The aim of the study was to evaluate the effects of Fluorothiazinone (FT), the inhibitor of type III secretion system (T3SS), on a model of a long-term infection in animal caused via the clinical isolate of Burkholderia cenocepacia acquired from the chronically infected cystic fibrosis patient. To simulate acute and long-term lung infection, DBA/2 mice were infected intranasally, the bacterial recovery from the lungs was assessed, and a histopathological study was performed. Intracellular survival and reproduction of bacteria in macrophages were evaluated on a monolayer of RAW264.7 cells. When simulating an acute infection, the isolate caused death of 50% of mice within 5 days, while combined preventive-therapeutic scheme with FT increased the survival rate to 100%. When simulating a prolonged infection, combined preventive-therapeutic scheme with FT resulted in decrease in lung colonization compared to the control group by several orders of magnitude on the 5th day after the infection and to complete eradication on the 10th day. Histopathological examination confirmed the efficacy of FT in reducing the intensity of lung damage. FT prevented survival of the isolate in RAW264.7 macrophages and reduced the ability of bacteria to replicate intracellularly. Thus the efficacy of the new non-traditional antibacterial agent FT has been shown in models of acute and in prevention of a long-term lung infections caused via a multidrug-resistant isolate B. cenocepacia. FT has reduced the ability of the isolate to survive intracellularly and replicate in macrophages, thereby affecting significant mechanisms of the development of chronic infections.
Antimicrobial consumption (AMC) measures the level and types of antibiotics consumed in a specific setting. Monitoring AMC is critical component of antimicrobial resistance (AMR) containment strategies. However, AMC data at both facility and national-levels are scarce in Africa, which limits our understanding of the rate, patterns and drivers of antibiotic consumption, and prevents the establishment of evidence-based antimicrobial stewardship. We determined facility and national-level rates and patterns of AMC from data retrospectively collected between 2016 and 2019 in 327 pharmacies from 14 countries AMC data collection followed a backfilling strategy leveraging from public and private central medical stores, wholesalers, distributors or import services as data sources. Participating hospital and community pharmacies were selected based on their location in or proximity to hospitals capable of producing AMR data. Levels of AMC were determined as defined daily dose (DDD) and DDD per inhabitant (DID). AMC patterns were analysed according to the WHO Access, Watch, and Reserve (AWaRe) Categories, the Anatomical Therapeutic Chemical (ATC) classes and the individual antibiotic molecules included in the Drug Utilisation 75% (DU75). The availability of antibiotics was examined against the WHO and the National Essential Medicine Lists (EML). National AMC data was available in 11 of the 14 participating countries, revealing a collective AMC of 8.42 DID varying from 2.8 to 115.5 at individual country level. AMC was also determined in 327 hospital and community pharmacies. Nine of 11 (82%) countries with national data available, and 219 of the 327 (72%) participating pharmacies achieved the WHO AWaRe target of at least 60% of antibiotic consumption from Access drugs. Eighty percent of country-level AMC was accounted for by five ATC sub-classes classes of antibacterial for systemic use. Facility-level antibiotic consumption was dominated by a narrow scope of less than five drugs, taking advantage of only 10% of all possible WHO-recommended Access drugs within ATC classes. Collectively, the 14 national EML included 70% of Access, 60% of Watch and less than 5% of Reserve antibiotics listed in the WHO EML. Forty-eight uncategorized and 50 categorized non-WHO-recommended drugs were included in national EMLs or documented to be circulating in countries. The relatively low AMC and the poorly diversified subset of antibiotics available in countries underscores that strategies to expand equitable access to adequate treatment of bacterial infections should complement current efforts to promote the judicious use of antimicrobials. Interventions to increase the volume of analysable data on AMU, AMC and AMR, should be prioritized in national AMR action plans as well as in wider infrastructural and economic development plans.
