous system of . FV2201 bacterium was isolated from soil in the Moscow region (growth optimum 28 °C). We showed that its luminescence significantly increases when the temperature rises to 34 °C. The increase in luminescence is associated with an increase in the transcription of genes, which was confirmed by RT-PCR. The promoter of the -operon of the related bacterium ZM1 from the forests of Moldova, being cloned in the heterologous system of , is activated when the temperature rises from room temperature to 42 °C. When heat shock is caused by ethanol addition, transcription of -operon increases only in the natural strain of , but not in the heterologous system of cells. In addition, the activation of the -operon of persists in strains deficient in both the and genes. These results indicate the presence of sigma 32 and sigma 24 independent heat-shock-like mechanism of regulation of the -operon of in the heterologous system.
PubMed: 36950606
DOI: 10.1016/j.heliyon.2023.e14527 -
PLoS Biology Nov 2020Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy....
Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.
Topics: Actins; Animals; Bacterial Toxins; Binding Sites; Binding, Competitive; Cofilin 1; Cryoelectron Microscopy; Fluorescent Dyes; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; In Vitro Techniques; Microfilament Proteins; Microscopy, Confocal; Models, Molecular; Myosins; Peptide Fragments; Protein Engineering; Protein Interaction Domains and Motifs; Rabbits; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins
PubMed: 33216759
DOI: 10.1371/journal.pbio.3000925 -
The Journal of Biological Chemistry Apr 2017Members of the gammaproteobacterial genus share mutualistic relationships with nematodes, and the pairs infect a wide swath of insect larvae. species produce a family...
Members of the gammaproteobacterial genus share mutualistic relationships with nematodes, and the pairs infect a wide swath of insect larvae. species produce a family of stilbenes, with two major components being 3,5-dihydroxy-4-isopropyl--stilbene (compound 1) and its stilbene epoxide (compound 2). This family of molecules harbors antimicrobial and immunosuppressive activities, and its pathway is responsible for producing a nematode "food signal" involved in nematode development. However, stilbene epoxidation biosynthesis and its biological roles remain unknown. Here, we identified an orphan protein (Plu2236) from that catalyzes stilbene epoxidation. Structural, mutational, and biochemical analyses confirmed the enzyme adopts a fold common to FAD-dependent monooxygenases, contains a tightly bound FAD prosthetic group, and is required for the stereoselective epoxidation of compounds 1 and 2. The epoxidase gene was dispensable in a nematode-infective juvenile recovery assay, indicating the oxidized compound is not required for the food signal. The epoxide exhibited reduced cytotoxicity toward its producer, suggesting this may be a natural route for intracellular detoxification. In an insect infection model, we also observed two stilbene-derived metabolites that were dependent on the epoxidase. NMR, computational, and chemical degradation studies established their structures as new stilbene-l-proline conjugates, prolbenes A (compound 3) and B (compound 4). The prolbenes lacked immunosuppressive and antimicrobial activities compared with their stilbene substrates, suggesting a metabolite attenuation mechanism in the animal model. Collectively, our studies provide a structural view for stereoselective stilbene epoxidation and functionalization in an invertebrate animal infection model and provide new insights into stilbene cellular detoxification.
Topics: Animals; Anti-Infective Agents; Biological Products; Catalysis; Chromatography, High Pressure Liquid; Crystallography, X-Ray; DNA Mutational Analysis; Epoxy Compounds; Gene Deletion; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Immunosuppressive Agents; Magnetic Resonance Spectroscopy; Molecular Conformation; Mutation; Photorhabdus; Protein Folding; Rhabditoidea; Stereoisomerism; Stilbenes; Symbiosis
PubMed: 28246174
DOI: 10.1074/jbc.M116.762542 -
MBio Jun 2022With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups...
With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured , a betaproteobacterium; odilorhabdins, from , are broad-spectrum inhibitors of protein synthesis, and darobactins from target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. and are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to and , kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on , gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express . To gain access to silent operons, we first fractionated supernatant from and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.
Topics: Animals; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Biological Products; Deoxyguanosine; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Guanosine; Guanosine Triphosphate; Nematoda; Operon; Photorhabdus; Prodrugs; Xenorhabdus
PubMed: 35575547
DOI: 10.1128/mbio.00700-22 -
Pathogens (Basel, Switzerland) Aug 2023Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the...
Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the transmission of infectious diseases such as West Nile disease and Lymphatic Filariasis. Synthetic insecticides have been the prime control method for many years to suppress populations. However, recently, the use of insecticides has begun to be questioned due to the detrimental impact on human health and the natural environment. Therefore, many authorities urge the development of eco-friendly control methods that are nontoxic to humans. The bacterial associates [ and spp. (Enterobacterales: Morganellaceae)] of entomopathogenic nematodes (EPNs) ( spp. and spp.) (Rhabditida: Heterorhabditidae and Steinernematidae) are one of the green approaches to combat a variety of insect pests. In the present study, the mosquitocidal activity of the cell-free supernatants and cell suspension (4 × 10 cells mL) of four different symbiotic bacteria (, , , and subsp. ) was assessed against different development stages of (The 1st/2nd and 3rd/4th instar larvae and pupa) under laboratory conditions. The bacterial symbionts were able to kill all the development stages with varying levels of mortality. The 1st/2nd instar larvae exhibited the highest susceptibility to the cell-free supernatants and cell suspensions of symbiotic bacteria and the efficacy of the cell-free supernatants and cell suspensions gradually declined with increasing phases of growth. The highest effectiveness was achieved by the KCS-4S strain inducing 95% mortality to the 1st/2nd instar larvae. The results indicate that tested bacterial symbionts have great potential as an eco-friendly alternative to insecticides.
