Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.     

Environmentally induced phenotypic plasticity and DNA methylation changes in a wild potato growing in two contrasting Andean experimental gardens

DNA methylation can be environmentally modulated and plays a role in phenotypic plasticity. To understand the role of environmentally induced epigenetic variation and its dynamics in natural populations and ecosystems, it is relevant to place studies in a real-world context. Our experimental model is the wild potato Solanum kurtzianum, a close relative of the cultivated potato S. tuberosum. It was evaluated in its natural habitat, an arid Andean region in Argentina characterised by spatial and temporal environmental fluctuations. The dynamics of phenotypic and epigenetic variability (with Methyl Sensitive Amplified Polymorphism markers, MSAP) were assayed in three genotypes across three growing seasons. These genotypes were cultivated permanently and also reciprocally transplanted between experimental gardens (EG) differing in ca. 1000 m of altitude. In two seasons, the genotypes presented differential methylation patterns associated to the EG. In the reciprocal transplants, a rapid epigenomic remodelling occurred according to the growing season. Phenotypic plasticity, both spatial (between EGs within season) and temporal (between seasons), was detected. The epigenetic and phenotypic variability was positively correlated. The lack of an evident mitotic epigenetic memory would be a common response to short-term environmental fluctuations. Thus, the environmentally induced phenotypic and epigenetic variation could contribute to populations persistence through time. These results have implications for understanding the great ecological diversity of wild potatoes.     

Cross-scale quality assessment of a mechanistic cation exchange chromatography model

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches.     

A depth-dependent model of the pericellular microenvironment of chondrocytes in articular cartilage

Experimental studies suggest that the magnitude of chondrocyte deformation is much smaller than expected based on the material properties of extracellular matrix (ECM) and cells, and that this result could be explained by a structural unit, the chondron, that is thought to protect chondrocytes from large deformations in situ. We extended an existing numerical model of chondrocyte, ECM and pericellular matrix (PCM) to include depth-dependent structural information. Our results suggest that superficial zone chondrocytes, which lack a pericellular capsule (PC), are relatively stiff, and therefore are protected from excessive deformations, whereas middle and deep zone chondrocytes are softer but are protected by the PC that limits cell deformations in these regions. We conclude that cell deformations sensitively depend on the immediate structural environment of the PCM in a depth-dependent manner, and that the functional stiffness of chondrocytes in situ is much larger than experiments on isolated cells would suggest.     

Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: Practical experiences

One of the most attractive segments in food and cosmetic industries is that of natural pigments. Since some synthetic pigments have been reported to be hazardous for humans, natural pigments obtained through biotechnological processes represent an attractive alternative. Our research group has previously worked on the development of an aqueous two-phase system (ATPS)-based prototype process for the recovery of B-phycoerythrin (BPE), a natural high-value pigment obtained from Porphyridium cruentum. Detailed studies describing the scaling up of ATPS processes from bench scale to pilot plant facilities are not common. In this paper experiences derived from the scale-up of a previously developed process for production and recovery of highly purified (purity defined as the absorbance ratio A₅₄₅/A₂₈₀ > 4) BPE are described, where a scale-up factor of 850× was implemented. Characterization of cell disruption with a pilot-scale bead mill allowed efficient BPE release at 2900 rpm, 10% (w/v) sample load, 60% (v/v) bead load and 0.5 mm glass beads and 22 min of residence time with a yield of 1.35 mg BPE/g of wet biomass. BPE was recovered and purified using a strategy comprising isoelectric precipitation, aqueous two-phase fractionation and ultrafiltration. A 54% global BPE recovery yield, with final purity of 4.1, was achieved under optimal process conditions. Considering total costs for raw materials and energy expenditures for one batch, it was determined that the production cost of BPE was of $1.17 USD/mg, which is underneath the commercial price of a BPE standard (>$30 USD/mg).     

Environmental gradients shift the direction of the relationship between native and alien plant species richness

Aim

To assess how environmental, biotic and anthropogenic factors shape native–alien plant species richness relationships across a heterogeneous landscape.

Location

Banks Peninsula, New Zealand.

Methods

We integrated a comprehensive floristic survey of over 1200 systematically located 6 × 6 m plots, with corresponding climate, environmental and anthropogenic data. General linear models examined variation in native and alien plant species richness across the entire landscape, between native‐ and alien‐dominated plots, and within separate elevational bands.

Results

Across all plots, there was a significant negative correlation between native and alien species richness, but this relationship differed within subsets of the data: the correlation was positive in alien‐dominated plots but negative in native‐dominated plots. Within separate elevational bands, native and alien species richness were positively correlated at lower elevations, but negatively correlated at higher elevations. Alien species richness tended to be high across the elevation gradient but peaked in warmer, mid‐ to low‐elevation sites, while native species richness increased linearly with elevation. The negative relationship between native and alien species richness in native‐dominated communities reflected a land‐use gradient with low native and high alien richness in more heavily modified native‐dominated vegetation. In contrast, native and alien richness were positively correlated in very heavily modified alien‐dominated plots, most likely due to covariation along a gradient of management intensity.

Main conclusions

Both positive and negative native–alien richness relationships can occur across the same landscape, depending on the plant community and the underlying human and environmental gradients examined. Human habitat modification, which is often confounded with environmental variation, can result in high alien and low native species richness in areas still dominated by native species. In the most heavily human modified areas, dominated by alien species, both native and alien species may be responding to similar underlying gradients.

     

Biology and ecology of Brazilian elodea (Egeria densa) and its specific herbivore, Hydrellia sp., in Argentina

Egeria densa (Hydrocharitaceae) is a submerged macrophyte from South America that is a weed in several countries. It crowds out native plants and hinders water use, causing economic and environmental damage. The leafminer fly Hydrellia sp. 1 (Diptera: Ephydridae), was found feeding in E. densa throughout its Argentine distribution, and is currently the only known specialist herbivore of E. densa. It was reared in the laboratory and tested on 25 plant species. This herbivore can cause heavy defoliation in the laboratory and in the field. Hydrellia sp. 1 was found only on E. densa, but in the laboratory it also developed on two other Hydrocharitaceae species in the same family; Egeria naias, and Elodea callitrichoides. Significant oviposition and feeding were only observed on its primary natural host, and to a lesser degree on E. naias. Field studies indicate Hydrellia sp. 1 is present in the field year round, unless the host plant is prostrate for long periods, or covered by floating macrophytes. These results indicate Hydrellia sp. 1 may be a suitable biocontrol candidate for E. densa.     

Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.