Benzoisochromanequinone (BIQ) polyketides including actinorhodin, medermycin, and granaticin, are biosynthesized via the intermediate 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho[2,3-c]pyran-3-acetic acid (DNPA), which serves as a critical branch point. The configuration at C-3 of DNPA is a key determinant of structural diversity, leading to either (S)- or (R)-DNPA. Actinorhodin and medermycin are generated via (S)-DNPA, whereas granaticin is produced via (R)-DNPA. In contrast to the ketoreductase ActVI-ORF1 involved in (S)-DNPA production, the enzymatic function of Gra-6, presumed to be associated with (R)-DNPA biosynthesis, remains uncharacterized. This study evaluated the DNPA-producing ability of Gra-6 using a recombinant enzyme system expressed in Escherichia coli, revealing that it catalyzes stereoselective (R)-DNPA production. Furthermore, the putative amino acid residues involved in the DNPA-producing ability of Gra-6 were predicted through molecular modeling, including homology modeling and docking simulations with the coenzyme and an acyl carrier protein (ACP)-free bicyclic intermediate (BI) as the substrate. The involvement of these residues was verified by employing Gra-6 mutants. Additionally, the stereoselective mechanisms underlying DNPA production by each enzyme were proposed by comparing the docking models of ActVI-ORF1 and Gra-6 with the coenzyme and ACP-free BI. These findings establish Gra-6 as a pivotal stereospecific branch-point enzyme in BIQ polyketide biosynthetic branching and provide new insights into the stereoselective catalytic mechanisms.
The objective of this study was to investigate the prevalence and phenotypic identification of antimicrobial resistance of K. pneumoniae and K. oxytoca recovered from backyard broiler chickens and their human contacts. The serotypes and genotypes of antibiotic resistance genes of K. pneumoniae isolates were investigated. A total of 80 samples were collected from backyard broiler chickens that showed signs of illness, sneezing, coughing, and diarrhea. Twenty stool samples were collected from individuals who had been in contact simultaneously. In total, 19 Klebsiella species were isolated, 12 of which were from broiler chicken samples and seven from human stool samples. Recovery rates of K. pneumoniae were 11.3% (n = 9/80) and 15% (n = 3/20) of broiler and human stool samples, respectively. K. oxytoca was detected in 3 of 80 (3.75%) broiler chicken and 4 of 20 (20%) human stool samples. Antimicrobial susceptibility showed that all 19 Klebsiella isolates were resistant to erythromycin and clindamycin (100%), followed by penicillin (94.7%) and ampicillin (84.3%). Within the 12 K. pneumoniae isolates, the most common serotype was K1. Antibiotic resistance gene profile of K. pneumoniae isolates was observed, with a high carrying rate of the macrolide gene ermB (91.7%), followed by the β-lactam genes blaTEM (75%) and blaCTX-M1 (66.7%). In conclusion, the emergence of multidrug-resistant (MDR) bacteria, K. pneumoniae and K. oxytoca in broiler backyard chickens and their human contacts is alarming, particularly for erythromycin and clindamycin. This underscores the need for alternatives like vaccination, probiotics, and better biosecurity to combat antimicrobial resistance.
Breast tumor kinase (Brk) is an intracellular kinase that initiates a downstream oncogenic signaling through phosphorylation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3). Demethylmycemycin A (DA) was isolated from a sponge-derived Dactylosporangium sp. OK1079. Though known as a microbial dibenzoxazepinone, its biological activity has never been investigated. Previous studies on the marine triterpene sipholenol A identified its perhydrobenzoxepine system as the key pharmacophore that mediated its Brk binding. The bioisosteric similarity of DA to the sipholenol A perhydrobenzoxepine motivated a molecular docking simulation of DA for potential Brk binding. The antiproliferative effect of DA was investigated against diverse prostate cancer (PC) cell lines including LNCaP (castration/hormone-sensitive primary adenocarcinoma), PC3, and 22Rv1 (castration-resistant), in addition to the androgen-independent DU145 cells. LNCaP cells were the most sensitive to the effects of DA, followed by PC3, showing IC50 values of 7.6 and 9.8 μM, respectively. DA treatments significantly reduced the migration and clonogenicity of the LNCaP cells. Western blot analysis indicated the ability of DA to reduce the expression levels of activated Brk, FAK and STAT3 in a dose-dependent manner in both cell lines. DA also decreased the expression levels of the total FAK but didn't affect the total level of Brk while the expression level of total STAT3 was only suppressed in LNCaP cells. These results highlight the PC proliferation and migration suppressive effects of DA through targeting Brk-FAK-STAT3 axis. DA is a potential prototype hit that can be developed particularly for Brk-expressing PC control.
