A useful strategy: Deciphering biosynthesis by trans‐AT polyketide synthases (PKS) is challenging due to their highly nonlinear organization. Our analysis of the trans‐AT PKS responsible for ...sorangicin production in the myxobacterium Sorangium cellulosum So ce12 reinforces the utility of a combined phylogenetic/retrobiosynthetic approach for understanding this complex mode of biosynthesis.
Lidars With Narrow FOV for Daylight Measurements Eixmann, Ronald; Gerding, Michael; Hoffner, Josef ...
IEEE transactions on geoscience and remote sensing,
2015-Aug., 2015-8-00, 20150801, Letnik:
53, Številka:
8
Journal Article
Recenzirano
Daytime lidar operation in the middle atmosphere requires a narrow field of view (FOV) of the receiving telescope for effective background reduction and a high-transmission narrow-band detection. The ...laser beam position in the atmosphere relative to the optical axis of the receiving telescope is subject to high-frequency disturbances such as turbulence, vibration, and wind as well as comparable slow drift (thermal effects of the laser, stability of the building, etc.). We developed a beam stabilization system (BSS) that ensured a pulse-to-pulse stabilization of the laser beam with ~ 3 μrad remaining jitter, allowing ~ 60 μrad FOV. With BSS and single-pulse data acquisition system, the optimal alignment of the laser and telescope can be controlled, and information on the FOV and laser divergence in the far field can be derived. The capability of the BSS is to stabilize the laser against all internal and external disturbances below the repetition rate of the laser.
The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of ...the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.
Temperature measurements by lidar are an important tool for the understanding of the mean state of the atmosphere as well as the propagation of gravity waves and thermal tides. Though, mesospheric ...lidar soundings are often limited to nighttime conditions (e.g., solar zenith angle > 96°) due to the low signal-to-noise ratio during the day. By this, examination of long-period gravity waves and tides is inhibited, as well as soundings in summer at polar latitudes. We developed a new daylight-capable Rayleigh–Mie–Raman (RMR) lidar at our site in Kühlungsborn, Germany (54° N, 12° E), that is in routine operation since 2010 for temperature soundings up to 90 km or ∼ 75 km (night or day) and soundings of noctilucent clouds. Here we describe the setup of the system with special emphasis on the daylight suppression methods like spatial and spectral filtering. The small bandwidth of the Fabry–Pérot etalons for spectral filtering of the received signal induces an altitude-dependent transmission of the detector. As a result, the signal is no longer proportional to the air density and the hydrostatic integration of the profile results in systematic temperature errors of up to 4 K. We demonstrate a correction method and the validity of correction by comparison with data obtained by our co-located, nighttime-only RMR lidar where no etalon is installed. As a further example a time series of temperature profiles between 20 and 80 km is presented for day and night of 9–10 March 2014. Together with the other data of March 2014 these profiles are used to calculate tidal amplitudes. It is found that tidal amplitudes vary between ∼ 1 and 5 K depending on altitude.
Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities.1, 2 The genus Sorangium is of special ...biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon-mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol-type tubulin destabilizers,3-5 S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors,6 bactericidal sorangiolides, and the antifungal chivosazoles.7, 8 To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol-negative mutants. This approach led to the identification of the corresponding trans-acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.
The anti-fungal leupyrrins are secondary metabolites produced by several strains of the myxobacterium Sorangium cellulosum. These intriguing compounds incorporate an atypically substituted ...γ-butyrolactone ring, as well as pyrrole and oxazolinone functionalities, which are located within an unusual asymmetrical macrodiolide. Previous feeding studies revealed that this novel structure arises from the homologation of four distinct structural units, nonribosomally-derived peptide, polyketide, isoprenoid and a dicarboxylic acid, coupled with modification of the various building blocks. Here we have attempted to reconcile the biosynthetic pathway proposed on the basis of the feeding studies with the underlying enzymatic machinery in the S. cellulosum strain So ce690. Gene products can be assigned to many of the suggested steps, but inspection of the gene set provokes the reconsideration of several key transformations. We support our analysis by the reconstitution in vitro of the biosynthesis of the pyrrole carboxylic starter unit along with gene inactivation. In addition, this study reveals that a significant proportion of the genes for leupyrrin biosynthesis are located outside the core cluster, a 'split' organization which is increasingly characteristic of the myxobacteria. Finally, we report the generation of four novel deshydroxy leupyrrin analogues by genetic engineering of the pathway.
