Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine production

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81_kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.     

Structural aspects of akinete germination in the cyanobacterium Anabaena variabilis

Electronmicroscopical investigations of light activated akinetes in different phases before outgrowth of the germinating cell showed two alterations in the akinete envelope, obviously in connection with the germination process. After induction of germination the akinetes show formation of an expanding more or less electron dense layer between the outer cell wall layer (outer membrane, L_IV) and the condensed part of the akinete coat (the transformed sheath of the vegetative cell). Between this new formed layer and the mentioned part of the akinete coat thick laminar layers are deposited which contain alternately electron dense and electron transparent strata. The expanding layer is assumed to be a mucous layer which acts as swelling body causing, after bursting of the layered shell, the expulsion of the germinating cell in the manner characteristic for Anabaena variabilis.     

Effects of temperature on the rates of oxygen consumption during morphogenesis and diapause in the egg stage of Letopterna dolobrata (Heteroptera,Miridae)

Summary Oxygen consumption at 25°C was measured continously throughout the egg stage of Leptopterna dolobrata (more than 9 months).The rate of O₂-uptake (l O₂/100 eggs · 1 h) is low in freshly laid eggs. Maintaining the eggs at a constant temperature of 16°C, respiration rises abruptly from the first day after oviposition and continues rising steadily for 3 days, reaching an average value of 1.4 l. Oxygen consumption persists at or near this high level during the developmental phase of prediapause, which lasts about 15 days. After some days of oscillating high and low values, respiration decreases, and from the 24th day a low level (0.3–0.4 l) is reached. If the eggs are incubated at 16°C continuously, this low diapause-level is maintained until the end of the experiments (42 weeks) and diapause is terminated in a few eggs only.A significant increase in the success of hatching is obtained by exposing the eggs to a sufficient period of chilling.24 groups of diapausing eggs were chilled at 5°C for certain periods (10, 18, 22, 26, 31, and 34 weeks) and afterwards transferred to 16°C and re-incubated. The changes of their O₂-uptake at 25°C were traced throughout their chilling and successive re-incubating periods.Oxygen consumption is greatly accelerated during the cold treatment of the eggs. The low values of the diapause-level are raised progressively during the first 6 weeks of chilling. After this primary rapid increase, respiration remains at a level 5-times as high as the diapause values over a period up to the 25th week at 5°C. This is almost exactly the duration of mesodiapause (6 months).The rates of O₂-uptake during the subsequent re-incubation at 16°C depend on the extent of chilling. The ability of diapause-breaking is correlated to the rates of O₂-uptake, measured after setting of re-incubation. If respiration never decreases by the onset of re-incubation, diapause is terminated in most of the eggs, and the rates of O₂-uptake increase as re-incubation goes on towards the emergence of the larvae (postdiapause period).A preliminary interpretation of the cold-stimulated O₂-uptake in diapausing Leptopterna-eggs is given.Dedicated to Prof. Dr. H. Precht (Kiel) on the occasion of his 65th birthday     

Local temperatures inferred from plant communities suggest strong spatial buffering of climate warming across Northern Europe

Recent studies from mountainous areas of small spatial extent (<2500 km²) suggest that fine‐grained thermal variability over tens or hundreds of metres exceeds much of the climate warming expected for the coming decades. Such variability in temperature provides buffering to mitigate climate‐change impacts. Is this local spatial buffering restricted to topographically complex terrains? To answer this, we here study fine‐grained thermal variability across a 2500‐km wide latitudinal gradient in Northern Europe encompassing a large array of topographic complexities. We first combined plant community data, Ellenberg temperature indicator values, locally measured temperatures (LmT) and globally interpolated temperatures (GiT) in a modelling framework to infer biologically relevant temperature conditions from plant assemblages within <1000‐m² units (community‐inferred temperatures: CiT). We then assessed: (1) CiT range (thermal variability) within 1‐km² units; (2) the relationship between CiT range and topographically and geographically derived predictors at 1‐km resolution; and (3) whether spatial turnover in CiT is greater than spatial turnover in GiT within 100‐km² units. Ellenberg temperature indicator values in combination with plant assemblages explained 46–72% of variation in LmT and 92–96% of variation in GiT during the growing season (June, July, August). Growing‐season CiT range within 1‐km² units peaked at 60–65°N and increased with terrain roughness, averaging 1.97 °C (SD = 0.84 °C) and 2.68 °C (SD = 1.26 °C) within the flattest and roughest units respectively. Complex interactions between topography‐related variables and latitude explained 35% of variation in growing‐season CiT range when accounting for sampling effort and residual spatial autocorrelation. Spatial turnover in growing‐season CiT within 100‐km² units was, on average, 1.8 times greater (0.32 °C km^?1) than spatial turnover in growing‐season GiT (0.18 °C km^?1). We conclude that thermal variability within 1‐km² units strongly increases local spatial buffering of future climate warming across Northern Europe, even in the flattest terrains.     

