-
Bio-protocol Jun 2023Cycloheximide (CHX) is a small molecule derived from that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein...
Cycloheximide (CHX) is a small molecule derived from that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay. Graphical overview.
PubMed: 37323633
DOI: 10.21769/BioProtoc.4690 -
FEBS Open Bio Dec 2023Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl amino acids is conventionally performed by the...
Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl amino acids is conventionally performed by the Schotten-Baumann reaction using fatty acyl chlorides, but aminoacylases have also been investigated for use in biocatalytic synthesis with free fatty acids. Aminoacylases and their properties are diverse; they belong to different peptidase families and show differences in substrate specificity and biocatalytic potential. Bacterial aminoacylases capable of synthesis have been isolated from Burkholderia, Mycolicibacterium, and Streptomyces. Although several proteases and peptidases from S. griseus have been described, no aminoacylases from this species have been identified yet. In this study, we investigated two novel enzymes produced by S. griseus DSM 40236 . We identified and cloned the respective genes and recombinantly expressed an α-aminoacylase (EC3.5.1.14), designated SgAA, and an ε-lysine acylase (EC3.5.1.17), designated SgELA, in S. lividans TK23. The purified aminoacylase SgAA was biochemically characterized, focusing on its hydrolytic activity to determine temperature- and pH optima and stabilities. The aminoacylase could hydrolyze various acetyl amino acids at the N -position with a broad specificity regarding the sidechain. Substrates with longer acyl chains, like lauroyl amino acids, were hydrolyzed to a lesser extent. Purified aminoacylase SgELA specific for the hydrolysis of N -acetyl-l-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. The pH optimum of SgELA was pH 8.0. While synthesis of acyl amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine.
Topics: Humans; Streptomyces lividans; Streptomyces griseus; Streptomyces; Amino Acids; Lysine
PubMed: 37879963
DOI: 10.1002/2211-5463.13723 -
Molecules (Basel, Switzerland) Jun 2021This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by isolate KJ623766, a recovered soil...
BACKGROUND
This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines.
METHODS
Production of cytotoxic metabolites from isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against 2 and cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis.
RESULTS
Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as β- and γ-rhodomycinone with CD of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against 2, and cell lines, respectively. Values were comparable to those of the positive control doxorubicin.
CONCLUSIONS
This is the first report about the production of β- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from .
Topics: Animals; Anthracyclines; Antibiotics, Antineoplastic; Biological Products; Caco-2 Cells; Chlorocebus aethiops; HeLa Cells; Humans; Streptomyces griseus; Vero Cells
PubMed: 34209170
DOI: 10.3390/molecules26134009 -
World Journal of Microbiology &... Nov 2022Following the discovery of streptomycin from Streptomyces griseus in the 1940s by Selman Waksman and colleagues, aminoglycosides were first used to treat tuberculosis... (Review)
Review
Following the discovery of streptomycin from Streptomyces griseus in the 1940s by Selman Waksman and colleagues, aminoglycosides were first used to treat tuberculosis and then numerous derivatives have since been used to combat a wide variety of bacterial infections. These bactericidal antibiotics were used as first-line treatments for several decades but were largely replaced by ß-lactams and fluoroquinolones in the 1980s, although widespread emergence of antibiotic-resistance has led to renewed interest in aminoglycosides. The primary site of action for aminoglycosides is the 30 S ribosomal subunit where they disrupt protein translation, which contributes to widespread cellular damage through a number of secondary effects including rapid uptake of aminoglycosides via elevated proton-motive force (PMF), membrane damage and breakdown, oxidative stress, and hyperpolarisation of the membrane. Several factors associated with aminoglycoside entry have been shown to impact upon bacterial killing, and more recent work has revealed a complex relationship between metabolic states and the efficacy of different aminoglycosides. Hence, it is imperative to consider the environmental conditions and bacterial physiology and how this can impact upon aminoglycoside entry and potency. This mini-review seeks to discuss recent advances in this area and how this might affect the future use of aminoglycosides.
Topics: Aminoglycosides; Anti-Bacterial Agents; Drug Resistance, Microbial; Bacteria; Streptomyces griseus
PubMed: 36350431
DOI: 10.1007/s11274-022-03445-8 -
Frontiers in Bioengineering and... 2022Bacteria belonging to have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex...