A highly flexible enzyme module system (EMS) was developed which allows for the first time the in situ regeneration of deoxythymidine 5′‐diphosphate (dTDP)‐activated deoxy sugars and furthermore ...enables us to produce novel sorangiosides in a combinatorial biocatalytic approach using three enzyme modules. The SuSy module with the recombinant plant enzyme sucrose synthase (SuSy) and the deoxy sugar module consisting of the enzymes RmlB (4,6‐dehydratase), RmlC (3,5‐epimerase) and RmlD (4‐ketoreductase) from the biosynthetic pathway of dTDP‐β‐L‐rhamnose were combined with the glycosyltransferase module containing the promiscuous recombinant glycosyltransferase SorF from Sorangium cellulosum So ce12. Kinetic data and the catalytic efficiency were determined for the donor substrates of SorF: dTDP‐α‐D‐glucose, dTDP‐β‐L‐rhamnose, uridine diphosphate (UDP)‐α‐D‐glucose (Glc), and dTDP‐6‐deoxy‐4‐keto‐α‐D‐glucose. The synthesis of glucosyl‐sorangioside with in situ regeneration of dTDP‐Glc was accomplished by combination of SuSy and SorF. The potential of the EMS is demonstrated by combining SuSy, RmlB, RmlC, RmlD with SorF in one‐pot for the in situ regeneration of dTDP‐activated (deoxy) sugars. The HPLC/MS analysis revealed the formation of rhamnosyl‐sorangioside and glucosyl‐sorangioside, demonstrating the in situ regeneration of dTDP‐β‐L‐rhamnose and dTDP‐α‐D‐glucose and a cycle number for dTDP higher than 9. Furthermore, NADH (reduced form of nicotinamdie adenine dinucleotide) regeneration with formate dehydrogenase in the reduction step catalyzed by the 4‐ketoreductase RmlD could be integrated in the one‐pot synthesis yielding similar conversion rates and cycle numbers. In summary, we have established the first in situ regeneration cycle for dTDP‐activated (deoxy) sugars by a highly flexible EMS which allows simple exchange of enzymes in the deoxy sugar module and exchange of glycosyltransferases as well as aglycones in the glycosyltransferase module to synthesize new hybrid glycosylated natural products in one‐pot.
Glycosylations are well‐established steps in numerous biosynthetic pathways, and the attached sugar moieties often influence the specificity or pharmacology of the modified compounds. The sorangicins ...belong to the polyketide family of natural products, and exhibit antibiotic activity through inhibition of bacterial RNA polymerase. We have identified the sorangicin biosynthetic gene cluster in the producing myxobacterium Sorangium cellulosum So ce12. Within the cluster, sorF encodes a putative glycosyltransferase. To determine its function in sorangicin biosynthesis, SorF was heterologously expressed as a fusion protein in Escherichia coli. After purification by affinity chromatography, SorF was found to glucosylate sorangicin A in vitro, utilizing UDP‐α‐D‐glucose as the natural donor substrate. Additionally, SorF showed high flexibility towards further UDP‐ and dTDP‐sugars and was able to transfer several other sugar moieties—α‐D‐galactose, α‐D‐xylose, β‐L‐rhamnose, and 6‐deoxy‐4‐keto‐α‐D‐glucose—onto the aglycon. SorF is therefore one of the rare glycosyltransferases able to transfer both D‐ and L‐sugars, and could thus be used to generate novel sorangiosides.