First bacterial chalcone isomerase isolated from Eubacterium ramulus

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.     

Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata

Nine isoforms of voltage-gated sodium channels (NaV) have been characterized and in excitable tissues they are responsible for the initiation and conduction of action potentials. For primary afferent neurons residing in dorsal root ganglia (DRG), individual neurons may express multiple NaV isoforms extending the neuron’s functional capabilities. Since expression of NaV isoforms can be differentially regulated by neurotrophic factors we have examined the functional consequences of exposure to either nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) on action potential conduction in outgrowing cultured porcine neurites of DRG neurons. Calcium signals were recorded using the exogenous intensity based calcium indicator Fluo-8®, AM. In 94 neurons, calcium signals were conducted along neurites in response to electrical stimulation of the soma. At an image acquisition rate of 25 Hz it was possible to discern calcium transients in response to individual electrical stimuli. The peak amplitude of electrically-evoked calcium signals was limited by the ability of the neuron to follow the stimulus frequency. The stimulus frequency required to evoke a half-maximal calcium response was approximately 3 Hz at room temperature. In 13 of 14 (93%) NGF-responsive neurites, TTX-r NaV isoforms alone were sufficient to support propagated signals. In contrast, calcium signals mediated by TTX-r NaVs were evident in only 4 of 11 (36%) neurites from somata cultured in GDNF. This establishes a basis for assessing action potential signaling using calcium imaging techniques in individual cultured neurites and suggests that, in the pig, afferent nociceptor classes relying on the functional properties of TTX-r NaV isoforms, such as cold-nociceptors, most probably derive from NGF-responsive DRG neurons.     

Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10

The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.     

Plasticity and acclimation to light reflected in temporal and spatial changes of small-scale macroalgal distribution in a stream

The small-scale distribution pattern of macroalgae in the river Ilm, in Germany was monitored. These patterns were then related to abiotic factors and tested to discover whether the distribution of the common macroalgae, Cladophora glomerata (L.) Kütz. and Vaucheria sp., was linked to differences in their photosynthetic plasticity. Cladophora glomerata revealed higher maximum photosynthetic electron transport rates after acclimation to high light (HL) compared with low light (LL) acclimated samples. By contrast, Vaucheria sp. did not acclimate to different growth light conditions. The photosynthetic performance of both algae also varied according to diurnal conditions. High light caused a reversible decrease of the dark-adapted quantum yield (F(v)/F(m)) in C. glomerata and a concomitant reversible decrease of the light-adapted quantum yield (DeltaF/F'(m)). In Vaucheria sp., F(v)/F(m) remained mostly unchanged over the day, whereas DeltaF/F'(m) decreased during the morning at low light. Photosynthetic pigments confirmed acclimational differences between the species. HL C. glomerata showed increased chlorophyll a:chlorophyll b ratios, and higher amounts of xanthophyll-cycle pigments compared with LL samples, whereas Vaucheria sp. did not reveal differences between the light treatments. While preferences for substrate size, water velocity, and depth are similar for C. glomerata and Vaucheria sp., the physiological responses to light conditions are different. It is concluded that light conditions significantly affect the small-scale spatial distribution of macroalgae and that fitness is enhanced in species with a higher plasticity in photosynthetic acclimation in unstable environments.