PubMed: 37764903
DOI: 10.3390/pathogens12091095 -
Beilstein Journal of Organic Chemistry 2020The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on...
The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera or . In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected and mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine () biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class.
PubMed: 32461774
DOI: 10.3762/bjoc.16.84 -
Pathogens (Basel, Switzerland) Mar 2023Symbiotic bacteria form a mutualistic relationship with nematodes and are pathogenic to many insect pests. They kill insects using various strategies to evade or...
Symbiotic bacteria form a mutualistic relationship with nematodes and are pathogenic to many insect pests. They kill insects using various strategies to evade or suppress their humoral and cellular immunity. Here we evaluate the toxic effects of these bacteria and their secondary metabolites on the survival and phenoloxidase (PO) activation of larvae using biochemical and molecular methods. The results show H06 and All treatments caused significant reductions in the number of larvae in a dose-dependent manner. Secondly, the immune system recognizes symbiotic bacteria at early and late stages of infection via the induction of C-type lectin. Live symbiotic bacteria significantly inhibit PO activity in whereas heat-treated bacteria strongly increase PO activity. Additionally, expression levels of four proPhenoloxidase genes following treatment with H06 and All were compared. We found that the expression levels of all proPhenoloxidase genes were significantly down-regulated at all-time points. Similarly, treatments of larvae with metabolites benzylideneacetone and oxindole significantly down-regulated the expression of the PPO gene and inhibited PO activity. However, the addition of arachidonic acid to metabolite-treated larvae restored the expression level of the PPO gene and increased PO activity. Our results provide new insight into the roles of symbiotic bacteria in countering the insect phenoloxidase activation system.
PubMed: 37111392
DOI: 10.3390/pathogens12040506 -
AMB Express Aug 2021Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics,...
Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics, yet several aspects of azasugars remain unknown including their distribution, structural diversity, and chemical ecology. The hallmark signature of bacterial azasugar biosynthesis is a three gene cluster (3GC) coding for aminotransferase, phosphatase, and dehydrogenase enzymes. Using the bioinformatics platform Enzyme Similarity Tool (EST), we identified hundreds of putative three gene clusters coding for azasugar production in microbial species. In the course of this work, we also report a consensus sequence for the aminotransferase involved in azasugar biosynthesis as being: SGNXFRXXXFPNXXXXXXXLXVPXPYCXRC. Most clusters are found in Bacillus and Streptomyces species which typically inhabit soil and the rhizosphere, but some clusters are found with diverse species representation such as Photorhabdus and Xenorhabdus which are symbiotic with entomopathogenic nematodes; the human skin commensal Cutibacterium acnes, and the marine Bacillus rugosus SPB7, a symbiont to the sea sponge Spongia officinalis. This pan-taxonomic survey of the azasugar 3GC signature may lead to the identification of new azasugar producers, facilitate studies of their natural functions, and lead to new potential therapeutics.
PubMed: 34424396
DOI: 10.1186/s13568-021-01279-5 -
Molekuliarnaia Biologiia 2018The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past... (Review)
Review
The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past decades. Here we discuss the mechanisms of bacterial bioluminescence. We have the main results from structural and functional analysis of the genes of lux operons, enzymes (luciferase), and mechanisms of bioluminescence in several species of marine bacteria, which belong to three genera, Vibrio, Aliivibrio, and Photobacterium (A. fischeri, V. harveyi, P. leiognathi, and P. phosphoreum), and in terrestrial bacteria of the genus Photorhabdus (Ph. luminescens). The structure and mechanisms for the regulation of the expression of the lux operons are discussed. The fundamental characteristics of luciferase and luciferase-catalyzed reactions (stages of FMNH2 and tetradecanal oxidation, dimensional structure, as well as folding and refolding of the macromolecule) are described. We also discuss the main concepts of the origin of bacterial bioluminescence and its role in the ecology of modern marine fauna, including its involvement in the processes of detoxification of the reactive oxygen species and DNA repair, as well as the bait hypothesis.
Topics: Aliivibrio; Bacterial Proteins; DNA, Bacterial; Genes, Bacterial; Luciferases; Luminescence; Operon; Photobacterium; Vibrio
PubMed: 30633237
DOI: 10.1134/S0026898418060186 -
Frontiers in Microbiology 2022Phylum Nematoda is of great economic importance. It has been a focused area for various research activities in distinct domains across the globe. Among nematodes, there... (Review)
Review
Phylum Nematoda is of great economic importance. It has been a focused area for various research activities in distinct domains across the globe. Among nematodes, there is a group called entomopathogenic nematodes, which has two families that live in symbiotic association with bacteria of genus and , respectively. With the passing years, researchers have isolated a wide array of bioactive compounds from these symbiotically associated nematodes. In this article, we are encapsulating bioactive compounds isolated from members of the family Heterorhabditidae inhabiting in its gut. Isolated bioactive compounds have shown a wide range of biological activity against deadly pathogens to both plants as well as animals. Some compounds exhibit lethal effects against fungi, bacteria, protozoan, insects, cancerous cell lines, neuroinflammation, etc., with great potency. The main aim of this article is to collect and analyze the importance of nematode and its associated bacteria, isolated secondary metabolites, and their biomedical potential, which can serve as potential leads for further drug discovery.
PubMed: 35422783
DOI: 10.3389/fmicb.2022.790339