Bacteria belonging to have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex networks activated after vegetative growth terminates. Despite considerable effort to understand the regulatory elements governing gene expression related to primary and secondary metabolism in , system-level information remains limited. In this study, we integrated four multi-omics datasets from NBRC 13350: RNA-seq, ribosome profiling, dRNA-seq, and Term-Seq, to analyze the regulatory elements of transcription and translation of differentially expressed genes during cell growth. With the functional enrichment of gene expression in different growth phases, one sigma factor regulon and four transcription factor regulons governing differential gene transcription patterns were found. In addition, the regulatory elements of transcription termination and post-transcriptional processing at transcript 3'-end positions were elucidated, including their conserved motifs, stem-loop RNA structures, and non-terminal locations within the polycistronic operons, and the potential regulatory elements of translation initiation and elongation such as 5'-UTR length, RNA structures at ribosome-bound sites, and codon usage were investigated. This comprehensive genetic information provides a foundational genetic resource for strain engineering to enhance secondary metabolite production in .
PubMed: 35284422
DOI: 10.3389/fbioe.2022.844200 -
The Journal of Biological Chemistry May 2022The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central...
The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3'',5'' double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3''-position than the 5''-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.
Topics: Antigens, Bacterial; Carbohydrate Epimerases; Coxiella burnetii; Nucleoside Diphosphate Sugars; Streptomyces griseus; Thymine Nucleotides
PubMed: 35398092
DOI: 10.1016/j.jbc.2022.101903 -
Plants (Basel, Switzerland) Oct 2022With this research, we aimed to determine the impact of grafting and rootstock seed treated with ( () or shikimic acid (SA) at a 60 ppm concentration on tomato ( L.)...
With this research, we aimed to determine the impact of grafting and rootstock seed treated with ( () or shikimic acid (SA) at a 60 ppm concentration on tomato ( L.) production grown under low-temperature conditions. Two open-field trials were performed during both winter seasons of 2020 and 2021 at the Experimental Farm, Faculty of Agriculture, Cairo University, Giza, Egypt. A tomato cultivar (Peto 86) was used as a scion and two tomato phenotypes were employed as rootstocks ( L and GS hybrid), as well as self-grafted as a control. Effects of sub-optimal temperature on vegetative growth, yield, and fruit quality were tested. The results indicate that, under cold stress, rootstock seed priming, especially with , enhanced plant growth, total yield, and fruit quality properties. GS hybrid rootstock was more effective than that of rootstock in terms of mitigating the negative effect of cold stress. GS hybrid, inoculated with increased the total yield per plant by 10.5% and 5.7% in the first and second seasons, respectively. Higher levels of GA3 and mineral content were noticed in leaves that were grafted and treated with compared to the control treatment. Additionally, the great enhancing effects of all anatomical features of tomato plants were recorded with GS hybrid rootstock, inoculated by . These results prove that grafting on GS hybrid rootstock treated with is a potential choice to alleviate the cold stress of commercial tomato varieties.
PubMed: 36365275
DOI: 10.3390/plants11212822 -
The Plant Pathology Journal Apr 2020S4-7, a well-characterized keystone taxon among strawberry microbial communities, shows exceptional disease-preventing ability. The whole-genome sequence, functional...
S4-7, a well-characterized keystone taxon among strawberry microbial communities, shows exceptional disease-preventing ability. The whole-genome sequence, functional genes, and bioactive secondary metabolites of the strain have been described in previous studies. However, proteomics studies of not only the S4-7 strain, but also the genus as a whole, remain limited to date. Therefore, in the present study, we created a proteomics reference map for S4-7. Additionally, analysis of differentially expressed proteins was performed against a 2 mutant, which was deficient in spore chain development and did not express an antifungal activity-regulatory transcription factor. We believe that our data provide a foundation for further in-depth studies of functional keystone taxa of the phytobiome and elucidation of the mechanisms underlying plant-microbe interactions, es-pecially those involving the genus.
PubMed: 32296298
DOI: 10.5423/PPJ.NT.02.2020.0037 -
International Journal of Molecular... Oct 2023The co-culturing of microorganisms is a well-known strategy to study microbial interactions in the laboratory. This approach facilitates the identification of new...
The co-culturing of microorganisms is a well-known strategy to study microbial interactions in the laboratory. This approach facilitates the identification of new signals and molecules produced by one species that affects other species' behavior. In this work, we have studied the effects of the interaction of nine species (, , , , , , , , and ) with the predator bacteria , five of which (, , , , and ) induce mound formation of on complex media (Casitone Yeast extract (CYE) and Casitone tris (CTT); media on which does not form these aggregates under normal culture conditions. An in-depth study on - interactions (the strain producing the strongest effect) has allowed the identification of two siderophores produced by , demethylenenocardamine and nocardamine, responsible for this grouping effect over . Experiments using pure commercial nocardamine and different concentrations of FeSO show that iron depletion is responsible for the behavior of . Additionally, it was found that molecules, smaller than 3 kDa, produced by can induce the production of DK-xanthenes by .
Topics: Myxococcus; Myxococcus xanthus; Streptomyces; Microbial Interactions; Iron
PubMed: 37958645
DOI: 10.3390/ijms242115659