Fungi have a potential to produce a variety of natural products for their survival by utilizing dozens of biosynthetic gene clusters (BGCs). Fungal BGCs are typically conserved in species but recent studies showed that many BGCs are maintained in limited number of strains. However, little is reported for the products of strain specific BGCs. In this study, we analyzed publicly available genomic data of human pathogenic fungus Aspergillus lentulus and identified seven strain-specific BGCs in addition to (-)- and (+)-auranthine BGCs. One of these BGCs, leq cluster, was found in nine of thirteen A. lentulus strains and a new compound named lentoquinazoline (1) appeared in the metabolites when non-ribosomal peptide synthetase (NRPS) gene leqA was heterologously expressed in Aspergillus nidulans. The structure of 1 was determined by the combination of 2D NMR analysis and advanced Marfey's method to be composed of anthranilic acid, L-leucine and L-asparagine. Whereas 6-6-6 tricyclic quinazoline-synthesizing NRPSs typically contain an epimerase domain and incorporate one molecule of D-amino acid to the product, LeqA was found to introduce only L-amino acids due to the mutations in the active site of the epimerase domain.
Antibiotic resistance poses a significant threat to global food safety, particularly in unpasteurised dairy products. In the locality of Bwegera (South Kivu, DRC), livestock management practices promote the emergence of resistant bacterial strains, constituting a risk to public health. This study aimed to assess the resistance profile of enteropathogenic bacteria isolated from raw cow's milk in Bwegera, and to identify farming practices associated with this antibiotic resistance. A total of 144 milk samples were collected from 12 farms and analyzed following the standards of the International Organization for Standardization and the Clinical and Laboratory Standards Institute / European Committee on Antimicrobial Susceptibility Testing to identify bacteria belonging to the Enterobacteriaceae family and to determine their susceptibility to various antibiotics. The analysis revealed a high prevalence of Escherichia coli (52.8%), followed by Klebsiella pneumoniae (10.4%) and Citrobacter diversus (10.4%). Resistance was notably high for tetracyclines and vancomycin, affecting up to 95% of isolates, while amikacin remained fully effective (100% of isolates susceptible). Farming practices were characterised by frequent self-medication (83.3%) and a lack of awareness regarding antibiotic resistance (66.7%). Three farmer profiles were identified: one well-trained group misusing antibiotics, and another using antibiotics as feed additives. The majority of samples exhibited polymicrobial contamination (71.5%), with microbial loads often exceeding 300 CFU/mL. Inadequate sanitary and pharmaceutical practices foster resistant bacterial strains. Improved farmer education, stricter antibiotic regulations, and alternative interventions such as probiotics are needed within a 'One Health' framework.
Actinomycetes have been a cornerstone species for the discovery of bioactive natural products with applications in pharmacotherapy and biotechnology. To expand the experimental evidence of their biosynthetic potential, we collected liquid-chromatography mass spectrometry untargeted metabolomics data on 948 microbial strains, mostly from Actinomycetes. This resulted in nearly two million MS/MS spectra, with an annotation rate of 13.3% corresponding to 2352 annotated molecules. Despite the efforts to link biosynthetic gene clusters to known molecules, most remain uncharacterized. This highlights the need for metabolomic data to bridge the gap between genomic potential and metabolite production. Although many unannotated spectra might correspond to different ion forms of the same molecule, the large amount of unknown molecules present in these datasets indicates that a significant number of natural products remain to be discovered, even within one of the most thoroughly studied sets of organisms. We provide a large metabolomics dataset as a public resource for data mining of microbial molecules and highlight its value by demonstrating the detection of edapochelins, recently discovered non-ribosomal peptides.
