Occurrence of vanHAX and related genes beyond the Actinobacteria phylum

Clinically relevant glycopeptide antibiotics remain among the most successful classes of natural antibacterials. This success, however, is endangered by the spread of glycopeptide resistance genes, also known as van genes. Thus, it is important to trace and comprehend possible routes of van gene dissemination. In the current work, we present a comprehensive bioinformatic analysis aimed at mapping the occurrence of van genes beyond the Actinobacteria phylum—the most likely natural reservoir of van genes. We show that two additional classes of Gram-positive bacteria, Erysipelotrichia and Ktedonobacteria, as well as one class of Gram-negative bacteria, Anaerolineae, carry van genes. Additionally, we demonstrate that various new genera belonging to the classes Clostridia and Bacilli also carry van genes. The majority of discovered van loci are co-localized with MGE-related genes of various types. Finally, we propose a phylogeny-based scenario for the spread of van genes, unraveling a network of consequential horizontal gene transfer events linking the phylum Actinobacteria with the five other bacterial classes carrying van genes

Heterologous expression reveals ancient properties of Tei3 - A VanS ortholog from the teicoplanin producer Actinoplanes teichomyceticus

Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus—the producer of the last-resort-drug teicoplanin—has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein ombining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A teichomyceticus was hereby proposed

Genetics Behind the Glycosylation Patterns in the Biosynthesis of Dalbaheptides

Glycopeptide antibiotics are valuable natural metabolites endowed with different pharmacological properties. Among them are dalbaheptides, used to treat different infections caused by multidrug-resistant Gram-positive pathogens. Dalbaheptides are produced by soil-dwelling high G-C Gram-positive actinobacteria. Their biosynthetic pathways are encoded within large biosynthetic gene clusters. A non-ribosomally synthesized heptapeptide aglycone is the common scaffold for all dalbaheptides. Different enzymatic tailoring steps, including glycosylation, are further involved in decorating it. Glycosylation of dalbaheptides is crucial conferring them specific biological activities. It is achieved by a plethora of glycosyltransferases, encoded within the corresponding biosynthetic gene clusters, able to install different sugar residues. These sugars might originate from primary metabolism, or, alternatively, their biosynthesis might be encoded within the biosynthetic gene clusters. Already installed monosaccharides might be further enzymatically modified or work as substrates for additional glycosylation. In the current minireview, we cover recent updates concerning the genetics and enzymology behind the glycosylation of dalbaheptides, building a detailed and consecutive picture of this process and of its biological evolution. A thorough understanding of how glycosyltransferases function in dalbaheptide biosynthesis might open new ways to use them in chemo-enzymes synthesis and/or in combinatorial biosynthesis for building novel glycosylated antibiotics

Role of GntR family regulatory gene SCO1678 in gluconate metabolism in streptomyces coelicolor M145

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed

SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors

Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed

Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNALeuUAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining

Properties of Multidrug-Resistant Mutants Derived from Heterologous Expression Chassis Strain Streptomyces albidoflavus J1074

Streptomyces albidoflavus J1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within gene rpoB for the β-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects of rpoB mutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneous rpoB mutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 different rpoB mutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. The rpoB mutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea that rpoB mutations are useful tools to enhance the ability of J1074 to produce specialized metabolites

The three-dimensional randomly dilute Ising model: Monte Carlo results

We perform a high-statistics simulation of the three-dimensional randomly dilute Ising model on cubic lattices $L^3$ with $L\le 256$. We choose a particular value of the density, x=0.8, for which the leading scaling corrections are suppressed. We determine the critical exponents, obtaining $\nu = 0.683(3)$, $\eta = 0.035(2)$, $\beta = 0.3535(17)$, and $\alpha = -0.049(9)$, in agreement with previous numerical simulations. We also estimate numerically the fixed-point values of the four-point zero-momentum couplings that are used in field-theoretical fixed-dimension studies. Although these results somewhat differ from those obtained using perturbative field theory, the field-theoretical estimates of the critical exponents do not change significantly if the Monte Carlo result for the fixed point is used. Finally, we determine the six-point zero-momentum couplings, relevant for the small-magnetization expansion of the equation of state, and the invariant amplitude ratio $R^+_\xi$ that expresses the universality of the free-energy density per correlation volume. We find $R^+_\xi = 0.2885(15)$.Comment: 34 pages, 7 figs, few correction