High promiscuity. Studies to evaluate the substrate specificity of the sorangicin glycosyltransferase SorF from Sorangium cellulosum revealed a highly flexible enzyme. Even the transfer of NDP‐activated D‐ and L‐deoxysugars onto the aglycon sorangicin A was possible in good yield, and resulted in several novel glycosylated sorangiosides.
Myxobacteria increasingly gain attention as a source of bioactive natural products. The genus
Sorangium produces almost half of the secondary metabolites isolated from these microorganisms. ...Nevertheless, genetic systems for
Sorangium strains are poorly developed, which makes the identification of the genes directing natural product biosynthesis difficult. Using biparental and triparental mating, we have developed methodologies for DNA transfer from
Escherichia coli via conjugation for the genome sequencing model strain So ce56 and the secondary metabolite multiproducing strain So ce12. The conjugation protocol developed for strain So ce56 is not applicable to other
Sorangium strains. Crucial points for the conjugation are the ratio of
E. coli and
Sorangium cellulosum cells, the choice of liquid or solid medium, the time used for the conjugation process and antibiotic selection in liquid medium prior to the plating of cells. A
mariner-based transposon containing a hygromycin resistance gene was generated and used as the selectable marker for
S. cellulosum. The transposon randomly integrates into the chromosome of both strains. As a proof of principle,
S. cellulosum So ce12 transposon mutants were screened using an overlay assay to target the chivosazole biosynthetic gene cluster.
Background: Despite the risk for complications, allograft surveillance after orthotopic heart transplantation (OHT) is performed by cardiac catheterization and biopsies. We investigated the ...diagnostic and prognostic value of a TDI-derived systolic wall motion analysis of the posterobasal wall of the left ventricle (Sm) as a screening modality in OHT aftercare. Methods: We examined data of 210 eligible patients who underwent OHT between 2010 and 2020. Forty-four patients who had died within the initial hospital stay were excluded. For 166 patients, baseline and follow-up data were analyzed. The mean age at OHT was 46.2 (±11.4) years; 76.5% were male. Results: Within the observational period, 22 (13.3%) patients died. In total, 170 episodes of acute cellular or humoral rejections occurred (84 ISHLT1R; 13 ISHLT2R; 8 ISHLT3R; 65 AMR), and 29 catheterizations revealed cardiac allograft vasculopathy (5 CAV1; 4 CAV2; 20 CAV3). Individual Sm radial/longitudinal remained stable within the follow-up period (11.5 ± 2.2 cm/s; 10.9 ± 2.1 cm/s). Patients with acute rejections and CAV3 showed significant Sm radial/longitudinal reductions (AMR1: 1.6 ± 1.9 cm/s, confidence interval (CI) 0.77–0.243, p < 0.001; 1.8 ± 2.0 cm/s, CI 0.92–0.267, p < 0.001. ISHLT1R: 1.7 ± 1.8 cm/s, CI 1.32–2.08, p < 0.001; 2.0 ± 1.6 cm/s, CI 1.66–2.34, p < 0.001. CAV3: 1.3 ± 2.5 cm/s, CI 0.23–2.43, p < 0.017; 1.4 ± 2.8 cm/s, CI 0.21–2.66, p < 0.021). Lower Sm was associated with a threefold increase in all-cause mortality (hazard ratio (HR) 3.24, CI 1.2–8.76, p = 0.020; HR 2.92, CI 1.19–7.18, p = 0.019). Overall, Sm-triggered surveillance led to 0.75 invasive diagnostics per patient post-OHT year. Conclusions: Sm remained stable in the post-OHT course. Reductions indicated ISHLT1R, AMR1 and CAV3 and were associated with higher all-cause mortality. Sm-triggered surveillance may be referred to as a safe, high-yield screening modality in OHT aftercare.
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