Gymnopilus orientispectabilis, a bitter and hallucinogenic mushroom found in East Asia, contains polyisoprenepolyol ester gymnopilins as active constituents. Detection of antibacterial activity against a solanaceous plant pathogen, Ralstonia solanacearum SUPP1541, prompted fractionation of an extract of this mushroom, which specified a mixture of gymnopilins responsible for the activity. Evaluation of antibacterial activity of purified gymnopilins A10 and A11, their deacylated metabolites gymnoprenols A10 and A11, and gymnopilene, a terminally desaturated derivative of gymnoprenol B10, all obtained from G. aeruginosus, revealed gymnopilin A10 to be active but the rest were not active at 200 μg/disk. Meanwhile, a new polyisoprenepolyol, gymnoprenol B13 (1), along with three known congeners, gymnoprenols B10, B11, and B12, was discovered from a fraction eluting faster than the active fraction in HPLC. The planar structure of 1 was determined by NMR and MS/MS fragmentation analysis. Consistent with the above observation, the four gymnoprenol B congeners were not antagonistic to R. solanacearum. The present study discovered antibacterial activity of a polyisoprenepolyol-class metabolite against R. solanacearum and revealed the importance of both the 3-hydroxy-3-methylglutaryl unit and a specific length of the polyisoprenyl chain or composition of hydroxylation in gymnopilin A10 for its activity.
Antibiotic resistance of Acinetobacter poses a serious threat to public health. Still, the antibiotic resistance features of marine Acinetobacter remain largely unexplored. Here, an Acinetobacter beijerinkii strain, named as MPE71, was isolated from the marine plastisphere. Phylogenetic analysis revealed close relationship of MPE71 with two human pathogenic strains of Acinetobacter. Minimum inhibitory concentration (MIC) assays of 10 antibiotics demonstrated the multidrug-resistance of MPE71. Notably, it showed a MIC of 200 µg/mL against polymyxin B. To the best of our knowledge, this is the first report demonstrating that Acinetobacter from the marine plastisphere exhibits multidrug resistance, including high-level resistance to polymyxin B. Genomic resistance gene analysis revealed numerous multidrug efflux pump genes encoded in MPE71. The resistance mechanism was further studied using transcriptomics. Enhanced membrane biosynthesis, multidrug efflux pumps, and periplasmic protein factors largely explained the resistance of MPE71 to polymyxin B, with up-regulation of relevant genes in a dose-dependent manner. Subsequently, inhibition of the proton motive force (PMF) by carbonyl cyanide 3-chlorophenylhydrazone diminished MPE71 resistance to polymyxin B, underscoring the key role of PMF-dependent efflux pumps. This study provides new evidence for the ecological risk of antibiotic resistance in the marine plastisphere.
Antibiotic resistance has emerged as a critical global public health challenge. Quorum sensing (QS), a density-dependent regulatory mechanism, plays a pivotal role in bacterial pathogenesis by coordinating virulence factor expression, making it a critical target for antivirulence therapy. Leveraging a drug repositioning strategy, this study investigated the antivirulence potential of drugs in the database of DrugBank on the common opportunistic pathogen Pseudomonas aeruginosa by virtual screening. Molecular docking analysis predicted that the antitumor drug, Tirazone, could bind to the core QS regulatory proteins, LasR, RhlR, and PqsR of P. aeruginosa with abundant active sites, whereas the binding free energies were higher than those of the native QS signals. In vitro experiments demonstrated that Tirazone significantly suppressed virulence factor secretion, cell motilities, and biofilm formation in the model P. aeruginosa strain PAO1, and downregulated the expression of a series of QS-related genes with low effective concentration (≤ 8 μM). A competitive binding model of QS signal molecules further elucidated that Tirazone interfered with QS signaling by competitively inhibiting the function of LasR, RhlR, and PqsR. Additionally, Tirazone treatment significantly protected Caenorhabditis elegans and mouse models from P. aeruginosa infection, and reduced the bacterial loads and pathological lesions in mouse lungs. Moreover, Tirazone demonstrated synergistic effects with polymyxin B, levofloxacin, and amikacin, significantly enhancing their bactericidal efficacy in treating P. aeruginosa. This study reveals the molecular mechanism underlying Tirazone's multi-target intervention in the QS system, and provides an experimental foundation for developing combination therapies based on antivirulence strategies.
