Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.     

Characterization of the antibiotic resistance plasmid ERL1 from Streptococcus pyogenes

Summary The streptococcal plasmid ERL1 determining inducible resistance to erythromycin, lincomycin, and staphylomycin S was isolated by dye-buoyant density centrifugation and shown to have a molecular weight of about 17.5 Mdal, as revealed by sedimentation through neutral sucrose gradients. In SM60 cells entering the stationary phase its covalently closed circular form was present to the extent of 5 copies per chromosomal genome equivalent. ERL1 was subject to the DNA restriction and modification mechanism discovered in strain 56188. It did not apear to exercise restriction of phage DNA but mediated a partial release of the restricted growth of A25.     

Wolframin deficiency is accompanied with metabolic inflexibility in rat striated muscles

The protein wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. Mutations in Wfs1 gene cause autosomal recessive disorder Wolfram syndrome (WS). The first symptom of the WS is diabetes mellitus, so accurate diagnosis of the disease as WS is often delayed. In this study we aimed to characterize the role of the Wfs1 deficiency on bioenergetics of muscles. Alterations in the bioenergetic profiles of Wfs1-exon-5-knock-out (Wfs1KO) male rats in comparison with their wild-type male littermates were investigated using high-resolution respirometry, and enzyme activity measurements. The changes were followed in oxidative (cardiac and soleus) and glycolytic (rectus femoris and gastrocnemius) muscles. There were substrate-dependent alterations in the oxygen consumption rate in Wfs1KO rat muscles. In soleus muscle, decrease in respiration rate was significant in all the followed pathways. The relatively small alterations in muscle during development of WS, such as increased mitochondrial content and/or increase in the OxPhos-related enzymatic activity could be an adaptive response to changes in the metabolic environment. The significant decrease in the OxPhos capacity is substrate dependent indicating metabolic inflexibility when multiple substrates are available.     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

Rock glaciers in crystalline catchments: Hidden permafrost‐related threats to alpine headwater lakes

A global warming‐induced transition from glacial to periglacial processes has been identified in mountainous regions around the world. Degrading permafrost in pristine periglacial environments can produce acid rock drainage (ARD) and cause severe ecological damage in areas underlain by sulfide‐bearing bedrock. Limnological and paleolimnological approaches were used to assess and compare ARDs generated by rock glaciers, a typical landform of the mountain permafrost domain, and their effects on alpine headwater lakes with similar morphometric features and underlying bedrock geology, but characterized by different intensities of frost action in their catchments during the year. We argue that ARD and its effects on lakes are more severe in the alpine periglacial belt with mean annual air temperatures (MAAT) between ?2°C and +3°C, where groundwater persists in the liquid phase for most of the year, in contrast to ARD in the periglacial belt where frost action dominates (MAAT < ?2°C). The findings clearly suggest that the ambient air temperature is an important factor affecting the ARD production in alpine periglacial environments. Applying the paleoecological analysis of morphological abnormalities in chironomids through the past millennium, we tested and rejected the hypothesis that unfavorable conditions for aquatic life in the ARD‐stressed lakes are largely related to the temperature increase over recent decades, responsible for the enhanced release of ARD contaminants. Our results indicate that the ARDs generated in the catchments are of a long‐lasting nature and the frequency of chironomid morphological deformities was significantly higher during the Little Ice Age (LIA) than during pre‐ or post‐LIA periods, suggesting that lower water temperatures may increase the adverse impacts of ARD on aquatic invertebrates. This highlights that temperature‐mediated modulations of the metabolism and life cycle of aquatic organisms should be considered when reconstructing long‐term trends in the ecotoxicological state of lakes.     

Wnt5a induces a tolerogenic phenotype of macrophages in sepsis and breast cancer patients

A well-orchestrated inflammatory reaction involves the induction of effector functions and, at a later stage, an active downregulation of this potentially harmful process. In this study we show that under proinflammatory conditions the noncanonical Wnt protein, Wnt5a, induces immunosuppressive macrophages. The suppressive phenotype induced by Wnt5a is associated with induction of IL-10 and inhibition of the classical TLR4-NF-κB signaling. Interestingly, this phenotype closely resembles that observed in reprogrammed monocytes in sepsis patients. The Wnt5a-induced feedback inhibition is active both during in vitro LPS stimulation of macrophages and in patients with sepsis caused by LPS-containing, gram-negative bacteria. Furthermore, using breast cancer patient tissue microarrays, we find a strong correlation between the expression of Wnt5a in malignant epithelial cells and the frequency of CD163(+) anti-inflammatory tumor-associated macrophages. In conclusion, our data point out Wnt5a as a potential target for an efficient therapeutic modality in severe human diseases as diverse as sepsis and malignancy.     

FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.     

Microbial metabolomics: toward a platform with full metabolome coverage

Achieving metabolome data with satisfactory coverage is a formidable challenge in metabolomics because metabolites are a chemically highly diverse group of compounds. Here we present a strategy for the development of an advanced analytical platform that allows the comprehensive analysis of microbial metabolomes. Our approach started with in silico metabolome information from three microorganisms-Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae-and resulted in a list of 905 different metabolites. Subsequently, these metabolites were classified based on their physicochemical properties, followed by the development of complementary gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry methods, each of which analyzes different metabolite classes. This metabolomics platform, consisting of six different analytical methods, was applied for the analysis of the metabolites for which commercial standards could be purchased (399 compounds). Of these 399 metabolites, 380 could be analyzed with the platform. To demonstrate the potential of this metabolomics platform, we report on its application to the analysis of the metabolome composition of mid-logarithmic E. coli cells grown on a mineral salts medium using glucose as the carbon source. Of the 431 peaks detected, 235 (=176 unique metabolites) could be identified. These include 61 metabolites that were not previously identified or annotated in existing E. coli databases.