Indoor Autonomous Airship Control and Navigation System

The paper presents an automatic control system for autonomous airship. The system is designed to organize autonomous flight of the mini-airship performing flight mission defined from ground control station. Structure, hardware and software implementation of indoor autonomous airship and its navigation and control system as well as experiment results are described

Розроблення умов глибинного біосинтезу антибіотика ландоміцину А

Background. Development of the process and biotechnology of production of antitumor antibiotic with microbial origin – landomycin A.Objective. Establishment of the rational parameters for the submerged biosynthesis of landomycin A during the cultivation in the laboratory conditions.Methods. Cultivation of the producer Streptomyces cyanogenus S136 was performed on shaking devices in the flasks. Antibiotic was extracted from culture fluid with ethyl acetate; the content of antibiotic was determined by measuring optical density of the extract.Results. Conditions and parameters for antibiotic submerged biosynthesis of the landomycin A by S. cyanogenus S136 were investigated. Alternative nutrient mediums for the biosynthesis of the landomycin A were presented. Mediums that are based on soybean’s flour extract and corn’s flour allow obtaining up to 80 mg/ml of antibiotic. The influence of mixing intensity and temperature on the rate of the product accumulation and its dynamics was established. Rational technological parameters of the producer’s submerged biosynthesis such as temperature 25 ± 1 °С mixing intensity 230–250 U/min, duration of the submerged fermentation 50–60 hours were established.Conclusions. Variants of the culture media, that were selected (based on the corn’s flour and the extract of the soybean’s flour) and the parameters of the landomycin A biosynthesis, that were set are the basis for optimization and design of the technology for obtaining the antibiotic in industrial conditions.Проблематика. Разработка способа и биотехнологии производства противоопухолевого антибиотика микробного происхождения – ландомицина А.Цель исследования. Установление рациональных параметров глубинного биосинтеза ландомицина А при культивировании в лабораторных условиях.Методика реализации. Культивирование продуцента Streptomyces cyanogenus S136 проводили на качалочных устройствах в колбах. Антибиотик экстрагировали из культуральной жидкости этилацетатом и определяли его содержание по оптической плотности экстракта.Результаты исследований. Исследованы условия и параметры глубинного биосинтеза антибиотика ландомицина А штаммом S. cyanogenus S136. Предложены альтернативные питательные среды для биосинтеза ландомицина А на основе экстракта соевой муки и на основе кукурузной муки, позволяющие получить до 80 мкг/мл антибиотика. Установлено влияние интенсивности перемешивания и температуры на уровень накопления продукта и его динамику. Определены рациональные технологические параметры глубинного культивирования продуцента, а именно: температура 25 ± 1 °С, интенсивность переме­ши­вания 230-250 об/мин, продолжительность глубинной ферментации 50-60 ч.Выводы. Подобранные варианты питательных сред (на основе кукурузной муки и экстракта соевой муки) и установленные параметры биосинтеза ландомицина А являются основой для оптимизации и разработки технологии получения антибиотика в промышленных условиях.Проблематика. Розробка способу та біотехнології виробництва протипухлинного антибіотика мікробного походження – ландоміцину А.Мета дослідження. Встановлення раціональних параметрів глибинного біосинтезу ландоміцину А при культивуванні у лабораторних умовах.Методика реалізації. Культивування продуцента Streptomyces cyanogenus S136 проводили на качалкових пристроях у колбах. Антибіотик екстрагували з культуральної рідини етилацетатом і визначали його вміст за оптичною густиною екстракту.Результати дослідження. Досліджено умови та параметри глибинного біосинтезу антибіотика ландоміцину А штамом S. cyanogenus S136. Запропоновано альтернативні живильні середовища для біосинтезу ландоміцину А на основі екстракту соє­вого борошна та на основі кукурудзяного борошна, що дає змогу отримати до 80 мкг/мл антибіотика. Встановлено вплив інтенсивності перемішування і температури на рівень накопичення продукту та його динаміку. Визначено раціональні технологічні параметри глибинного культивування продуцента, а саме: температура 25 ± 1 °С, інтенсивність перемішування 230-250 об/хв, тривалість глибинної ферментації 50-60 год.Висновки. Підібрані варіанти живильних середовищ (на основі кукурудзяного борошна та екстракту соєвого борошна) і встановлені параметри біосинтезу ландоміцину А є основою для оптимізації та розробки технології отримання антибіотика в промислових умовах