A novel actinomycete, designated as NUM-3379T, was isolated from seeds of Anabasis brevifolia collected from Urgun Soum, Dornogovi Province, Mongolia. The cells of the strain were aerobic, Gram-positive, motile, non-spore-forming, and coccus-shaped. 16S rRNA gene sequence analysis indicated that strain NUM-3379T belonged to the genus Kineococcus and showed the highest similarity to Kineococcus glutinatus DSM 26692T and Kineococcus xinjiangensis DSM 22857T, with both sharing 98.87% sequence similarity. The chemotaxonomic characteristics of the strain NUM-3379T were consistent with those of the genus Kineococcus. The diagnostic diamino acid for peptidoglycans was meso-diaminopimelic acid. The whole-cell sugars included glucose, galactose, mannose, and arabinose. The major menaquinone was MK-9(H2). The polar lipid profile was comprised of phosphatidylglycerol, diphosphatidylglycerol, and two unidentified phospholipids. The major fatty acids were anteiso-C15:0 and iso-C17:0 3-OH. In contrast, digital DNA-DNA hybridization and average nucleotide identity values revealed low relatedness between strain NUM-3379T and other type strains of the genus Kineococcus. Furthermore, strain NUM-3379T exhibited several phenotypic properties that distinguished it from other closely related species. Based on the genotypic and phenotypic data, strain NUM-3379T represents a novel species within the genus Kineococcus, for which the name Kineococcus anabasis sp. nov. is proposed. The type strain is NUM-3379T (=NBRC 117252T = TBRC 21100T).
Indole derivatives possess various biological activities, including antifungal, antibacterial, anticancer, antioxidant, antimalarial, antidiabetic, antitubercular, and anticholinesterase activities. This study focused on the synthesis of indole and its derivatives using the Fischer indole synthesis method and the Ullmann condensation reaction. The synthesized compound was characterized by melting point determination, UV-spectroscopy, thin layer chromatography, Fourier-transform infrared spectroscopy, and proton nuclear magnetic resonance. The zone of inhibition and the minimum Inhibitory concentration were used to determine antifungal activity against Candida albicans and Aspergillus niger. The parent molecule 2-methyl-1H-indole (B), with a yield of 44% was synthesized by Fischer indole synthesis and Ullmann condensation. Three derivatives of the parent molecules, 3-(2-methyl-1H-indol-1-yl)phenol (A1) with a yield of 68%, 1-(3-(2-methyl-1H-indol-1-yl)phenyl)ethan-1-one (A2) with a yield of 60%, and 2-methyl-1-(3-methylphenyl)-1H-indole (A3) with a yield of 84% were synthesized. Molecular docking revealed that all derivatives bound lanosterol 14α-demethylase more strongly than fluconazole (-7.1 kcal/mol), with A2 showing the highest affinity (-8.1 kcal/mol). (PDBID: 5TZ1). Furthermore, other computational studies through Pass analysis server, Drug likeness prediction by SwissADME, Toxicity prediction by Stoptox, and pkCSM server were performed on synthesized compounds to predict the biological activity. Collectively, these findings highlight indole derivatives, particularly A1 and A2, as promising antifungal leads with potent in vitro efficacy and strong molecular interactions with a clinically validated target.
This article aims to elucidate the potential role of a comprehensive tooth extraction procedure in preventing mdication-related osteonecrosis of the jaw (MRONJ) through a prospective cohort study. By systematically assessing clinical outcomes following this procedure, the study seeks to provide evidence regarding its effectiveness in MRONJ prevention, thereby contributing to improved clinical guidelines and patient care related to dental extractions in at-risk populations. Patients using anti-resorptive agents (ARAs) who required extraction of at least one tooth were included in the study. Patients' medical history, medication history, and intraoral dental conditions were documented, and CBCT scans were performed. Following a standardized treatment protocol, patients received professional oral cleaning and antibiotics preoperatively. During surgery, minimally invasive extraction and concentrated growth factor (CGF) filling were performed with meticulous suturing whenever possible. Postoperatively, mouthwash was used within one month. Follow-up visits were scheduled at 10, 30, and 90 days to monitor and analyze MRONJ incidence and surgical outcomes. A total of 101 patients were included in the study, with 20 receiving oral ARAs for osteoporosis, 57 receiving intravenous ARAs for osteoporosis, 13 undergoing combination therapy for osteoporosis, and 11 using ARAs for malignancy. Zoledronic acid and denosumab were the most commonly used drugs. Increased bone density was observed on preoperative CBCT in 26 patients, and on postoperative CBCT at 90 days in 31 patients. In total, 248 teeth were extracted, mostly due to severe defects; periapical periodontitis and periodontitis were also major reasons for extraction. Most patients could not achieve complete and tight suturing. All patients remained free of MRONJ during the 90‑day postoperative period, with complete mucosal healing in every case. This prospective cohort study provides evidence that implementing an effective and rational treatment protocol during tooth extractions significantly benefits high-risk MRONJ patients. Adherence to such protocols minimizes the risk of postoperative infection, fosters improved healing of extraction sites, and maximizes the prevention of MRONJ. Not applicable.
