Successful predation of filamentous bacteria by a nanoflagellate challenges current models of flagellate bacterivory

Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate, Ochromonas sp. strain DS, to test these assumptions. The five strains, including one spirochete and four Betaproteobacteria strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 micro m. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.     

Studies on atrophic change of soleus muscle and its countermeasures in suspended rat

In the environment of microgravity, the disused atrophy of skeletal muscle, especially leg's muscle, would occur. The three purposes of this study were: 1. To observe the dynamic changes of disused atrophy of skeletal muscle under simulated weightlessness; 2. To approach the mechanism of disused atrophy of muscle; 3. To approach the countermeasures for reducing the degree of atrophy of muscle.     

The passive yet successful way of planktonic life: genomic and experimental analysis of the ecology of a free-living polynucleobacter population

Background

The bacterial taxon Polynucleobacter necessarius subspecies asymbioticus represents a group of planktonic freshwater bacteria with cosmopolitan and ubiquitous distribution in standing freshwater habitats. These bacteria comprise <1% to 70% (on average about 20%) of total bacterioplankton cells in various freshwater habitats. The ubiquity of this taxon was recently explained by intra-taxon ecological diversification, i.e. specialization of lineages to specific environmental conditions; however, details on specific adaptations are not known. Here we investigated by means of genomic and experimental analyses the ecological adaptation of a persistent population dwelling in a small acidic pond.

Findings

The investigated population (F10 lineage) contributed on average 11% to total bacterioplankton in the pond during the vegetation periods (ice-free period, usually May to November). Only a low degree of genetic diversification of the population could be revealed. These bacteria are characterized by a small genome size (2.1 Mb), a relatively small number of genes involved in transduction of environmental signals, and the lack of motility and quorum sensing. Experiments indicated that these bacteria live as chemoorganotrophs by mainly utilizing low-molecular-weight substrates derived from photooxidation of humic substances.

Conclusions

Evolutionary genome streamlining resulted in a highly passive lifestyle so far only known among free-living bacteria from pelagic marine taxa dwelling in environmentally stable nutrient-poor off-shore systems. Surprisingly, such a lifestyle is also successful in a highly dynamic and nutrient-richer environment such as the water column of the investigated pond, which was undergoing complete mixis and pronounced stratification in diurnal cycles. Obviously, metabolic and ecological versatility is not a prerequisite for long-lasting establishment of abundant bacterial populations under highly dynamic environmental conditions. Caution should be exercised when generalizing the obtained insights into the ecology and adaptation of the investigated lineage to other Polynucleobacter lineages.     

Optimization of riboflavin production by recombinant <Emphasis Type="Italic">Bacillus subtilis</Emphasis> RH44 using statistical designs

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett–Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO₃, K₂HPO₄, ZnSO₄, and MnCl₂ were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.     

Recombinant expression of alpha-bungarotoxin in Pichia pastoris facilitates identification of mutant toxins engineered to recognize neuronal nicotinic acetylcholine receptors

A snake venom-derived alpha-neurotoxin, alpha-bungarotoxin (alphaBgtx), is the classic competitive antagonist of nicotinic acetylcholine receptors (nAChRs). The very high specificity and essentially irreversible binding of alphaBgtx to various nAChRs make alphaBgtx the prime candidate for studying the molecular determinants of specificity for nAChR-ligand interactions. To facilitate site-directed mutagenesis of alphaBgtx for functional analysis, we have developed a recombinant expression system for alphaBgtx using the methylotropic yeast Pichia pastoris. A synthetic gene coding for alphaBgtx was subcloned into an expression vector that directs secretion of the recombinant alphaBgtx (rBgtx) when stably integrated into the yeast genome. Expression of rBgtx was induced by growth of yeast cultures with methanol as the sole carbon source. The activity of the rBgtx in the cell-free medium was measured by competition with 1251-Bgtx for binding to Torpedo nAChR-enriched membranes. The rBgtx, purified to homogeneity by standard HPLC, has the correct predicted amino terminal sequence and molecular mass. Its circular dichroism spectrum is very similar to that of authentic venom-derived alphaBgtx, and the biological activity of the rBgtx is identical to that of authentic alphaBgtx. We have used the Pichia expression system to study a double point mutation of alphaBgtx, rBgtx-K38P/L42Q, that has a high affinity for alpha3beta2 neuronal nAChRs. This is the first demonstration of engineering an alpha-neurotoxin to recognize non-alpha7 neuronal nicotinic receptors.     

Effects of shark hepatic stimulator substance on the function and antioxidant capacity of liver mitochondria in an animal model of acute liver injury

This study was carried out to investigate whether shark hepatic stimulator substance （HSS） can prevent acute liver injury and affect mitochondrial function and antioxidant defenses in a rat model of thioacetamide （TAA）-induced liver injury. The acute liver injury was induced by two intraperitoneal injections of TAA （400 mg/kg） in a 24 h interval. In the TAA plus shark HSS group, rats were treated with shark HSS （80 mg/kg） 1 h prior to each TAA injection. In this group, serum liver enzyme activities were significantly lower than those in the TAA group. The mitochondrial respiratory control ratio was improved, and the mitochondrial respiratory enzyme activities were increased in the TAA plus shark HSS group. The mitochondrial antioxidant enzyme activities and glutathione level were higher in the TAA plus shark HSS group than in the TAA group. These results suggest that the protective effect of shark HSS against TAA-induced acute liver injury may be a result of the restoration of the mitochondrial respiratory function and antioxidant defenses and decreased oxygen stress.     

Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.