Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells

Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPo1 expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximately 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximately 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximately 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AlkB.     

Petrobactin is the Primary Siderophore Synthesized by <Emphasis Type="BoldItalic">Bacillus anthracis</Emphasis> Str. <Emphasis Type="BoldItalic">Sterne</Emphasis> under Conditions of Iron Starvation

The siderophores of Bacillus anthracis are critical for the pathogen’s proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of ¹H and ¹³C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.     

Epiphytic vegetation on pollarded trunks of Fraxinus excelsior in four different habitats at Grinde,Leikanger, western Norway

The epiphytic vegetation on 24 pollarded trees of Fraxinus excelsior at the farm Grinde, Leikanger, western Norway was investigated. Each trunk was divided into a basal zone, a middle zone and a top zone. In each zone the four different aspects were analysed (12 sampling units from each trunk). Within a total of 276 sampling units, 162 taxa were recorded (99 lichens, 56 bryophytes, 7 vascular plants). The trunks were covered mainly by an old, thick and occasionally swollen bark, but decaying wood did not occur. Their habitats were different, and each trunk was classified into one of four categories: open meadow, wooded hay meadow, deciduous wood, and spruce plantation. A climate station was established in each habitat to measure important parameters. The floristic and environmental data were analysed by canonical correspondence analysis (CCA). The floristic data were classified into eight TWINSPAN groups that have been taken into account in the CCA diagrams. At Grinde all the pollarded trunks grew under fairly homogeneous conditions during a more extensive agricultural period until about 1962. The deciduous wood developed by tree colonization on old meadows and wooded hay meadows, whilst spruce has been planted in a small part of the area. Floristic differences in the epiphytic vegetation between the four different habitats were found, which suggests that changes in the vegetation have developed during the last two or three decades. The spruce plantation was the most shady habitat having a very sparse epiphytic vegetation, mainly remnants from vegetation established during more open area conditions.     

Migration and stress during reproduction govern telomere dynamics in a seabird

Changes in telomere length are believed to reflect changes in physiological state and life expectancy in animals. However, much remains unknown about the determinants of telomere dynamics in wild populations, and specifically the influence of conditions during highly mobile life-history stages, for example migration. We tested whether telomere dynamics were associated with migratory behaviour and/or with stress during reproduction in free-living seabirds. We induced short-term stress during reproduction in chick-rearing, black-legged kittiwakes (Rissa tridactyla), tracked winter migration with geolocators and measured telomere length before and after winter migration. We found that time spent at wintering grounds correlated with reduced telomere loss, while stress during reproduction accelerated telomere shortening. Our results suggest that different life-history stages interact to influence telomere length, and that migratory patterns may be important determinants of variation in an individual''s telomere dynamics.     

Termites, Large Herbivores, and Herbaceous Plant Dominance Structure Small Mammal Communities in Savannahs

Large herbivores and termites are important functional groups in African savannahs. Both groups affect small mammals, which are also important determinants for savannah structure and function. Because vegetation on Macrotermes mounds are preferentially grazed by large herbivores, and mounds represent resource-rich distinct habitat patches for small mammals in relatively resource-poor savannahs, termite mounds are ideal sites for studies of how grazing by large mammals and productivity affect communities of small mammals. We conducted an experiment in Lake Mburo National Park, Uganda, with four treatments: large vegetated Macrotermes mounds (with and without large herbivores) and adjacent savannah areas (with and without large herbivores). We replicated the treatment blocks nine times and trapped small mammals regularly over a period of almost 2 years. Small mammal species assemblages differed considerably between mounds and savannah areas. Grazing had a substantial effect on small mammal species assemblages in the resource-poor savannah, but not in the relatively resource-rich termitaria. Small mammal species abundance, biomass, and richness were higher on termite mounds than adjacent savannah areas. Excluding large herbivores caused a major increase in species abundance, biomass, and richness both on savannah and termitaria. Herbaceous plant species evenness was an important determinant of the small mammal community. Small mammal biomass increased with high plant dominance, indicating that a few dominant plant species are important for biomass production of small mammals. Small mammal diversity was not related to any of the treatments, but increased with plant species evenness as well as richness. Fencing increased species dominance in the small mammal community on both savannah and termitaria, probably because competitive patterns shift from inter-guild (that is, between large and small mammals) to intra-guild (that is, between small mammals) when large mammals are excluded. The study highlights the complex interactions among large herbivores, termites, herbaceous plants, and small mammals in African savannahs. When studying the structure and function of small mammal communities it is therefore important to consider several coexisting functional groups.     

The photosynthetic performance of a cultivated Japanese green alga Caulerpa lentillifera in response to three different stressors,temperature, irradiance,and desiccation

The effects of temperature, irradiance, and desiccation on the photosynthesis of a cultivated Japanese green alga Caulerpa lentillifera (Caulerpaceae) were determined by a pulse amplitude modulation (PAM)-chlorophyll fluorometer and dissolved oxygen sensors. The photochemical efficiency in the photosystem II (F_v/F_m and ΔF/F_m') during the 72-h temperature exposures (8, 12, 16, 20, 24, 28, 32, 36, and 40°C) was generally stable at 16–32°C but quickly dropped at lower and higher temperatures. The photosynthesis–temperature curve at 200 μmol photons m^?2 s^?1 also revealed that the maximum gross photosynthesis (GP_max) occurred at 30.7°C (30.5–30.9, 95% highest density credible intervals). Photosynthesis–irradiance curves at 16, 24, and 32°C quickly saturated, then expressed photoinhibition, and revealed that the maximum net photosynthetic rates (NP_max) and saturation irradiance (E_k) were highest at 32°C and lowest at 16°C. Continuous 6-h exposure to irradiances of 200 (low) and 400 (high) μmol photons m^?2 s^?1 at 16, 24, and 32°C expressed greater declines in their ΔF/F_m' at 16°C, revealing chronic chilling-light stress. The response to continuous desiccation (~480 min) under 50% humidity at 24°C showed that ΔF/F_m' dropped to zero at 480-min aerial exposure, and the treatments of more than 60-min desiccation did not return to the initial level even after 24-h subsequent rehydration in seawater. Likewise, ΔF/F_m' fell when the absolute water content (AWC) of the frond dropped below AWC of 90% and mostly did not return to the initial level even after 24-h subsequent rehydration in seawater, signifying a low tolerance to desiccation.

     

Spatial regulation of MCAK promotes cell polarization and focal adhesion turnover to drive robust cell migration

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.