The past decade has witnessed the recognition of antibiotic resistance as an emerging threat to public health. Polymyxin B (PMB) is a last-resort antibiotic against multidrug-resistant (MDR) Gram-negative bacteria, but it has nephrotoxicity. A cocktail with molecules to sensitize microbes to PMB is an effective approach to rescue and expand its clinical application range. Tuspetinib (TUS) is a selective FMS-like tyrosine kinase 3 (FLT3) inhibitor with activity against acute myeloid leukemia (AML) and a favorable safety profile. In this study, we found that TUS can enhance the antibacterial activity of PMB against Klebsiella pneumoniae and its clinical PMB-resistant isolates. We also revealed the mechanism by which TUS inhibits the activity of GlcNAc6P deacetylase (NagA) and clarified the association between NagA and PMB resistance. Finally, we demonstrated the efficacy of TUS plus PMB therapy in a mouse model of pulmonary infection with a clinical PMB-resistant K. pneumoniae isolate. In all, this work discovers a promising drug combination strategy based on PMB and TUS and underlies the special mode of action that involves inhibiting the activity of NagA.
Rapamycin, a 'wonder drug', was first reported 50 years ago in two papers in the Journal of Antibiotics, which described an antifungal compound purified from a bacterium isolated from soil samples collected on Easter Island (Rapa Nui). What began as the characterization of a natural product led to the identification of an entirely new signaling cascade, the target of rapamycin (TOR) pathway, and the emergence of a new field in biomedical research. Here, we review the historical and molecular foundations of the TOR field, from the discovery of rapamycin to current models of nutrient sensing, pathway regulation, clinical applications, and future perspectives.
Sepsis is a leading cause of neonatal mortality in sub-Saharan Africa (SSA), where microbiological diagnostic capacity and antibiotic access are limited. High antimicrobial resistance (AMR) rates limit the effectiveness of current treatment guidelines, with concern that available antibiotics are rarely adequate treatment for neonatal sepsis in the region. A cross-sectional online survey was electronically distributed in English, French and Portuguese to neonatal clinicians across SSA between April and June 2025. Questions focused on the management of neonatal sepsis including diagnostic, antibiotic and guideline use. Responses were analysed descriptively and presented as percentages of the total number of responses. Of 169 responses (40/48 countries; 83.3%) from SSA, 71.6% were senior doctors, 88.8% managed neonatal sepsis at least weekly and 58.0% worked in central healthcare facilities.14.9% (95% CI 10.3% to 21.0%) of respondents never and 28.6% (CI 22.3% to 35.8%) less than half of the time received blood culture results in time to impact patient care. Guidelines were almost universally used (97.6% (CI 94.0% to 99.1%)). The most common guideline for early-onset neonatal sepsis advised amoxicillin/ampicillin plus aminoglycosides (46.4% of responses (CI 39.1% to 54.0%)). 50.3% (CI 42.8% to 57.7%) of respondents had difficulties accessing antibiotics, with carbapenems and piperacillin-tazobactam least accessible. 45.4% (CI 38.0% to 53.1%) had attempted to author local guidelines with insufficient local AMR data (45.6% (CI 35.7% to 55.8%)) the most common barrier to guideline development. This large survey highlighted widespread challenges in diagnostic and antibiotic access for neonatal sepsis in SSA. We find that clinicians rely on guidelines to guide starting antibiotics and to guide agent choice. Their practices reflect advice in global guidelines. Attempts to author locally applicable guidelines are hindered by insufficient AMR data. These findings strengthen calls to improve microbiological diagnostic access and support data sharing to generate evidence-based, locally appropriate guidelines.
The taxonomic status of strain KL5T, a novel actinobacterium isolated from the rhizosphere soil of Dryopteris championii, was described using a polyphasic approach. Strain KL5T contained rich meso and ll-diaminopimelic acids in the cell-wall peptidoglycan. Whole-cell hydrolysates of strain KL5T were galactose and ribose. The major fatty acids were iso-C15:0, anteiso-C15:0, iso-C16:0, C16:0 and anteiso-C17:0. The polar lipids mainly consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. 16S rRNA gene sequence analysis indicated that this strain shared the highest sequence similarity to Kitasatospora arboriphila HKI 0189 T (99.29%). Phylogenetic analysis based on 16S rRNA gene sequences revealed strain KL5T was most related to K. arboriphila HKI 0189 T, as was also confirmed by phylogenomic analysis. But strain KL5T exhibited low average nucleotide identity and digital DNA-DNA hybridization values with K. arboriphila JCM 13002 T (90.40% and 37.2%, respectively), suggesting that strain KL5T was a new Kitasatospora species. The results of phenotypic and chemotaxonomic analyses further verified the above conclusion. Consequently, strain KL5T represents a novel species of the genus Kitasatospora, for which the name Kitasatospora dryopteridis sp. nov. is proposed. The type strain is KL5T ( = MCCC 1K10066T = KCTC 59627 T). In addition, the comparative genomic analysis suggested that Kitasatospora cinereorecta and Kitasatospora paracochleata should belong to the same species. Based on the principle of priority, it is proposed Kitasatospora paracochleata corrig. (Nakagaito et al. 1993) Zhang et al. [1] is a later heterotypic synonym of Kitasatospora cinereorecta (Terekhova and Preobrazhenskaya 1986) Labeda et al. [2].
Genome mining is a powerful strategy for finding biosynthetic gene clusters (BGCs) for unprecedented natural products and their biosynthetic mechanisms. In this study, to obtain novel type polyketides, we performed genome mining focusing on three perspectives for novelty: 1) amino acid sequence-based classification, 2) unique domain architectures of polyketide synthase, and 3) predicted structural features from protein modeling. As a result, we discovered a BGC consisting of a highly reducing polyketide synthase and a non-reducing polyketide synthase (NR-PKS), which harbors noncanonical tandem acyl carrier protein (ACP) domains and a structurally characteristic thioesterase (TE) domain. Heterologous expression revealed that the cyrl cluster produces a novel dimer of long-chain alkylresorcinolic acid (1). Furthermore, site-directed mutagenesis of each ACP domain revealed that both domains are essential for efficient dimerization. This study provides the first example of a fungal dimer-forming NR-PKS in which the tandem ACP domains work "in-series" with nonredundant roles to construct a dimeric long-chain alkylresorcinolic acid. The in silico analysis suggested that the TE domain led the substrate to a dimerizable form. Since 1 is structurally related to integracins with HIV-1 integrase inhibition, our findings provide insight into integracins biosynthesis and offer a basis for generating new integracin derivatives.
There is an urgent need for the development of new antibacterial drugs, caused by the increasing number of resistances. The first step for the development of new antibacterial compounds is usually a high-throughput screen of naturally occurring or synthetic compounds. Here, we propose to screen compound libraries on the Euglenoid Euglena gracilis. This protozoan has obtained a chloroplast via secondary endosymbiosis of an alga, but still maintains its ability to metabolise organic carbon sources. Importantly, the chloroplast has preserved some bacterial features and can be targeted by a range of antibiotics, resulting in organelle loss and Euglena bleaching, while the mixotrophic metabolism ensures growth of Euglena. Therefore, Euglena allows simultaneous screening of a compound library for (i) antibacterial activity (Euglena bleaches), (ii) absence of toxicity (Euglena grows) and (iii) membrane permeability across at least three membranes of both eukaryotic and prokaryotic origin. We have established and optimised all the parameters needed for such an Euglena-based antibacterial drug screen. We define absorbance parameters to distinguish the three possible outcomes (bleaching, growing and dying) in an automatised way. We have successfully tested the screen on both a commercially available compound library from MedChemExpress and on a self-assembled library of rare natural compounds from myxobacteria and fungi. Our Euglena-based screening platform provides a novel high-throughput method to screen for new compounds with antibiotic properties, in a cost-effective way and with any library. While some classes of antibiotics will be missed, the screen is unbiased and has the potential to discover novel antibiotic targets.